Research ArticleComparison of Signaling Pathways Gene Expression in CD34minus

Umbilical Cord Blood and Bone Marrow Stem Cells

RafaB Stojko12 Monika Bojdys-Szyndlar2 Agnieszka Drosdzol-Cop12

Andrzej Madej1 and Krzysztof Wilk2

1School of Health Sciences in Katowice Chair of Womanrsquos Health Medical University of Silesia Ulicy Medykow 1240-752 Katowice Poland2Department of Obstetrics and Gynecology The Boni Fratres Catoviensis Hospital Ulica Księdza Leopolda Markiefki 8740-211 Katowice Poland

Correspondence should be addressed to Agnieszka Drosdzol-Cop cor111pocztaonetpl

Received 12 July 2015 Revised 30 August 2015 Accepted 16 September 2015

Academic Editor Maria A Rueger

Copyright copy 2016 Rafał Stojko et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The aim of the study was to compare the biological activity of the total pool of genes in CD34minus umbilical cord blood and bonemarrow stem cells and to search for the differences in signaling pathway gene expression responsible for the biological processesThe introductory analysis revealed a big similarity of gene expression among stem cells When analyzing GO terms for biologicalprocesses we observed an increased activity of JAK-STAT signaling pathway calcium-mediated cytokine-mediated integrin-mediated signaling pathway and MAPK in a cluster of upregulating genes in CD34minus umbilical cord blood stem cells At the sametime we observed a decreased activity of BMP signaling pathways TGF-beta pathway and VEGF receptor signaling pathway in acluster of downregulating genes in CD34minus umbilical cord blood stem cells In accordance with KEGG classification the cytokine-cytokine receptor interaction toll-like receptor signaling pathway and JAK-STAT signaling pathway are overrepresented in CD34minusumbilical cord blood stem cells A similar gene expression in both CD34minus UCB and BM stem cells was characteristic for suchbiological processes as cell division cell cycle gene expression mitosis telomere maintenance with telomerase RNA and DNAtreatment processes during cell division and similar genes activity of Notch and Wnt signaling pathways

1 Introduction

In recent years the scientific environment has expressed agreat interest in the nonhematopoietic stem cells (CD34minus andCD45minus) These stem cells are capable of replicating in vitrowithout adding any growth factors in the period of morethan 10 passages and when induced properly differentiateinto at least three types of mesoderm layer cells osteoblastsadipocytes and chondrocytes [1 2] They are frequentlyreferred to as the mesenchymal stem cells (MSCs) Due totheir role in tissue repair processes their clinical potential forsystemic and local transplantation procedures is significantboth as a carrier in gene therapy and for generating tissuesand organs in tissue engineering procedures

Studies published to date have stressed that the MSCsof bone marrow and of fetal origin are very similar inimmunophenotypical and immunohistochemical function

The analysis of surface antigen markers by flow cytometrydid not reveal any significant differences [3ndash5] among bonemarrow and fetal MSCs Panepucci et al [5] showed thatthe MSCs of bone marrow and umbilical cord blood revealsimilarities among a thousand of most expressed transcriptsassayed However differences are seen at the molecularlevel in gene expression profiles of MSCs coming fromdifferent sources For example a distinct expression profilewas characteristic for genes related to antimicrobial activityand to osteogenesis and this distinct expression profile wasmore common in the MSC population from bone marrowIn the umbilical cord blood MSCs higher expression wasobserved for signaling pathway genes that participate inmatrix remodeling through metalloproteinases and genesrelated to angiogenesis Similar results were demonstratedin studies assessing the differentiation ability in comparablein vitro conditions The umbilical cord blood MSCs showed

Hindawi Publishing CorporationStem Cells InternationalVolume 2016 Article ID 5395261 13 pageshttpdxdoiorg10115520165395261

2 Stem Cells International

higher possibility of differentiation into osteogenic lineageand had little or no differentiation into adipocytes Thiscontrasted with bone marrow MSCs where expression ofmarkers characteristic for adipocytes was more frequentlydemonstrated [3 6] In the critical processes of regulatingself-renewal and the cellular purpose stem cells use thesignaling pathways which appear to be quite conservativefrom the evolutionary perspective such as Notch Wntand JAK-STAT Although the signaling proteins expressionis believed to be a highly restrictive process it appearsthat different stem cell types demonstrate varied rates ofexpression of these three families of signaling molecules Theglobal gene expression profile is commonly used to identifythe transcription signature of specific stem cells This signa-ture gives insight into the signaling mechanisms regulatingthe self-renewal and cellular purpose program especiallyin embryonic and hematopoietic stem cells Moreover bycomparing the gene expression profiles in different stemcell groups a common pool of genes were identified thatserve either as stem cells markers for self-renewal or directthe cells through differentiation [7ndash11] In comparison witha great number of studies carried out on the embryonichematopoietic or neural stem cells there are much fewerstudies of molecular mechanisms of MSC self-renewal anddifferentiation control mainly due to their diversified genesignature and the lack of agreement on typical markersantigens as far as some MSC markers are concerned [12ndash15]

This paper provides a comparison of the expression of theentire gene pool ofMSCmarkers with a special considerationto the signaling pathway genes in CD34minus stem cells whichphenotypically correspond with MSCs from the umbilicalcord blood and bone marrow The cells were extracted bymeans of the same single-bed room method on the basisof the same antigen phenotype Each cell population wasmultiplied three times in the same culture conditions Geneactivity was defined through the oligonucleotide microarrayswith the use of GO and KEGG databases We analyzed thenonhematopoietic stem cell signature based on the geneactivity of the conservative signaling pathways includingWnt and Notch We then asked the question whether dif-ferences in the signaling pathways for gene activity may beevidence of different populations of origin for theMSCs (egfetal verses adult origin) and consequently the predominanceof one population over the other Does a source of populationwhich undoubtedly shapes the cell epigenetic conformationhave a significant impact on its subsequent biological activity

2 Materials and Methods

Material for the study of gene expression by means ofoligonucleotide microarrays was sampled from CD34minus stemcell cultures from the bonemarrow and umbilical cord bloodComplete RNA was extracted from the abovementionedcultures

21 Obtaining Umbilical Blood Samples Samples of umbilicalblood were obtained after the delivery and cord clamping inthe cases of normal course of pregnancy and delivery after the

37th week The blood was obtained intranatally that is afteromphalotomy and before placenta expulsion

22 Obtaining Bone Marrow Samples For the purpose ofthe study approximately 6mL of bone marrow was obtainedfrom healthy donors during the procedure of collectingmaterial for hematological purposes

The research program was approved by the BioethicalCommittee of the Medical University of Silesia in KatowicePoland All participants provided their written informedconsent to participate in this study

23 Isolation of CD34minus Cells from Umbilical Cord Bloodand Bone Marrow Freshly collected umbilical cord bloodand bone marrow were diluted with phosphate-bufferedsaline (PBS Gibco) layered on Ficoll (Sigma) at 2 1 andcentrifuged for 40min at 400timesg at 20∘C The lymphocyticskin obtained after centrifugation was suspended in the basewith an addition of serum and centrifuged again for 10min at200timesg at 20∘C All the cell sample preparation activities wererepeated twice The prepared cells were divided into CD34+and CD34minus fractions by means of the CD34 Progenitor CellIsolation Kit and the MiniampMidiMACS Starting Kit Cellsviability was determined in the fluorescent microscope studyafter staining the cells with ethidium bromide and acridineorange

24 CD34minus Cell Culture

241 Cell Labeling The cells were suspended in the appro-priate volume of buffer minus03mL per each 108 of cells 01mLof FcR Blocking Reagent was added to the cell suspension of3 times 107mL density with the ratio of 01mL FcR BR03mLcell suspension For each 03mL of cell suspension 01mL ofCD34 Microbeads was then added The material was mixedthoroughly and incubated for 30min at 6ndash12∘C 20mL ofbuffer was added and mixed thoroughly again The mixturewas centrifuged for 10min at 400 g The supernatant wasdiscarded and the sediment was suspended in 1mL of buffer

242 Cell Separation in Magnetic Column LS column wasplaced in the magnetic field The column was rinsed with3mL of buffer and the cell mixture as prepared abovewas added to the column After the suspension had passedthrough the column the CD34minus fraction was obtained Thecolumn was washed with buffer (2x 05mL) removed fromthemagnetic field and then placed in a 5mL tube Next 1mLof buffer was applied to the column The cells were elutedby means of a piston In this way the CD34+ fraction wasobtained

243 Starting the Culture The separated CD34minus cell frac-tions were used for cell cultures The IMDM-ISCOVErsquoSmodified Dulbeccorsquos medium with penicillin-streptomycinand 5 human albumin were used for culturing

244 Total RNA Extraction from Cell Culture RNA wasextracted from the CD34minus cell cultures with the Total RNA

Stem Cells International 3

PrepPlus (AampABiotechnology) based on themodifiedChom-czynski and Sacchirsquos method At the initial stage the cellswere lysed while the endogenous RNases were inactivatedwith fenozol After adding chloroform the mixture wascentrifuged and the collected RNA was precipitated withisopropanol Next the mixture was centrifuged for 20minat 13 000 rmin the supernatant was decanted and the RNAsediment was washed twice with 75 ethanol After dryingthe sediment was stored at minus70∘C for the analysis

25 Qualitative Assessment of RNA Extracts RNA extractsunderwent qualitative assessment via the agarose gel elec-trophoresis in 08 agarose gel with ethidium bromide(05mgmL) in the SUBMINI apparatus 5120583L of extract wasmixed with 3 120583L of gel-loading dye (005 wv bromophenolblue 60 glycerol) and heated at 65∘C for 4min After theseparation the electropherograms were assessed with the UVtransilluminator (120582 = 260 nm)

26 Quantitative Assessment of RNA Extracts RNA extractsunderwent quantitative assessment via spectrophotomet-ric measurement of RNA concentration (Gene Quant IIPharmacia) The spectrophotometric assessment of extractscovered the absorbance measurement at the wave lengthsof 230 260 280 and 320mn and then the A

260

A280

ratio and protein content were determined for each sampleAbsorbance value at the 260 nm wave length was used tocalculate RNA concentration based on the assumption thatresults of cuvette measurements using the 1 cm optical pathequaling 1 OD

260

correspondwith the concentration of 40mgRNA in 1 cm3 of extract

27 Gene Expression Analysis with the OligonucleotideMicroarrays Technique

271 General Method Assumptions The material for thestudy consisted of total RNA extracted from the cell cultureswith the TRIzol reagent (Invitrogen) The isolated RNA waspurified with the RNeasy Total RNA Mini Kit and digestedwith DNase I (Qiagen) in order to eliminate any possiblecontamination with DNA Approximately 8 120583g of total RNAwas used for the double-stranded cDNA synthesis Biotin-labeled cRNA was synthesized and biotinylated cRNA wasthen fragmented and hybridized with the Test3 microarrayand HG U133A (Affymetrix) and labeled twice with thestreptavidin-phycoerythrin conjugate and biotinylated strep-tavidin antibodies

The fluorescence intensity was analyzedwith theGeneAr-ray Scanner G2500A The quantity and quality of total RNAcDNA and cRNA were assessed spectrophotometrically andby means of the electrophoresis technique in 1 agarose gel

272 Purification of Total RNA Extract with RNasy Mini KitAfter a prior concentration measurement 100120583L of the RNAextract was obtained with RNase-free water 350 120583L of RLTbuffer with 120573-mercaptoethanol and 250120583L of 96 ethanolwere added mixed by pipetting and then applied to thecolumn and centrifuged for 15 seconds at 12 000 rmin The

filtrate was discarded and the column was moved to a newtube and 350 120583L of RW1 was added and then centrifugedfor 15 seconds at 10ndash12 000 rmin For digestion 70 120583L ofRDD buffer was added to a tube with 10120583L of DNase Thismixture was applied on the column membrane and left todigest for 15min at room temperature Then 350 120583L of RW1solution was added the old flow-through was discarded andthe column was centrifuged for 15 sec at 12 000 rmin Theflow-throughwas again discarded and 500120583L of RPE solutionwas added to the column The extracts were centrifuged for1min at 14 000 rmin then the column was turned upsidedown and centrifuged again for the same time and rate Aftercentrifugation the column was placed in a new tube and30 120583L of H

2

O was added and was left for 10min Then thecolumn was centrifuged and 20120583L of H

2

O was added andleft for a few minutes and then centrifuged again After finalcleanup the RNA concentration in extracts was measured

28 cDNA Synthesis

281 First Strand cDNA Synthesis In order to obtain the firststrand of cDNA 2 120583L 100 pM of T7 starter-oligo(dT)

24

(51015840-GCCAGTGAATTGTAATACGACTCACTATAG GGAG-GCGG-31015840) was added to 11 120583L of RNA extract the mixturewas incubated at 70∘C for 10minutes and placed on iceThen4 120583L of 5x First Strand Buffer 2120583L of 01M DTT and 1 120583L of10mM dNTPs (Invitrogen) were added After 2 minutes ofpreincubation at 45∘C 1 120583L (200U) of reverse transcriptaseSupercscript II (Life Technologies) was added to the reactionmixture and incubation continued for another hour

282 Second Strand cDNA Synthesis In order to obtain thesecond strand of cDNA the following were added to themixture obtained after the first strand synthesis 30 120583L of5x Second Strand Buffer 91 120583L of RNase-free water 3 120583L of10mM dNTPs 4 120583L (40U) of E coli DNA Polymerase I1 120583L (10U) of E coli DNA Ligase and 1 120583L (2U) of Rnase H(Invitrogen) The mixture was incubated for 2 hours at 16∘C

Next 25 120583L (10U) of T4 DNA Polymerase I was addedto the mixture and the mixture was incubated for another5 minutes at 16∘C The composition of the reaction mixtureused for the second strand cDNA synthesis is presentedin Table 2 The reaction was stopped by adding 10 120583L of05M EDTA and the double-stranded cDNA was extractedusing the phenolchloroformisoamyl alcohol mixture Theaqueous phase was divided bymeans of Phase Lock Gel Light15mL tubes (Eppendorf) 05 volume of 75M ammoniumacetate (Sigma) and 25 volume of 96 ethyl alcohol wereadded to the aqueous phase and it was left for 12 h at minus20∘C

29 Purification of the Obtained cDNA After freezing for theentire night at minus20∘C 03 120583L of Palet Point (Merc) was addedto the reaction mixture and it was centrifuged for 20min at12 000 rmin at room temperature supernatant was discardedand the sediment was rinsed with 2x 70 ethanol (500120583L)and centrifuged for 5min at 12 000 rmin the sediment wasdried thoroughly and dissolved in 12 120583L of RNase-free water

4 Stem Cells International

210 Biotinylated cRNA Synthesis The template for thebiotinylated cRNA synthesis consisted of 10 120583L ds cDNAto which the following were added 12 120583L of RNase-freewater 4 120583L of 2x hybridization buffer 4 120583L of biotin-labeledribonucleotides 4 120583L of DTT 4 120583L RNase inhibitor and2 120583L of T7 polymerase (BioArray High Yield RNA TranscriptLabeling Kit Enzo) The whole was left at 37∘C for 5 h andmixed every 30 minutes

211 Biotinylated cRNA Purification After a 5-hour incuba-tion cRNA was cleaned up using 60 120583L of H

2

O 350 120583L ofRLT buffer and 250 120583L of 96 ethanol added to the reactionmixture The mixture was applied onto the RNAeasy columnTotal RNA Mini Kit (Qiagen) and was centrifuged twice for15 seconds at 10 000 rmin In the last stage 2x 20120583L of H

2

Owas added to the column membrane it was centrifuged at12 000 rmin and then the cRNA concentration was mea-sured

212 Biotinylated cRNA Fragmentation For fragmentationpurposes 16 120583g of cRNA and 8 120583L of fragmentation buffer(Affymetrix) were used and the mixture was supplementedwith up to 40 120583L of RNase-free water After completion of thisstage the mixture was centrifuged and left for 35min at 94∘Cand then placed on iceThe fragmentation result was checkedon 1 agarose gel

213 Assessment of cDNA Biotinylated cRNA and cRNASynthesis after Fragmentation via Electrophoresis in AgaroseGel Evidence of proper cDNA biotinylated cRNA andcRNA synthesis after fragmentation was assessed throughelectrophoresis in agarose gel with ethidium bromide Theresulting separation electrophoregram of the abovemen-tioned products verified that samples were acceptable to usein the following analysis stage

214 Hybridization of the Fragmented BiotinylatedcRNA with Microarray

2141 Preparation of Hybridization Cocktail In order toprepare the hybridization cocktail 375 120583L of fragmentedbiotinylated cRNA was used and the following were added5 120583L of control oligonucleotide B2 15 120583L of eukaryotichybridization control (Affymetrix) 3120583L of Herring Sperm(Invitrogen) 3 120583L of acetylated BSA (Invitrogen) 150 120583L of2x hybridization buffer and 865 120583L of RNase-free water Thecocktail was heated for 5min at 99∘C and for 5min at 45∘Cand centrifuged for 5min at 14 000 rmin

2142 Application of Cocktail on Microarray HG-U133Amicroarray (Affymetrix)was heated to room temperature andthen 100 120583L of 1x hybridization buffer was applied The chipwas placed in hybridization oven for 10min Next the bufferwas removed from the chip and 200120583L of hybridizationcocktail was applied The microarray was placed in thehybridization oven for 16 h at 45∘C at 55 rmin

2143 Washing and Scanning of Microarrays After a 16-hour hybridization the HG U133A microarray was washedwith buffer and stained with solutions (SAPE solutionstreptavidin antibody solution) and then scanned with theGeneArray Scanner G2500A (Affymetrix)

215 Statistical Analysis Data regarding expression levelsobtained directly from the HGU133A array were reannotatedwith CDF control files developed by Dai et al [16] Thenext step consisted of microarray normalization with theRMA algorithm [17] Three repetitions were replaced withan average value with a prior detection of outliers by meansof the Dean and Dixon method [18] Determination of mea-surement noise threshold was conducted through empiricaldecomposition of probability density function of expressionlevels into normal components [19] The component of thelowest mean value unchanged across all the microarrays wasassumed to be the noise model The threshold value of thefilter was constructed at level 28 (in the field of logarithmsto base 2) The mixture model of normal distributions con-structed for the average SRL (Signal Log Ratio) of CD34minus cellsfromumbilical cord blood versusCD34minus of bonemarrowwasused to define the affinity of certain genes to subgroups of anincreased decreased or unchanged value of expression levelMoreover thismixturemodel was used to determine the FDR(False Discovery Rate) value for each of them

In order to define qualitatively the processes in stemcells from umbilical cord blood in comparison with stemcells from bonemarrow an unsupervised gene clustering wasconducted Again the GaussianMixture Model for empiricaldecomposition of expression levels constituted the algorithmenabling such a division However in this analysis additionallimitations based on the gene composition of each compo-nent that were the same across the experimental conditionswere added to the algorithm [20] Clustersclasses obtainedin this manner were compared to cellular processes in whichthey are involved The categories of cellular processes weredefined by gene ontology terms that are commonly appliedfor this purpose A number of conditioning hypergeometrictests were conducted verifying the one-sided hypothesisabout overrepresentation For each ontological term thatwas found to be significantly overrepresented statisticallyin a given gene cluster the OR (Odds Ratio) value wasdetermined

Gene pathways whose components are genes belongingto specific clusters underwent an additional analysis whichwas independent of a direct description of cellular processesFor this purpose we used data from the KEGG databaseFor each pathway the hypothesis of overrepresentation of aspecified cluster of genes as compared with the whole scopeof the analyzed genes was verified Precise 119901 values weredetermined

Statistically significant values were those of FDR lt 010 or119901 lt 005

3 Results

The first stage of the study consisted of data reannotation forexpression levels through the CDF control files developed

Stem Cells International 5

by Dai et al [16] For many years the platform for geneexpression profile was the Affymetrix GeneChip databaseHowever their probe section was created on the basis ofprior gene and transcriptome annotations which significantlydiffer from the modern expertise A comparative analysisbetween many popular Gene Chips and redefined probesbased on updated gene data with consideration to geneticpolymorphism showed a 30ndash50 discrepancy [16] thereforeit was decided to reannotate data as the first step in ourstudyThe assessment of noise threshold was then completedcreating a mixture model of normal distributions for themean SLR of CD34minus cells from umbilical cord blood versusCD34minus from bone marrow

The next step comprised microarrays normalizationthrough the RMA algorithm [17] Three repetitions werereplaced with a mean value with a prior detection of outliersby means of the Dean and Dixon method [18] Determina-tion of the measurement noise threshold was conducted byempirical decomposition of probability density function ofexpression levels into normal components [19] The noisemodel was assumed to be the component of the lowest meanvalue which was unchanged across all the microarrays Thethreshold value of the filter was constructed at level 28 (inthe field of logarithms to base 2)

As a result of the abovementioned procedures a data setwas obtained which described 11 979 genes which underwentfurther analysis The first stage consisted of creating the mix-ture model of normal distributions constructed for a meanSLR (Signal Log Ratio) of CD34minus stem cells from umbilicalcord blood versus CD34minus stem cells from bone marrowwhich allowed defining the affinity criteria of specific genes tosubgroups of an increased decreased and unchanged valueof expression levels This also enabled us to determine a FDRvalue (False Discovery Rate) for each of them (Figure 1)

This method is based on decomposition that is thehistogram decomposition into (K)mdashGaussrsquos components aswell as modeling the probability density function distribu-tions (pdf) for given gene expression levels The method wasused for analysis of a histogram reflecting the frequency ofgenes of a specific expression level in the tested preparationThis initial analysis showed considerable similarity of geneexpression of CD34minus cells from umbilical cord blood ascompared with CD34minus cells from bone marrow

31 Unsupervised Gene Clustering During the next stage ofdata analysis a qualitative determination of the processes inCD34minus stem cells from umbilical cord blood in comparisonwith CD34minus stem cells from bone marrow was conductedusing unsupervised gene clustering Again the GaussianMixture Model for empirical decomposition of expressionlevels constituted the algorithm enabling such a divisionHowever in this analysis additional limitations based onthe gene composition of each component that were thesame across the experimental conditions were added to thealgorithm [20] As a result 15 gene clusters were obtainedtwo of which (numbers 9 and 13 in this study) containeddiscriminatory genes exhibiting a statistically significantincrease (cluster number 9) and decrease of gene expression(cluster number 13) (Table 1)

1600

1400

1200

1000

800

600

400

200

00 2 4 6 8

Num

ber o

f obs

erva

tions

Gene expressionminus10 minus8 minus6 minus4 minus2

SLR CD34H adnotacje Dai ver12

Figure 1Mixturemodel of normal distributions (GaussianMixtureModel) for a mean SLR (Signal Log Ratio) of CD34minus cells fromumbilical cord blood versus CD34minus cells from bone marrow Log(CD34-P)( CD34-Sz)

The remaining clusters differing in terms of the numberof genes within the cluster aggregated the genes of similarexpression values Differences betweenmean gene expressionlevels of CD34minus stem cells from bone marrow and CD34minusstem cells from umbilical cord blood in clusters numbered1ndash8 10ndash12 14 and 15 are small or nonexistent Thus all theseclusters were classified as the groups of genes exhibiting thesame expression levels

Cluster number 9 included 493 genes which show anincreased activity in CD34minus stem cells from umbilical cordblood of the mean SRL (Signal Log Ratio) equaling 306 Incontrast cluster number 13 included a lower number of genes(119899 = 387) of decreased expression in CD34minus stem cells fromumbilical cord blood for which SRL equals minus343

At the next stage gene clustersgroups obtained in thismanner were compared to biological processes in whichthey are involved as defined by gene ontology (GO) termsthat are commonly applied for this purpose A number ofconditioning hypergeometric tests were conducted verifyingthe one-sided hypothesis about overrepresentation For eachontological term that was found to be significantly overrep-resented statistically in a given gene cluster the OR (OddsRatio) value was determined All the clusters were classifiedthrough the GO classification system that is the clusterswith genes discriminating both cell populations as well as theclusters grouping genes of a similar expression

Four hundred ninety-three genes of cluster 9 classifiedvia the GO system determined the terms of biological pro-cesses (119901 lt 005) overrepresented in CD34minus stem cells fromumbilical cord blood The classification system determined256 GO terms of biological processes out of which GO termsof 119901 value below 004 underwent further analysis As a resultof this analysis the majority of GO terms represented only byone or two genes were rejected The GO terms for biologicalprocesses in cluster 9 were assigned to one of six func-tional groups including metabolism biological processes

6 Stem Cells International

Table 1 15 clusters obtained as a result of the unsupervised gene clusteringMean CD34minus BM (mean of CD34minus stem cells from bonemarrow)Mean CD34minus UCB (mean of CD34minus stem cells from umbilical cord blood)119873 (a number of genes in a cluster) SRL (Signal Log Ratio)

Cluster MeanCD34minus BM

MeanCD34minusUCB

SDCD34minusBM

SDCD34minusUCB

119873

119901 value MeanSLR

minus95CISLR

+95CISLR

Conclusion

1497 491 008 009

1102

000000023 minus006 minus008 minus004

No change2

1157 1149 035 047

54

026488907 minus008 minus022 006

No change3

802 805 030 032

592

063190010 001 minus005 007

No change4

445 440 006 005

999

000000001 minus006 minus008 minus004

No change5

702 714 024 021

898

000000009 011 007 016

No change6

554 550 012 013

1133

000000889 minus006 minus009 minus003

No change7

353 346 004 005

960

451861119864 minus 13 minus007 minus009 minus005

No change8

476 483 071 088

961

003102164 011 001 020

No change9 564 715 158 270 493 p lt 00000001 306 295 316 Up10

311 309 004 004

872

000325256 minus003 minus005 minus001

No change11

276 278 002 002

647

000002947 003 001 004

No change12

398 390 006 007

1133

544009119864 minus 15 minus008 minus010 minus006

No change13 739 486 285 101 387 p lt 00000001 minus343 minus357 minus329 Down14

621 621 026 021

1353

044902052 minus001 minus005 002

No change15

930 936 064 084

395

015369595 008 minus003 020

No change

0 001 002 003 004

Signal transduction (GO0007165)

Activation of MAPK activity (GO0000187)

Protein kinase cascade (GO0007243)

Regulation of MAP kinase activity (GO0043405)

Integrin-mediated signaling pathway (GO0007229)

Positive regulation of tyrosine phosphorylation ofSTAT protein (GO0042531)

Positive regulation of calcium-mediated signaling(GO0050850)

JAK-STAT cascade (GO0007259)

Cytokine-mediated signaling pathway (GO0019221)

Positive regulation of cell proliferation (GO0008284)

Calcium-mediated signaling (GO0019722)

Regulation of JAK-STAT cascade (GO0046425)

p value

Signaling pathway

Figure 2 Functional annotations 12 GO classification terms for biological processes in the group signaling pathways in cluster 9 dependingon the 119901 value (119901 lt 004)

regulation cellular homeostasis cellular defensive responseand response to stimuli cytokine production and signalingpathways The GO terms regarding signaling pathways arepresented in Figure 2

Thenext stage consisted of a similar analysis of genes fromcluster 13 GO system classification for 387 genes of cluster 13determined 119 terms of biological processes (119901 lt 005) of

a decreased activity in CD34minus cells fromumbilical cord bloodas compared with CD34minus cells from bone marrow The GOterms of 119901 value below 004 underwent further analysis As aresult of this analysis the majority of GO terms representedonly by one or two genes were rejected The GO terms forbiological processes in cluster 13 were assigned to one ofthree functional groups including metabolism biological

Stem Cells International 7

0000 0010 0020 0030

Negative regulation of signal transduction (GO0009968)

Vascular endothelial growth factor receptor signalingpathway (GO0048010)

Transmembrane receptor protein tyrosine kinase signalingpathway (GO0007169)

Transmembrane receptor protein serinethreonine kinasesignaling pathway (GO0007178)

Regulation of transforming growth factor beta receptorsignaling pathway (GO0017015)

Cell surface receptor linked signal transduction(GO0007166)

BMP signaling pathway (GO0030509)

p value

Signaling pathway

Figure 3 Functional annotations 7 GO classification terms for biological processes in the group signaling pathways in cluster 13 dependingon the 119901 value (119901 lt 004)

Table 2 Gene pathways based on the KEGG classification of an increased expression in CD34minus stem cells from umbilical cord blood

KEGGID 119901 value Odds ratio Exp count Count Size Term4060 0001 2947 14 33 223 Cytokine-cytokine receptor interaction4620 0001 3672 6 17 92 Toll-like receptor signaling pathway5120 0001 3748 4 12 63 Epithelial cell signaling inHelicobacter pylori infection4630 0012 2067 8 15 130 JAK-STAT signaling pathway

processes regulation and signaling pathways In CD34minus cellsfrom umbilical cord blood as compared with CD34minus cellsfrom bone marrow a decreased gene activity was observedof the BMP signaling pathway TGF beta transmembranereceptor of protein serinethreonine kinase transmembranereceptor of tyrosine kinase and VEGF (Figure 3)

Gene pathways whose components are genes belongingto specific clusters underwent an additional analysis whichwas independent of a direct description of cellular processesFor this purpose data from the KEGG database (KyotoEncyclopedia of Genes and Genomes) were used For eachpathway the hypothesis of overrepresentation of specificclusters of genes as compared with the whole scope ofanalyzed genes was verified and precise 119901 values weredetermined For the purpose of this study gene pathwaysfor clusters 9 and 13 were analyzed because they containedgenes discriminating both gene populations On the basisof KEGG database cytokine-cytokine receptor interactionpathway toll-like receptor signaling pathway epithelial cellsignaling in Helicobacter pylori infection and JAK-STATsignaling pathway are overrepresented in CD34minus cells fromumbilical cord blood (Table 2)

In cluster 13 on the basis of KEGG classification theCD34minus cells from umbilical cord blood show a decreasedactivity of focal adhesion signaling pathway cell junctionsand TGF-beta signaling pathway (Table 3)

At the next stage the genes of the remaining clustersnumbered 1ndash8 10ndash12 and 14 of a similar gene expression were

analyzed The analysis was based on the GO classification foreach of the abovementioned clusters The criteria used forchoosing the clusters included the biological processes con-nected with cellular cycle as well as the processes connectedwith signaling pathways activity A similar gene expression inCD34minus cells from umbilical cord blood and CD34minus cells frombone marrow was characteristic for such biological processesas cell division mitosis maintaining telomere endings RNAandDNA processing and a similar activity of Notch andWntsignaling pathways genes

An independent analysis was also carried out for genes ofa similar activity in clusters 1ndash8 10ndash12 and 14 on the basis ofthe KEGG database CD34minus cells from umbilical cord bloodand CD34minus cells from bone marrow showed similar activityof Wnt and Notch signaling pathways genes p53 signalingpathway genes cell cycle PPAR and mTOR (Table 4)

CD34minus cells from umbilical cord blood and CD34minus cellsfrom bone marrow showed similar gene activity of Wntand Notch signaling pathways both on the basis of the GOclassification system and the KEGG database Therefore atthe last stage of the study the activity of single genes of Wntand Notch as well as JAK-STAT signaling pathways chosenfrom 11976 sampled genes was analyzed

In accordance with the KEGG database the JAK-STATsignaling pathway is defined by 155 genes out of whichthere were 128 active genes in the studied material Genesexhibiting increased activity in CD34minus cells from umbilicalcord blood included SPRY2 CISH CSF2 GRB2 IFNGR1

8 Stem Cells International

Table 3 Gene pathways based on the KEGG classification of a decreased expression in CD34minus stem cells from umbilical cord blood

KEGGID 119901 value Odds ratio Exp count Count Size Term4510 0001 7517 7 33 188 Focal adhesion1430 0001 8157 4 22 109 Cell junctions4350 0001 4038 3 10 82 TGF-beta signaling pathway

Table 4 Gene pathways based on the KEGG classification of a similar expression in CD34minus stem cells from umbilical cord blood and CD34minuscells from bone marrow for clusters 1ndash8 10ndash12 and 14-15

KEGGID 119901 value Odds ratio Exp count Count Size Term04310 0018 1880 10 17 131 Wnt signaling pathway04330 0025 2722 3 7 39 Notch signaling pathway04115 0038 2157 5 9 61 p53 signaling pathway04110 0009 2028 10 18 100 Cell cycle03320 0020 2791 3 7 60 PPAR signaling pathway04150 0035 2220 5 9 46 mTOR signaling pathway

IL2RB IL2RG IL7R IL10RA IL15RA PIM1 PTPN6 STAT1STAT5A and CCND3 Genes exhibiting decreased activityincluded PIAS3 IL11RA and CBLB

On the basis of the KEGG database the Wnt signalingpathway is defined by 151 genes out of which there were132 active genes in the studied material Genes exhibit-ing increased activity in CD34minus cells from umbilical cordblood include MMP7 PRKCB PSEN1 RAC2 and CCND3Genes exhibiting decreased activity include DKK1 WNT5AWNT5B FZD7 and TCF7L1

On the basis of the KEGG database the Notch signalingpathway is defined by 47 genes out of which there were38 active genes in the studied material Genes exhibitingincreased activity in CD34minus cells from umbilical cord bloodincluded DTX4 and PSEN1

4 Discussion

Nonhematopoietic stem cells were first identified by Frieden-stein et al who described the progenitor cells of rat bonemarrow [21] Currently the nonhematopoietic stem cellswhich are frequently referred to although not quite correctlyas the mesenchymal stem cells are isolated from varioustissues including the umbilical cord blood placenta andumbilical cord A constantly increasing number of studieson MSCs have proved that in appropriate environmentalconditions they are capable of differentiating into special-ized mesodermal endodermal and even ectodermal celllineages [2 22ndash24] In natural conditions they play an impor-tant role in cell regeneration processes and in repairingthe tissues damaged by degenerating processes Anotherintriguing characteristic of MSCs is their ability to avoidimmunological identification and response therefore theyare referred to as the hypoimmunogenic cells Despite greatattention paid to mesenchymal stem cells their biology isnot yet fully understood Introducing multipotential stemcells into clinical application requires a full understandingof the mechanisms which control key properties of thecell such as cell self-replication versus differentiation and

mobilization versus tissue homing The understanding ofthese mechanisms must be based on strict definitions ofhuman multipotential stem cells at the molecular level Inaddition understanding of molecular mechanisms of self-replicating pluripotentiality plasticity and differentiation ofstem cells not only is a necessary condition to overcomelimitations of stem cells application as a therapeutic factorbut also is key to understand a fundamental mystery of thedevelopment of human organism

This paper presents a comparison of gene activity of thenonhematopoietic cells of umbilical cord blood and bonemarrow using oligonucleotidemicroarrays Gene activity wastested in stem cells CD34 and included 11 979 genes obtainedafter completing procedures to increase the purity of cellsamples Cell populations included in the study were alsolimited based on the noise threshold revealed in the finalcomparative analysis The gene expression profiles of thecompared populations of CD34minus stem cells from umbilicalcord blood and CD34minus stem cells from bone marrow werevery similar (Figure 1) For a better illustration of the biolog-ical molecular activity and the activity of signaling pathwaysin the studied cell populations a transcription profile analysiswith the GO and KEGG databases was completed In termsof fundamental signaling pathways for biological activityof stem cells like JAK-STAT Wnt and Notch we recordeddiscrete differences among expressed genes from umbilicalcord blood cells as compared to bone marrow cells

The analysis regarding signaling pathways showedincreased activity of the JAK-STAT pathway in CD34minusstem cells from umbilical cord blood both on the basis ofthe gene ontology classification and the KEGG database(Figure 2 and Table 2) The increased activity regarded 15genes which take part in the JAK-STAT cascade that isSPRY2 CISH CSF2 GRB2 IFNGR1 IL2RB IL2RG IL7RIL10RA IL15RA PIM1 PTPN6 and CCND3 including thegenes which code transcription factors STAT1 and STAT5AIn previous publications the significance of the JAK-STATsignaling pathway is best described in the embryonic andhematopoietic stem cells Ramalho-Santos et al searchedfor a transcription profile describing both the embryonic

Stem Cells International 9

stem cells and the stem cells of an adult organism on thebasis of the study of embryonic cells neural stem cellsand hematopoietic stem cells activity [25] These studiesattempted to create a molecular signature of the stem cells inrelation to the self-regeneration and differentiation abilities ofthe cells The active JAK-STAT pathway belongs to the basicattributes defining the so-called ldquostemnessrdquo phenomenon Inthe embryonic stem cells the JAK-STAT signaling pathwaypromotes proliferation in the form of self-replicationThe activating factor LIF (Leukemia Inhibitory Factor)provokes phosphorylation and dimerisation of STAT3which after translocation to the cell nucleus stimulates theexpression of gene group responsible for self-replication ofthe embryonic stem cell However the JAK-STAT signalingpathway is not the only factor responsible for proliferationregulation of the embryonic stem cells Internal processesregulating the multiplication processes independent of theSTAT3 activity such as the Nanog and Oct-4 activity arealso important [15 17 26 27] In our study material theNanog and Oct-4 genes were not active in both types ofnonhematopoietic stem cells which is in agreement withother publications devoted to this subject [28 29] TheSTAT1 transcription factor overactive in our study in CD34minuscells of umbilical cord blood is responsible among othersfor the expression of interferon-activated genes involved incell defense against pathogenic factors [30] Moreover itis believed that STAT1 is a medium in expression of genegroup which is important for cell viability in response tostimulating factors In 2006 Kimrsquos team described STAT1as a highly overactive gene in human mesenchymal stemcells [31] In comparison the transcription factor STAT5which functions in the form of two isoforms STAT5a andSTAT5b is involved in the cellular proproliferation responsesand antiapoptosis processes [32 33] STAT5 is activatedby various cytokine receptors In our study the isoformSTAT5A showed increased activity It is believed that theSTAT5 factor activates specifically the cellular cycle genesthat is cyclins [34 35] In human stem cells the STAT factorsactivate the expression of cyclin Dwhich codes the regulatingsubunit of the CYCDCDK4 complex driving the G1S phasetransition which in turn activates the cell cycle progression[32 36] STAT5a and STAT5b also play an important rolein expression of genes promoting hematopoietic stem cellssurvival [37] Gene expression profiling over a range ofSTAT5 activities in human CB cells revealed subsets of genesthat are associated with the self-renewal and long-termexpansion phenotype Taking into account the fact thatin STAT5minusminus mice myelopoiesis appears to be relativelyunaffected it can be assumed that STAT5 can play a keyrole in assessing the quality of cells [38] Additionally notonly is the JAK-STAT signaling pathway a significant signaltransduction component which controls cell proliferationand purpose but it also regulates cell migration which isimportant both in the primary processes of gastrulationand in the tissue regeneration For instance the JAK-STATsignaling pathway is connected with cardioprotection [39]as well as with processes of regeneration and remodeling ofskeleton [40 41] The active JAK-STAT signaling pathwayhelps to regulate cell organization within cell grouping

[42] The above results may suggest a greater self-renewalpotential of CD34minus stem cells of umbilical cord bloodlong-term expansion phenotype and their higher ability interms of tissue regeneration

The Wnt signaling pathway is perceived as a signalingcascade involved in the control of fundamental processesof nondifferentiated mesenchymal stem cells such as cellreproduction and directed differentiation Both methods ofgene activity classification that is GO and KEGG showeda similar activity of the majority of Wnt signaling pathwaygenes (Table 4) In addition the analysis of separate genestaking part in the Wnt signaling pathway and coding 19glycosylated molecules of extracellular signaling of the Wntfamily cellular surface receptors including Frizzled receptorsintracellular signaling molecules and genes involved in cellcycle and taking part in regulation of proliferation growthand modification of proteins showed that the expressionprofilewas very similar except for single genes of an increasedand decreased expression in CD34minus stem cells of umbilicalcord blood in our study that is MMP7 PRKCB PSEN1RAC2 and CCND3 respectively as well as DKK1 WNT5AWNT5B FZD7 and TCF7L1 The Wnt5a ligand is involvedin the noncanonical Wnt signaling pathway transmissionand it is assumed that it may inhibit the canonical Wntpathway in mouse embryonic cells similarly to Dkk1 Therole of the noncanonical Wnt signaling pathway which inour results seemed to prevail in CD34minus stem cells of bonemarrow is not clear It is believed that the noncanonical Wntsignaling pathway controls the reorganization of cytoskeletonmuscle proteins tissue polarity and cell movement [43] Incontrast the canonical signaling pathway which activatestarget gene transcription through the increase of beta-cateninconcentration seems to be more active in CD34minus stemcells from umbilical cord cells Its role is to control thetissue-specific cell purpose during embryogenesis as well asregulation of proliferation in adult tissues [44] Additionallyhigh activity of WNT5A gene is noticeable in the humanMSCs differentiation into osteoblasts as well as in early stagesof adipogenesis [45 46] According to the publication ofYang et al the Wnt5a and Wnt5b glycoproteins are alsoresponsible for coordination of chondrocyte proliferation anddifferentiation [47] Wnt5a is believed to be an importantfactor taking part in the positive autocrinic regulation ofHSC repopulation ability [48] In their publication Prockopet al suggest that a high WNT5A expression in MSCs ischaracteristic for the end of the exponential and stationarygrowth phases in cell cultures in in vitro conditions [49]Slight changes in theWnt gene activity especially inWNT5Aactivity may result from the role played by MSCs in theirenvironmental niche Bone marrow MSCs seem to be moreinvolved in the renewal processes of the progenitor cells poolwhich reside in bone marrow The researches show that theexpression of Wnt ligands and Fz receptors in MSCs showssome correlation of WntFz pairs such as Wnt5aFz5 [50]The above discussed study results showed that there wasa coexpression of WNT5AFZD7 Their higher expressionwas characteristic for the bone marrow cells while thepreviously published data suggested differences in WntFzactivity depending on the origin-mouse versus humanMSCs

10 Stem Cells International

[51] Currently available data suggest differences which maybe characteristic not only for proliferation and differentiationprograms but also for the phenotype of MSCs from umbilicalcord blood and bone marrow which is in agreement withthe results published by Baksh et al [7] The remaining genestake part in theWnt signaling cascade of increased activity inCD34minus from umbilical cord blood such as MMP7 belongingto metalloproteinases which play a role in the extracellulararray remodeling and wound healing [5]

The Notch signaling pathway belongs to the processes ofintercellular communication which regulate gene expressionpatterns defining a dualistic character of stem cells reflectedby the asymmetric cell division [52] Additionally the Notchsignaling cascade controls various cells biological processesnot only proliferation and differentiation but also apoptosisand regeneration The signaling activity of Notch pathwaygenes determined in the study of 38 genes showed theirsimilar expression as defined by the KEGG classification(Table 4) A similar activity level was noted for all four Notch1ndash4 receptors as well as for JAG1 and JAG2 ligands The dif-ferences were related to two genes DTX4 and PSEN1 whichwere overactive in CD34minus cells from umbilical cord bloodThe function of the PSEN1 gene is mainly developmentalprocesses regarding cell differentiation during neurogenesisAnd the lack of this gene leads to a premature directeddifferentiation of neural progenitor cells [53] The activity ofthe Notch gene cascade plays an important role at all levelsof stem cell development starting from the embryonic stemcell through fetal cells to adult organism stem cells Notchis significantly involved in the asymmetric segregation offactors determining cell purpose during replication divisionsof the stem cell poolThe significance of receptors and ligandsis best described in the hematopoietic stem cells Expressionof the constitutively active Notch1 gene in hematopoieticstem cells induces the immortalized cell lineages which canreproduce the myeloid and lymphoid cell lineage in a long-term repopulation in animal studies [54 55] Apart from thatit delays differentiation of human hematopoietic progenitorstem cells in vitro [56] The studies suggest that Notch1 is animportant factor inHSC self-renewal and can also participatein the inhibition of stem cell differentiation The scientificfindings reveal that a suppression of the Notch signalingpathway via deletion of key genes in HSC did not cause cellsto lose their ability of long-term hematopoiesis renewal inanimal models [57] Therefore the role of ligands and Notchreceptors in HSC has not been precisely defined yet andcurrent publications do not provide precise data regardingthe role played by particular Notch signaling pathway genesin mesenchymal stem cells The only phenomenon whichappears to be superior and characteristic for all stem cellsincluding MSCs is the significance of the Notch cascadein asymmetrical cell divisions This is a significant com-ponent of stem cells population self-renewal as well asthe functionality of the cascade in the stem niche Thisphenomenon constitutes an integral part of the hypothesisabout stem cell population aging However further inves-tigation will be required to determine if the differentiallyexpressed genes described in the studies above could serve asstemness genes

Remaining signaling pathways which revealed increasedactivity in the studied CD34minus cells of umbilical cord bloodincludedMAPK (Mitogen-Activated Protein Kinase Figure 2)which regulates a wide range of cell biological activities suchas gene expression mitosis proliferation differentiationmigration and apoptosis processes [58]TheMAPKpathwaystake part in regulation of early embryonic development andin defining the role of embryonic stem cells from the earlydevelopmental phases until the moment of formation ofdifferentiated adult cells However this precise role played bythe MAPK pathways in various stem cell types has not yetbeen defined

Transmission via calcium ions was also singled out inthe group of the overactive signaling pathways of the studiedCD34minus stem cells of umbilical cord blood versus CD34minus stemcells of bone marrow (Figure 2) The ions are a vital andcommonly present mediator in the process of extracellularsignals transmission and conversion into a cellular responseVariations of calcium ions concentrations regulate geneexpression through an impact on the activity of numeroussignaling pathways Intracellular increase of calcium ionsconcentration also regulates cell activities including adhe-sion motility gene expression and proliferation [59] Thesignificance of transmission via calcium ions and the ways oftheir concentration changes in mesenchymal stem cells stillhave not been studied Kawano et al were the first to reportthat in the nondifferentiated MSCs there are spontaneouschanges of calcium ions concentrations despite the lack ofany stimulating factors [60] As reported the calcium iontransmission through the activation of different gene groupsmay direct the cells onto specific developmental pathways[61]

The integrin-mediated signaling pathway takes part inextracellular array remodeling which means that it con-trols cell behavior and tissue organization Its increasedactivity was observed in our study in CD34minus stem cells ofumbilical cord blood (Figure 2) In recent years the role ofintegrins and selectins has been stressed in the adhesion andtransendothelial migration which has a significant meaningin the processes of repopulation of tissues damaged bydiseases and their regeneration via the circulating MSCs Forinstance Ip et al showed that blocking of beta 1-integrinwhich constitutes a component of the adhesive moleculeVLA-4 in MSC weakened the ability of mesenchymal stemcells to engraft into the infarction-changedmyocardium [62]The above results may suggest a better migration abilityof CD34minus stem cells of umbilical cord blood into the areaof pathologic tissue MSCs own cytokinetic activity thathas similar significance in terms of tissue regenerationThe study of cytokinetic activity of CD34minus stem cells ofumbilical cord blood showed increased activity of signalingpathways activated via cytokines (Figure 2 Table 2) On theother hand the TLR signaling pathway (toll-like receptorsignaling) which in our own results revealed a significantincreased activity in CD34minus stem cells of umbilical cordblood (Figure 2) increases the immunosuppressive charac-teristics of MSCs Weakening of the immunological responsegenerated by the TLR pathway depends on the produc-tion of immunosuppressive kynurenines by the indoleamine

Stem Cells International 11

23-dioxygenase enzyme which degrades tryptophan Induc-tion of indoleamine 23-dioxygenase by TLR entails autocrinesignaling loop of interferon-beta which plays a role in MSCsimmunomodulative effects [63]

According to the GO and KEGG classifications carriedout for cluster 13 with the gene pool of a decreased expressiona statistically significant decrease of gene activity was shownfor the TGF-beta and BMP pathways (Figure 3 Table 3) Itis believed that the family of TGF-beta members whichalso includes BMP plays an important role in initiating theprocess of a directed MSCs differentiation TGF-beta sig-naling transmission runs through specific serine-threoninekinase receptors and their nuclear receptors called the Smadproteins TGF-beta signaling transmission promotes theprocess of early differentiation stages into the chondrogenicand osteogenic lineages [64 65] However cell differentiatinginto the adipogenic lineage results in inhibition of the TGF-beta signaling pathway This was confirmed in experimentalstudies developed by Ng et al where the inhibition of TGF-beta transmission resulted in an increase of adipogenesisand a decrease of differentiating ability towards chondrocytes[66] The BMP signaling pathway strongly promotes differ-entiation towards osteoblasts but it also leads to inhibitionof myogenesis and adipogenesis Such a gene expressionprofile may suggest that the MSCs biological characteristicsare strongly influenced by the significance of stem cell nicheTherefore the mesenchymal stem cells from bone marrowmay be more involved in the promotion of the phenomenawhich maintain the stability of bone marrow environment

5 Conclusions

Multipotentiality of the mesenchymal stem cells their highproliferation ability and not too-demanding culturing con-ditions reveal their potential for wide application in variousmedical areas These are the reasons for the common pursuitto understand their biology based onmore andmore sublimebiomolecular procedures Scientists have been searchingfor an unequivocal marker which would characterize themesenchymal stem cells and simultaneously would enable aseparation of a homogenous MSC population Understand-ing of molecular mechanisms controlling self-replication anddifferentiation is becoming a fundamental issue for clinicalapplication of MSC In this respect the activity of signalingpathways is important yet the interpretation of the pathwaysespecially their interdependence constitutes a great challengefor the scientific world Moreover the answer to the questionregarding MSC innidation and its survival ability in hosttissues after transplantation is yet to be found Explanationof the abovementioned issues will require a more globalunderstanding of biological activity of these cells especiallyon the basis of the significance of the mesenchymal nicheenvironment of stem cells and its impact on the gene andepigenetic activity It should be assumed that continuedprogress in the understanding of MSCs will finally lead totheir common therapeutic and preventive application andconsequently to the improvement of the quality and lengthof human life

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] R J Deans andA BMoseley ldquoMesenchymal stem cells biologyand potential clinical usesrdquo Experimental Hematology vol 28no 8 pp 875ndash884 2000

[2] M F Pittenger A M Mackay S C Beck et al ldquoMultilineagepotential of adult human mesenchymal stem cellsrdquo Science vol284 no 5411 pp 143ndash147 1999

[3] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006

[4] Y-J Chang D T-B Shih C-P Tseng T-B Hsieh D-C Leeand S-M Hwang ldquoDisparate mesenchyme-lineage tendenciesin mesenchymal stem cells from human bone marrow andumbilical cord bloodrdquo Stem Cells vol 24 no 3 pp 679ndash6852006

[5] R A Panepucci J L C Siufi W A Silva Jr et al ldquoComparisonof gene expression of umbilical cord vein and bone marrowndashderived mesenchymal stem cellsrdquo STEM CELLS vol 22 no 7pp 1263ndash1278 2004

[6] C K Rebelatto A M Aguiar M P Moretao et al ldquoDissimilardifferentiation of mesenchymal stem cells from bone marrowumbilical cord blood and adipose tissuerdquo Experimental Biologyand Medicine vol 233 no 7 pp 901ndash913 2008

[7] D Baksh R Yao and R S Tuan ldquoComparison of proliferativeand multilineage differentiation potential of human mesenchy-mal stem cells derived from umbilical cord and bone marrowrdquoStem Cells vol 25 no 6 pp 1384ndash1392 2007

[8] D L Clarke C B Johansson J Wilbertz et al ldquoGeneralizedpotential of adult neural stem cellsrdquo Science vol 288 no 5471pp 1660ndash1663 2000

[9] D J Anderson F H Gage and I L Weissman ldquoCan stem cellscross lineage boundariesrdquo Nature Medicine vol 7 no 4 pp393ndash395 2001

[10] D L Stemple and D J Anderson ldquoIsolation of a stem cell forneurons and glia from the mammalian neural crestrdquo Cell vol71 no 6 pp 973ndash985 1992

[11] R L Rietze H Valcanis G F Brooker T Thomas A K Vossand P F Bartlett ldquoPurification of a pluripotent neural stem cellfrom the adult mouse brainrdquoNature vol 412 no 6848 pp 736ndash739 2001

[12] R M Seaberg S R Smukler T J Kieffer et al ldquoClonalidentification of multipotent precursors from adult mousepancreas that generate neural and pancreatic lineagesrdquo NatureBiotechnology vol 22 no 9 pp 1115ndash1124 2004

[13] A P Beltrami L Barlucchi D Torella et al ldquoAdult cardiac stemcells are multipotent and support myocardial regenerationrdquoCell vol 114 no 6 pp 763ndash776 2003

[14] L Yang S Li H Hatch et al ldquoIn vitro trans-differentiation ofadult hepatic stem cells into pancreatic endocrine hormone-producing cellsrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 99 no 12 pp 8078ndash80832002

[15] I Chambers D Colby M Robertson et al ldquoFunctional expres-sion cloning of Nanog a pluripotency sustaining factor inembryonic stem cellsrdquo Cell vol 113 no 5 pp 643ndash655 2003

12 Stem Cells International

[16] M Dai P Wang A D Boyd et al ldquoEvolving genetranscriptdefinitions significantly alter the interpretation of GeneChipdatardquo Nucleic Acids Research vol 33 pp 175ndash179 2005

[17] RA Irizarry BHobbs F Collin et al ldquoExploration normaliza-tion and summaries of high density oligonucleotide array probelevel datardquo Biostatistics vol 4 no 2 pp 249ndash264 2003

[18] R B Dean and W J Dixon ldquoSimplified statistics for smallnumbers of observationsrdquo Analytical Chemistry vol 23 no 4pp 636ndash638 1951

[19] G J McLachlan and D Peel ldquoOn computational aspectsof clustering via mixtures of normal and t-componentsrdquo inProceedings of the American Statistical Association (BayesianStatistical Science Section) American Statistical AssociationIndianapolis Ind USA 2000

[20] J Polanska P Widlak J Rzeszowska-Wolny M Kimmel andA Polanski ldquoGaussian mixture decomposition of time-courseDNA microarray datardquo in Mathematical Modeling of BiologicalSystems Volume I pp 351ndash359 Birkhauser Boston Mass USA2007

[21] A J Friedenstein U F Gorskaja and N N Kulagina ldquoFibrob-last precursors in normal and irradiated mouse hematopoieticorgansrdquo Experimental Hematology vol 4 no 5 pp 267ndash2741976

[22] S Wakitani T Saito and A I Caplan ldquoMyogenic cells derivedfrom rat bone marrow mesenchymal stem cells exposed to 5-azacytidinerdquoMuscle ampNerve vol 18 no 12 pp 1417ndash1426 1995

[23] D Woodbury E J Schwarz D J Prockop and I B BlackldquoAdult rat and human bone marrow stromal cells differentiateinto neuronsrdquo Journal of Neuroscience Research vol 61 no 4pp 364ndash370 2000

[24] M Reyes T Lund T Lenvik D Aguiar L Koodie and CM Verfaillie ldquoPurification and ex vivo expansion of postnatalhumanmarrowmesodermal progenitor cellsrdquoBlood vol 98 no9 pp 2615ndash2625 2001

[25] M Ramalho-Santos S Yoon Y Matsuzaki R C Mulliganand D A Melton ldquolsquoStemnessrsquo transcriptional profiling ofembryonic and adult stem cellsrdquo Science vol 298 no 5593 pp597ndash600 2002

[26] KMitsui Y Tokuzawa H Itoh et al ldquoThe homeoprotein nanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003

[27] S Okumura-Nakanishi M Saito H Niwa and F IshikawaldquoOct-34 and Sox2 regulate Oct-34 gene in embryonic stemcellsrdquo The Journal of Biological Chemistry vol 280 no 7 pp5307ndash5317 2005

[28] F Ulloa-Montoya B L Kidder K A Pauwelyn et al ldquoCompar-ative transcriptome analysis of embryonic and adult stem cellswith extended and limited differentiation capacityrdquo GenomeBiology vol 8 no 8 article R163 2007

[29] L Song N EWebb Y Song and R S Tuan ldquoIdentification andfunctional analysis of candidate genes regulating mesenchymalstem cell self-renewal and multipotencyrdquo STEMCELLS vol 24no 7 pp 1707ndash1718 2006

[30] A H H Van Boxel-Dezaire M R S Rani and G R StarkldquoComplexmodulation of cell type-specific signaling in responseto type I interferonsrdquo Immunity vol 25 no 3 pp 361ndash372 2006

[31] C G Kim J J Lee D Y Jung et al ldquoProfiling of differentiallyexpressed genes in human stem cells by cDNA microarrayrdquoMolecules and Cells vol 21 no 3 pp 343ndash355 2006

[32] T Bowman R Garcia J Turkson and R Jove ldquoSTATs inoncogenesisrdquo Oncogene vol 19 no 21 pp 2474ndash2488 2000

[33] A Stephanou andD S Latchman ldquoOpposing actions of STAT-1and STAT-3rdquo Growth Factors vol 23 no 3 pp 177ndash182 2005

[34] A Martino J H Holmes J D Lord J J Moon and B HNelson ldquoStat5 and Sp1 regulate transcription of the cyclin D2gene in response to IL-2rdquo The Journal of Immunology vol 166no 3 pp 1723ndash1729 2001

[35] I Matsumura T Kitamura H Wakao et al ldquoTranscriptionalregulation of the cyclin D1 promoter by STAT5 its involvementin cytokine-dependent growth of hematopoietic cellsrdquo TheEMBO Journal vol 18 no 5 pp 1367ndash1377 1999

[36] V Calo M Migliavacca V Bazan et al ldquoSTAT proteins fromnormal control of cellular events to tumorigenesisrdquo Journal ofCellular Physiology vol 197 no 2 pp 157ndash168 2003

[37] M Socolovsky A E J Fallon S Wang C Brugnara and HF Lodish ldquoFetal anemia and apoptosis of red cell progenitorsin Stat5aminusminus5bminusminus mice a direct role for stat5 in Bcl-X(L)inductionrdquo Cell vol 98 no 2 pp 181ndash191 1999

[38] G Li Z Wang Y Zhang et al ldquoSTAT5 requires the N-domainto maintain hematopoietic stem cell repopulating function andappropriate lymphoid-myeloid lineage outputrdquo ExperimentalHematology vol 35 no 11 pp 1684ndash1694 2007

[39] S Negoro K Kunisada E Tone et al ldquoActivation of JAKSTATpathway transduces cytoprotective signal in rat acute myocar-dial infarctionrdquo Cardiovascular Research vol 47 no 4 pp 797ndash805 2000

[40] H C Blair L J Robinson and M Zaidi ldquoOsteoclast signallingpathwaysrdquo Biochemical and Biophysical Research Communica-tions vol 328 no 3 pp 728ndash738 2005

[41] C J Malemud ldquoProtein kinases in chondrocyte signaling andosteoarthritisrdquo Clinical Orthopaedics and Related Research no427 supplement pp S145ndashS151 2004

[42] E M Pinheiro and D J Montell ldquoRequirement for Par-6 andBazooka inDrosophila border cell migrationrdquoDevelopment vol131 no 21 pp 5243ndash5251 2004

[43] D Strutt ldquoFrizzled signalling and cell polarisation inDrosophilaand vertebratesrdquo Development vol 130 no 19 pp 4501ndash45132003

[44] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[45] J Guo J Jin and L F Cooper ldquoDissection of sets of genes thatcontrol the character of wnt5a-deficient mouse calvarial cellsrdquoBone vol 43 no 5 pp 961ndash971 2008

[46] M Nishizuka A Koyanagi S Osada and M Imagawa ldquoWnt4and Wnt5a promote adipocyte differentiationrdquo FEBS Lettersvol 582 no 21-22 pp 3201ndash3205 2008

[47] Y Yang L Topol H Lee and J Wu ldquoWnt5a andWnt5b exhibitdistinct activities in coordinating chondrocyte proliferation anddifferentiationrdquo Development vol 130 no 5 pp 1003ndash10152003

[48] D J Van Den Berg A K Sharma E Bruno and R Hoff-man ldquoRole of members of the Wnt gene family in humanhematopoiesisrdquo Blood vol 92 no 9 pp 3189ndash3202 1998

[49] D J Prockop C A Gregory and J L Spees ldquoOne strategy forcell and gene therapy harnessing the power of adult stemcells torepair tissuesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 1 pp 11917ndash119232003

[50] X He J-P Saint-Jeannet Y Wang J Nathans I Dawid and HVarmus ldquoA member of the Frizzled protein family mediatingaxis induction byWnt-5Ardquo Science vol 275 no 5306 pp 1652ndash1654 1997

Stem Cells International 13

[51] D G Phinney G Kopen R L Isaacson and D J ProckopldquoPlastic adherent stromal cells from the bone marrow ofcommonly used strains of inbred mice variations in yieldgrowth and differentiationrdquo Journal of Cellular Biochemistryvol 72 no 4 pp 570ndash585 1999

[52] N Sasaki T Sasamura H O Ishikawa et al ldquoPolarized exo-cytosis and transcytosis of Notch during its apical localizationin Drosophila epithelial cellsrdquo Genes to Cells vol 12 no 1 pp89ndash103 2007

[53] M Handler X Yang and J Shen ldquoPresenilin-1 regulatesneuronal differentiation during neurogenesisrdquo Developmentvol 127 no 12 pp 2593ndash2606 2000

[54] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006

[55] A W Duncan F M Rattis L N DiMascio et al ldquoIntegrationof Notch andWnt signaling in hematopoietic stem cell mainte-nancerdquo Nature Immunology vol 6 no 3 pp 314ndash322 2005

[56] N Carlesso J C Aster J Sklar and D T Scadden ldquoNotch1-induced delay of human hematopoietic progenitor cell differen-tiation is associated with altered cell cycle kineticsrdquo Blood vol93 no 3 pp 838ndash848 1999

[57] I Maillard U Koch A Dumortier et al ldquoCanonical notch sig-naling is dispensable for the maintenance of adult hematopoi-etic stem cellsrdquo Cell Stem Cell vol 2 no 4 pp 356ndash366 2008

[58] G Pearson F Robinson T B Gibson et al ldquoMitogen-activatedprotein (MAP) kinase pathways regulation and physiologicalfunctionsrdquo Endocrine Reviews vol 22 no 2 pp 153ndash183 2001

[59] R E Dolmetsch R S Lewis C C Goodnow and J I HealyldquoDifferential activation of transcription factors induced by Ca2+response amplitude and durationrdquoNature vol 386 no 6627 pp855ndash858 1997

[60] S Kawano S Shoji S Ichinose K Yamagata M Tagami andM Hiraoka ldquoCharacterization of Ca2+ signaling pathways inhuman mesenchymal stem cellsrdquo Cell Calcium vol 32 no 4pp 165ndash174 2002

[61] B Ye ldquoCa2+ oscillations and its transporters in mesenchymalstem cellsrdquo Physiological Research vol 59 no 3 pp 323ndash3292010

[62] J E Ip Y Wu J Huang L Zhang R E Pratt and V J DzauldquoMesenchymal stem cells use integrin 1205731 not CXC chemokinereceptor 4 for myocardial migration and engraftmentrdquoMolecu-lar Biology of the Cell vol 18 no 8 pp 2873ndash2882 2007

[63] C A Opitz U M Litzenburger C Lutz et al ldquoToll-like recep-tor engagement enhances the immunosuppressive propertiesof human bone marrow-derived mesenchymal stem cells byinducing indoleamine-23-dioxygenase-1 via Interferon-120573 andprotein kinase Rrdquo Stem Cells vol 27 no 4 pp 909ndash919 2009

[64] B Kulterer G Friedl A Jandrositz et al ldquoGene expressionprofiling of human mesenchymal stem cells derived from bonemarrow during expansion and osteoblast differentiationrdquo BMCGenomics vol 8 article 70 2007

[65] W G Li and X X Xu ldquoThe expression of N-cadherinfibronectin during chondrogenic differentiation of MSCinduced by TGF-1205731rdquo Chinese Journal of Traumatology vol 8no 6 pp 349ndash351 2005

[66] F Ng S Boucher S Koh et al ldquoPDGFTGF-120573 and FGF signal-ing is important for differentiation and growth of mesenchymalstem cells (mscs) transcriptional profiling can identify markersand signaling pathways important in differentiation of MSCsinto adipogenic chondrogenic and osteogenic lineagesrdquo Bloodvol 112 no 2 pp 295ndash307 2008

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Microbiology

2 Stem Cells International

higher possibility of differentiation into osteogenic lineageand had little or no differentiation into adipocytes Thiscontrasted with bone marrow MSCs where expression ofmarkers characteristic for adipocytes was more frequentlydemonstrated [3 6] In the critical processes of regulatingself-renewal and the cellular purpose stem cells use thesignaling pathways which appear to be quite conservativefrom the evolutionary perspective such as Notch Wntand JAK-STAT Although the signaling proteins expressionis believed to be a highly restrictive process it appearsthat different stem cell types demonstrate varied rates ofexpression of these three families of signaling molecules Theglobal gene expression profile is commonly used to identifythe transcription signature of specific stem cells This signa-ture gives insight into the signaling mechanisms regulatingthe self-renewal and cellular purpose program especiallyin embryonic and hematopoietic stem cells Moreover bycomparing the gene expression profiles in different stemcell groups a common pool of genes were identified thatserve either as stem cells markers for self-renewal or directthe cells through differentiation [7ndash11] In comparison witha great number of studies carried out on the embryonichematopoietic or neural stem cells there are much fewerstudies of molecular mechanisms of MSC self-renewal anddifferentiation control mainly due to their diversified genesignature and the lack of agreement on typical markersantigens as far as some MSC markers are concerned [12ndash15]

This paper provides a comparison of the expression of theentire gene pool ofMSCmarkers with a special considerationto the signaling pathway genes in CD34minus stem cells whichphenotypically correspond with MSCs from the umbilicalcord blood and bone marrow The cells were extracted bymeans of the same single-bed room method on the basisof the same antigen phenotype Each cell population wasmultiplied three times in the same culture conditions Geneactivity was defined through the oligonucleotide microarrayswith the use of GO and KEGG databases We analyzed thenonhematopoietic stem cell signature based on the geneactivity of the conservative signaling pathways includingWnt and Notch We then asked the question whether dif-ferences in the signaling pathways for gene activity may beevidence of different populations of origin for theMSCs (egfetal verses adult origin) and consequently the predominanceof one population over the other Does a source of populationwhich undoubtedly shapes the cell epigenetic conformationhave a significant impact on its subsequent biological activity

2 Materials and Methods

Material for the study of gene expression by means ofoligonucleotide microarrays was sampled from CD34minus stemcell cultures from the bonemarrow and umbilical cord bloodComplete RNA was extracted from the abovementionedcultures

21 Obtaining Umbilical Blood Samples Samples of umbilicalblood were obtained after the delivery and cord clamping inthe cases of normal course of pregnancy and delivery after the

37th week The blood was obtained intranatally that is afteromphalotomy and before placenta expulsion

22 Obtaining Bone Marrow Samples For the purpose ofthe study approximately 6mL of bone marrow was obtainedfrom healthy donors during the procedure of collectingmaterial for hematological purposes

The research program was approved by the BioethicalCommittee of the Medical University of Silesia in KatowicePoland All participants provided their written informedconsent to participate in this study

23 Isolation of CD34minus Cells from Umbilical Cord Bloodand Bone Marrow Freshly collected umbilical cord bloodand bone marrow were diluted with phosphate-bufferedsaline (PBS Gibco) layered on Ficoll (Sigma) at 2 1 andcentrifuged for 40min at 400timesg at 20∘C The lymphocyticskin obtained after centrifugation was suspended in the basewith an addition of serum and centrifuged again for 10min at200timesg at 20∘C All the cell sample preparation activities wererepeated twice The prepared cells were divided into CD34+and CD34minus fractions by means of the CD34 Progenitor CellIsolation Kit and the MiniampMidiMACS Starting Kit Cellsviability was determined in the fluorescent microscope studyafter staining the cells with ethidium bromide and acridineorange

24 CD34minus Cell Culture

241 Cell Labeling The cells were suspended in the appro-priate volume of buffer minus03mL per each 108 of cells 01mLof FcR Blocking Reagent was added to the cell suspension of3 times 107mL density with the ratio of 01mL FcR BR03mLcell suspension For each 03mL of cell suspension 01mL ofCD34 Microbeads was then added The material was mixedthoroughly and incubated for 30min at 6ndash12∘C 20mL ofbuffer was added and mixed thoroughly again The mixturewas centrifuged for 10min at 400 g The supernatant wasdiscarded and the sediment was suspended in 1mL of buffer

242 Cell Separation in Magnetic Column LS column wasplaced in the magnetic field The column was rinsed with3mL of buffer and the cell mixture as prepared abovewas added to the column After the suspension had passedthrough the column the CD34minus fraction was obtained Thecolumn was washed with buffer (2x 05mL) removed fromthemagnetic field and then placed in a 5mL tube Next 1mLof buffer was applied to the column The cells were elutedby means of a piston In this way the CD34+ fraction wasobtained

243 Starting the Culture The separated CD34minus cell frac-tions were used for cell cultures The IMDM-ISCOVErsquoSmodified Dulbeccorsquos medium with penicillin-streptomycinand 5 human albumin were used for culturing

244 Total RNA Extraction from Cell Culture RNA wasextracted from the CD34minus cell cultures with the Total RNA

Stem Cells International 3

PrepPlus (AampABiotechnology) based on themodifiedChom-czynski and Sacchirsquos method At the initial stage the cellswere lysed while the endogenous RNases were inactivatedwith fenozol After adding chloroform the mixture wascentrifuged and the collected RNA was precipitated withisopropanol Next the mixture was centrifuged for 20minat 13 000 rmin the supernatant was decanted and the RNAsediment was washed twice with 75 ethanol After dryingthe sediment was stored at minus70∘C for the analysis

25 Qualitative Assessment of RNA Extracts RNA extractsunderwent qualitative assessment via the agarose gel elec-trophoresis in 08 agarose gel with ethidium bromide(05mgmL) in the SUBMINI apparatus 5120583L of extract wasmixed with 3 120583L of gel-loading dye (005 wv bromophenolblue 60 glycerol) and heated at 65∘C for 4min After theseparation the electropherograms were assessed with the UVtransilluminator (120582 = 260 nm)

26 Quantitative Assessment of RNA Extracts RNA extractsunderwent quantitative assessment via spectrophotomet-ric measurement of RNA concentration (Gene Quant IIPharmacia) The spectrophotometric assessment of extractscovered the absorbance measurement at the wave lengthsof 230 260 280 and 320mn and then the A

260

A280

ratio and protein content were determined for each sampleAbsorbance value at the 260 nm wave length was used tocalculate RNA concentration based on the assumption thatresults of cuvette measurements using the 1 cm optical pathequaling 1 OD

260

correspondwith the concentration of 40mgRNA in 1 cm3 of extract

27 Gene Expression Analysis with the OligonucleotideMicroarrays Technique

271 General Method Assumptions The material for thestudy consisted of total RNA extracted from the cell cultureswith the TRIzol reagent (Invitrogen) The isolated RNA waspurified with the RNeasy Total RNA Mini Kit and digestedwith DNase I (Qiagen) in order to eliminate any possiblecontamination with DNA Approximately 8 120583g of total RNAwas used for the double-stranded cDNA synthesis Biotin-labeled cRNA was synthesized and biotinylated cRNA wasthen fragmented and hybridized with the Test3 microarrayand HG U133A (Affymetrix) and labeled twice with thestreptavidin-phycoerythrin conjugate and biotinylated strep-tavidin antibodies

The fluorescence intensity was analyzedwith theGeneAr-ray Scanner G2500A The quantity and quality of total RNAcDNA and cRNA were assessed spectrophotometrically andby means of the electrophoresis technique in 1 agarose gel

272 Purification of Total RNA Extract with RNasy Mini KitAfter a prior concentration measurement 100120583L of the RNAextract was obtained with RNase-free water 350 120583L of RLTbuffer with 120573-mercaptoethanol and 250120583L of 96 ethanolwere added mixed by pipetting and then applied to thecolumn and centrifuged for 15 seconds at 12 000 rmin The

filtrate was discarded and the column was moved to a newtube and 350 120583L of RW1 was added and then centrifugedfor 15 seconds at 10ndash12 000 rmin For digestion 70 120583L ofRDD buffer was added to a tube with 10120583L of DNase Thismixture was applied on the column membrane and left todigest for 15min at room temperature Then 350 120583L of RW1solution was added the old flow-through was discarded andthe column was centrifuged for 15 sec at 12 000 rmin Theflow-throughwas again discarded and 500120583L of RPE solutionwas added to the column The extracts were centrifuged for1min at 14 000 rmin then the column was turned upsidedown and centrifuged again for the same time and rate Aftercentrifugation the column was placed in a new tube and30 120583L of H

2

O was added and was left for 10min Then thecolumn was centrifuged and 20120583L of H

2

O was added andleft for a few minutes and then centrifuged again After finalcleanup the RNA concentration in extracts was measured

28 cDNA Synthesis

281 First Strand cDNA Synthesis In order to obtain the firststrand of cDNA 2 120583L 100 pM of T7 starter-oligo(dT)

24

(51015840-GCCAGTGAATTGTAATACGACTCACTATAG GGAG-GCGG-31015840) was added to 11 120583L of RNA extract the mixturewas incubated at 70∘C for 10minutes and placed on iceThen4 120583L of 5x First Strand Buffer 2120583L of 01M DTT and 1 120583L of10mM dNTPs (Invitrogen) were added After 2 minutes ofpreincubation at 45∘C 1 120583L (200U) of reverse transcriptaseSupercscript II (Life Technologies) was added to the reactionmixture and incubation continued for another hour

282 Second Strand cDNA Synthesis In order to obtain thesecond strand of cDNA the following were added to themixture obtained after the first strand synthesis 30 120583L of5x Second Strand Buffer 91 120583L of RNase-free water 3 120583L of10mM dNTPs 4 120583L (40U) of E coli DNA Polymerase I1 120583L (10U) of E coli DNA Ligase and 1 120583L (2U) of Rnase H(Invitrogen) The mixture was incubated for 2 hours at 16∘C

Next 25 120583L (10U) of T4 DNA Polymerase I was addedto the mixture and the mixture was incubated for another5 minutes at 16∘C The composition of the reaction mixtureused for the second strand cDNA synthesis is presentedin Table 2 The reaction was stopped by adding 10 120583L of05M EDTA and the double-stranded cDNA was extractedusing the phenolchloroformisoamyl alcohol mixture Theaqueous phase was divided bymeans of Phase Lock Gel Light15mL tubes (Eppendorf) 05 volume of 75M ammoniumacetate (Sigma) and 25 volume of 96 ethyl alcohol wereadded to the aqueous phase and it was left for 12 h at minus20∘C

29 Purification of the Obtained cDNA After freezing for theentire night at minus20∘C 03 120583L of Palet Point (Merc) was addedto the reaction mixture and it was centrifuged for 20min at12 000 rmin at room temperature supernatant was discardedand the sediment was rinsed with 2x 70 ethanol (500120583L)and centrifuged for 5min at 12 000 rmin the sediment wasdried thoroughly and dissolved in 12 120583L of RNase-free water

4 Stem Cells International

210 Biotinylated cRNA Synthesis The template for thebiotinylated cRNA synthesis consisted of 10 120583L ds cDNAto which the following were added 12 120583L of RNase-freewater 4 120583L of 2x hybridization buffer 4 120583L of biotin-labeledribonucleotides 4 120583L of DTT 4 120583L RNase inhibitor and2 120583L of T7 polymerase (BioArray High Yield RNA TranscriptLabeling Kit Enzo) The whole was left at 37∘C for 5 h andmixed every 30 minutes

211 Biotinylated cRNA Purification After a 5-hour incuba-tion cRNA was cleaned up using 60 120583L of H

2

O 350 120583L ofRLT buffer and 250 120583L of 96 ethanol added to the reactionmixture The mixture was applied onto the RNAeasy columnTotal RNA Mini Kit (Qiagen) and was centrifuged twice for15 seconds at 10 000 rmin In the last stage 2x 20120583L of H

2

Owas added to the column membrane it was centrifuged at12 000 rmin and then the cRNA concentration was mea-sured

212 Biotinylated cRNA Fragmentation For fragmentationpurposes 16 120583g of cRNA and 8 120583L of fragmentation buffer(Affymetrix) were used and the mixture was supplementedwith up to 40 120583L of RNase-free water After completion of thisstage the mixture was centrifuged and left for 35min at 94∘Cand then placed on iceThe fragmentation result was checkedon 1 agarose gel

213 Assessment of cDNA Biotinylated cRNA and cRNASynthesis after Fragmentation via Electrophoresis in AgaroseGel Evidence of proper cDNA biotinylated cRNA andcRNA synthesis after fragmentation was assessed throughelectrophoresis in agarose gel with ethidium bromide Theresulting separation electrophoregram of the abovemen-tioned products verified that samples were acceptable to usein the following analysis stage

214 Hybridization of the Fragmented BiotinylatedcRNA with Microarray

2141 Preparation of Hybridization Cocktail In order toprepare the hybridization cocktail 375 120583L of fragmentedbiotinylated cRNA was used and the following were added5 120583L of control oligonucleotide B2 15 120583L of eukaryotichybridization control (Affymetrix) 3120583L of Herring Sperm(Invitrogen) 3 120583L of acetylated BSA (Invitrogen) 150 120583L of2x hybridization buffer and 865 120583L of RNase-free water Thecocktail was heated for 5min at 99∘C and for 5min at 45∘Cand centrifuged for 5min at 14 000 rmin

2142 Application of Cocktail on Microarray HG-U133Amicroarray (Affymetrix)was heated to room temperature andthen 100 120583L of 1x hybridization buffer was applied The chipwas placed in hybridization oven for 10min Next the bufferwas removed from the chip and 200120583L of hybridizationcocktail was applied The microarray was placed in thehybridization oven for 16 h at 45∘C at 55 rmin

2143 Washing and Scanning of Microarrays After a 16-hour hybridization the HG U133A microarray was washedwith buffer and stained with solutions (SAPE solutionstreptavidin antibody solution) and then scanned with theGeneArray Scanner G2500A (Affymetrix)

215 Statistical Analysis Data regarding expression levelsobtained directly from the HGU133A array were reannotatedwith CDF control files developed by Dai et al [16] Thenext step consisted of microarray normalization with theRMA algorithm [17] Three repetitions were replaced withan average value with a prior detection of outliers by meansof the Dean and Dixon method [18] Determination of mea-surement noise threshold was conducted through empiricaldecomposition of probability density function of expressionlevels into normal components [19] The component of thelowest mean value unchanged across all the microarrays wasassumed to be the noise model The threshold value of thefilter was constructed at level 28 (in the field of logarithmsto base 2) The mixture model of normal distributions con-structed for the average SRL (Signal Log Ratio) of CD34minus cellsfromumbilical cord blood versusCD34minus of bonemarrowwasused to define the affinity of certain genes to subgroups of anincreased decreased or unchanged value of expression levelMoreover thismixturemodel was used to determine the FDR(False Discovery Rate) value for each of them

In order to define qualitatively the processes in stemcells from umbilical cord blood in comparison with stemcells from bonemarrow an unsupervised gene clustering wasconducted Again the GaussianMixture Model for empiricaldecomposition of expression levels constituted the algorithmenabling such a division However in this analysis additionallimitations based on the gene composition of each compo-nent that were the same across the experimental conditionswere added to the algorithm [20] Clustersclasses obtainedin this manner were compared to cellular processes in whichthey are involved The categories of cellular processes weredefined by gene ontology terms that are commonly appliedfor this purpose A number of conditioning hypergeometrictests were conducted verifying the one-sided hypothesisabout overrepresentation For each ontological term thatwas found to be significantly overrepresented statisticallyin a given gene cluster the OR (Odds Ratio) value wasdetermined

Gene pathways whose components are genes belongingto specific clusters underwent an additional analysis whichwas independent of a direct description of cellular processesFor this purpose we used data from the KEGG databaseFor each pathway the hypothesis of overrepresentation of aspecified cluster of genes as compared with the whole scopeof the analyzed genes was verified Precise 119901 values weredetermined

Statistically significant values were those of FDR lt 010 or119901 lt 005

3 Results

The first stage of the study consisted of data reannotation forexpression levels through the CDF control files developed

Stem Cells International 5

by Dai et al [16] For many years the platform for geneexpression profile was the Affymetrix GeneChip databaseHowever their probe section was created on the basis ofprior gene and transcriptome annotations which significantlydiffer from the modern expertise A comparative analysisbetween many popular Gene Chips and redefined probesbased on updated gene data with consideration to geneticpolymorphism showed a 30ndash50 discrepancy [16] thereforeit was decided to reannotate data as the first step in ourstudyThe assessment of noise threshold was then completedcreating a mixture model of normal distributions for themean SLR of CD34minus cells from umbilical cord blood versusCD34minus from bone marrow

The next step comprised microarrays normalizationthrough the RMA algorithm [17] Three repetitions werereplaced with a mean value with a prior detection of outliersby means of the Dean and Dixon method [18] Determina-tion of the measurement noise threshold was conducted byempirical decomposition of probability density function ofexpression levels into normal components [19] The noisemodel was assumed to be the component of the lowest meanvalue which was unchanged across all the microarrays Thethreshold value of the filter was constructed at level 28 (inthe field of logarithms to base 2)

As a result of the abovementioned procedures a data setwas obtained which described 11 979 genes which underwentfurther analysis The first stage consisted of creating the mix-ture model of normal distributions constructed for a meanSLR (Signal Log Ratio) of CD34minus stem cells from umbilicalcord blood versus CD34minus stem cells from bone marrowwhich allowed defining the affinity criteria of specific genes tosubgroups of an increased decreased and unchanged valueof expression levels This also enabled us to determine a FDRvalue (False Discovery Rate) for each of them (Figure 1)

This method is based on decomposition that is thehistogram decomposition into (K)mdashGaussrsquos components aswell as modeling the probability density function distribu-tions (pdf) for given gene expression levels The method wasused for analysis of a histogram reflecting the frequency ofgenes of a specific expression level in the tested preparationThis initial analysis showed considerable similarity of geneexpression of CD34minus cells from umbilical cord blood ascompared with CD34minus cells from bone marrow

31 Unsupervised Gene Clustering During the next stage ofdata analysis a qualitative determination of the processes inCD34minus stem cells from umbilical cord blood in comparisonwith CD34minus stem cells from bone marrow was conductedusing unsupervised gene clustering Again the GaussianMixture Model for empirical decomposition of expressionlevels constituted the algorithm enabling such a divisionHowever in this analysis additional limitations based onthe gene composition of each component that were thesame across the experimental conditions were added to thealgorithm [20] As a result 15 gene clusters were obtainedtwo of which (numbers 9 and 13 in this study) containeddiscriminatory genes exhibiting a statistically significantincrease (cluster number 9) and decrease of gene expression(cluster number 13) (Table 1)

1600

1400

1200

1000

800

600

400

200

00 2 4 6 8

Num

ber o

f obs

erva

tions

Gene expressionminus10 minus8 minus6 minus4 minus2

SLR CD34H adnotacje Dai ver12

Figure 1Mixturemodel of normal distributions (GaussianMixtureModel) for a mean SLR (Signal Log Ratio) of CD34minus cells fromumbilical cord blood versus CD34minus cells from bone marrow Log(CD34-P)( CD34-Sz)

The remaining clusters differing in terms of the numberof genes within the cluster aggregated the genes of similarexpression values Differences betweenmean gene expressionlevels of CD34minus stem cells from bone marrow and CD34minusstem cells from umbilical cord blood in clusters numbered1ndash8 10ndash12 14 and 15 are small or nonexistent Thus all theseclusters were classified as the groups of genes exhibiting thesame expression levels

Cluster number 9 included 493 genes which show anincreased activity in CD34minus stem cells from umbilical cordblood of the mean SRL (Signal Log Ratio) equaling 306 Incontrast cluster number 13 included a lower number of genes(119899 = 387) of decreased expression in CD34minus stem cells fromumbilical cord blood for which SRL equals minus343

At the next stage gene clustersgroups obtained in thismanner were compared to biological processes in whichthey are involved as defined by gene ontology (GO) termsthat are commonly applied for this purpose A number ofconditioning hypergeometric tests were conducted verifyingthe one-sided hypothesis about overrepresentation For eachontological term that was found to be significantly overrep-resented statistically in a given gene cluster the OR (OddsRatio) value was determined All the clusters were classifiedthrough the GO classification system that is the clusterswith genes discriminating both cell populations as well as theclusters grouping genes of a similar expression

Four hundred ninety-three genes of cluster 9 classifiedvia the GO system determined the terms of biological pro-cesses (119901 lt 005) overrepresented in CD34minus stem cells fromumbilical cord blood The classification system determined256 GO terms of biological processes out of which GO termsof 119901 value below 004 underwent further analysis As a resultof this analysis the majority of GO terms represented only byone or two genes were rejected The GO terms for biologicalprocesses in cluster 9 were assigned to one of six func-tional groups including metabolism biological processes

6 Stem Cells International

Table 1 15 clusters obtained as a result of the unsupervised gene clusteringMean CD34minus BM (mean of CD34minus stem cells from bonemarrow)Mean CD34minus UCB (mean of CD34minus stem cells from umbilical cord blood)119873 (a number of genes in a cluster) SRL (Signal Log Ratio)

Cluster MeanCD34minus BM

MeanCD34minusUCB

SDCD34minusBM

SDCD34minusUCB

119873

119901 value MeanSLR

minus95CISLR

+95CISLR

Conclusion

1497 491 008 009

1102

000000023 minus006 minus008 minus004

No change2

1157 1149 035 047

54

026488907 minus008 minus022 006

No change3

802 805 030 032

592

063190010 001 minus005 007

No change4

445 440 006 005

999

000000001 minus006 minus008 minus004

No change5

702 714 024 021

898

000000009 011 007 016

No change6

554 550 012 013

1133

000000889 minus006 minus009 minus003

No change7

353 346 004 005

960

451861119864 minus 13 minus007 minus009 minus005

No change8

476 483 071 088

961

003102164 011 001 020

No change9 564 715 158 270 493 p lt 00000001 306 295 316 Up10

311 309 004 004

872

000325256 minus003 minus005 minus001

No change11

276 278 002 002

647

000002947 003 001 004

No change12

398 390 006 007

1133

544009119864 minus 15 minus008 minus010 minus006

No change13 739 486 285 101 387 p lt 00000001 minus343 minus357 minus329 Down14

621 621 026 021

1353

044902052 minus001 minus005 002

No change15

930 936 064 084

395

015369595 008 minus003 020

No change

0 001 002 003 004

Signal transduction (GO0007165)

Activation of MAPK activity (GO0000187)

Protein kinase cascade (GO0007243)

Regulation of MAP kinase activity (GO0043405)

Integrin-mediated signaling pathway (GO0007229)

Positive regulation of tyrosine phosphorylation ofSTAT protein (GO0042531)

Positive regulation of calcium-mediated signaling(GO0050850)

JAK-STAT cascade (GO0007259)

Cytokine-mediated signaling pathway (GO0019221)

Positive regulation of cell proliferation (GO0008284)

Calcium-mediated signaling (GO0019722)

Regulation of JAK-STAT cascade (GO0046425)

p value

Signaling pathway

Figure 2 Functional annotations 12 GO classification terms for biological processes in the group signaling pathways in cluster 9 dependingon the 119901 value (119901 lt 004)

regulation cellular homeostasis cellular defensive responseand response to stimuli cytokine production and signalingpathways The GO terms regarding signaling pathways arepresented in Figure 2

Thenext stage consisted of a similar analysis of genes fromcluster 13 GO system classification for 387 genes of cluster 13determined 119 terms of biological processes (119901 lt 005) of

a decreased activity in CD34minus cells fromumbilical cord bloodas compared with CD34minus cells from bone marrow The GOterms of 119901 value below 004 underwent further analysis As aresult of this analysis the majority of GO terms representedonly by one or two genes were rejected The GO terms forbiological processes in cluster 13 were assigned to one ofthree functional groups including metabolism biological

Stem Cells International 7

0000 0010 0020 0030

Negative regulation of signal transduction (GO0009968)

Vascular endothelial growth factor receptor signalingpathway (GO0048010)

Transmembrane receptor protein tyrosine kinase signalingpathway (GO0007169)

Transmembrane receptor protein serinethreonine kinasesignaling pathway (GO0007178)

Regulation of transforming growth factor beta receptorsignaling pathway (GO0017015)

Cell surface receptor linked signal transduction(GO0007166)

BMP signaling pathway (GO0030509)

p value

Signaling pathway

Figure 3 Functional annotations 7 GO classification terms for biological processes in the group signaling pathways in cluster 13 dependingon the 119901 value (119901 lt 004)

Table 2 Gene pathways based on the KEGG classification of an increased expression in CD34minus stem cells from umbilical cord blood

KEGGID 119901 value Odds ratio Exp count Count Size Term4060 0001 2947 14 33 223 Cytokine-cytokine receptor interaction4620 0001 3672 6 17 92 Toll-like receptor signaling pathway5120 0001 3748 4 12 63 Epithelial cell signaling inHelicobacter pylori infection4630 0012 2067 8 15 130 JAK-STAT signaling pathway

processes regulation and signaling pathways In CD34minus cellsfrom umbilical cord blood as compared with CD34minus cellsfrom bone marrow a decreased gene activity was observedof the BMP signaling pathway TGF beta transmembranereceptor of protein serinethreonine kinase transmembranereceptor of tyrosine kinase and VEGF (Figure 3)

Gene pathways whose components are genes belongingto specific clusters underwent an additional analysis whichwas independent of a direct description of cellular processesFor this purpose data from the KEGG database (KyotoEncyclopedia of Genes and Genomes) were used For eachpathway the hypothesis of overrepresentation of specificclusters of genes as compared with the whole scope ofanalyzed genes was verified and precise 119901 values weredetermined For the purpose of this study gene pathwaysfor clusters 9 and 13 were analyzed because they containedgenes discriminating both gene populations On the basisof KEGG database cytokine-cytokine receptor interactionpathway toll-like receptor signaling pathway epithelial cellsignaling in Helicobacter pylori infection and JAK-STATsignaling pathway are overrepresented in CD34minus cells fromumbilical cord blood (Table 2)

In cluster 13 on the basis of KEGG classification theCD34minus cells from umbilical cord blood show a decreasedactivity of focal adhesion signaling pathway cell junctionsand TGF-beta signaling pathway (Table 3)

At the next stage the genes of the remaining clustersnumbered 1ndash8 10ndash12 and 14 of a similar gene expression were

analyzed The analysis was based on the GO classification foreach of the abovementioned clusters The criteria used forchoosing the clusters included the biological processes con-nected with cellular cycle as well as the processes connectedwith signaling pathways activity A similar gene expression inCD34minus cells from umbilical cord blood and CD34minus cells frombone marrow was characteristic for such biological processesas cell division mitosis maintaining telomere endings RNAandDNA processing and a similar activity of Notch andWntsignaling pathways genes

An independent analysis was also carried out for genes ofa similar activity in clusters 1ndash8 10ndash12 and 14 on the basis ofthe KEGG database CD34minus cells from umbilical cord bloodand CD34minus cells from bone marrow showed similar activityof Wnt and Notch signaling pathways genes p53 signalingpathway genes cell cycle PPAR and mTOR (Table 4)

CD34minus cells from umbilical cord blood and CD34minus cellsfrom bone marrow showed similar gene activity of Wntand Notch signaling pathways both on the basis of the GOclassification system and the KEGG database Therefore atthe last stage of the study the activity of single genes of Wntand Notch as well as JAK-STAT signaling pathways chosenfrom 11976 sampled genes was analyzed

In accordance with the KEGG database the JAK-STATsignaling pathway is defined by 155 genes out of whichthere were 128 active genes in the studied material Genesexhibiting increased activity in CD34minus cells from umbilicalcord blood included SPRY2 CISH CSF2 GRB2 IFNGR1

8 Stem Cells International

Table 3 Gene pathways based on the KEGG classification of a decreased expression in CD34minus stem cells from umbilical cord blood

KEGGID 119901 value Odds ratio Exp count Count Size Term4510 0001 7517 7 33 188 Focal adhesion1430 0001 8157 4 22 109 Cell junctions4350 0001 4038 3 10 82 TGF-beta signaling pathway

Table 4 Gene pathways based on the KEGG classification of a similar expression in CD34minus stem cells from umbilical cord blood and CD34minuscells from bone marrow for clusters 1ndash8 10ndash12 and 14-15

KEGGID 119901 value Odds ratio Exp count Count Size Term04310 0018 1880 10 17 131 Wnt signaling pathway04330 0025 2722 3 7 39 Notch signaling pathway04115 0038 2157 5 9 61 p53 signaling pathway04110 0009 2028 10 18 100 Cell cycle03320 0020 2791 3 7 60 PPAR signaling pathway04150 0035 2220 5 9 46 mTOR signaling pathway

IL2RB IL2RG IL7R IL10RA IL15RA PIM1 PTPN6 STAT1STAT5A and CCND3 Genes exhibiting decreased activityincluded PIAS3 IL11RA and CBLB

On the basis of the KEGG database the Wnt signalingpathway is defined by 151 genes out of which there were132 active genes in the studied material Genes exhibit-ing increased activity in CD34minus cells from umbilical cordblood include MMP7 PRKCB PSEN1 RAC2 and CCND3Genes exhibiting decreased activity include DKK1 WNT5AWNT5B FZD7 and TCF7L1

On the basis of the KEGG database the Notch signalingpathway is defined by 47 genes out of which there were38 active genes in the studied material Genes exhibitingincreased activity in CD34minus cells from umbilical cord bloodincluded DTX4 and PSEN1

4 Discussion

Nonhematopoietic stem cells were first identified by Frieden-stein et al who described the progenitor cells of rat bonemarrow [21] Currently the nonhematopoietic stem cellswhich are frequently referred to although not quite correctlyas the mesenchymal stem cells are isolated from varioustissues including the umbilical cord blood placenta andumbilical cord A constantly increasing number of studieson MSCs have proved that in appropriate environmentalconditions they are capable of differentiating into special-ized mesodermal endodermal and even ectodermal celllineages [2 22ndash24] In natural conditions they play an impor-tant role in cell regeneration processes and in repairingthe tissues damaged by degenerating processes Anotherintriguing characteristic of MSCs is their ability to avoidimmunological identification and response therefore theyare referred to as the hypoimmunogenic cells Despite greatattention paid to mesenchymal stem cells their biology isnot yet fully understood Introducing multipotential stemcells into clinical application requires a full understandingof the mechanisms which control key properties of thecell such as cell self-replication versus differentiation and

mobilization versus tissue homing The understanding ofthese mechanisms must be based on strict definitions ofhuman multipotential stem cells at the molecular level Inaddition understanding of molecular mechanisms of self-replicating pluripotentiality plasticity and differentiation ofstem cells not only is a necessary condition to overcomelimitations of stem cells application as a therapeutic factorbut also is key to understand a fundamental mystery of thedevelopment of human organism

This paper presents a comparison of gene activity of thenonhematopoietic cells of umbilical cord blood and bonemarrow using oligonucleotidemicroarrays Gene activity wastested in stem cells CD34 and included 11 979 genes obtainedafter completing procedures to increase the purity of cellsamples Cell populations included in the study were alsolimited based on the noise threshold revealed in the finalcomparative analysis The gene expression profiles of thecompared populations of CD34minus stem cells from umbilicalcord blood and CD34minus stem cells from bone marrow werevery similar (Figure 1) For a better illustration of the biolog-ical molecular activity and the activity of signaling pathwaysin the studied cell populations a transcription profile analysiswith the GO and KEGG databases was completed In termsof fundamental signaling pathways for biological activityof stem cells like JAK-STAT Wnt and Notch we recordeddiscrete differences among expressed genes from umbilicalcord blood cells as compared to bone marrow cells

The analysis regarding signaling pathways showedincreased activity of the JAK-STAT pathway in CD34minusstem cells from umbilical cord blood both on the basis ofthe gene ontology classification and the KEGG database(Figure 2 and Table 2) The increased activity regarded 15genes which take part in the JAK-STAT cascade that isSPRY2 CISH CSF2 GRB2 IFNGR1 IL2RB IL2RG IL7RIL10RA IL15RA PIM1 PTPN6 and CCND3 including thegenes which code transcription factors STAT1 and STAT5AIn previous publications the significance of the JAK-STATsignaling pathway is best described in the embryonic andhematopoietic stem cells Ramalho-Santos et al searchedfor a transcription profile describing both the embryonic

Stem Cells International 9

stem cells and the stem cells of an adult organism on thebasis of the study of embryonic cells neural stem cellsand hematopoietic stem cells activity [25] These studiesattempted to create a molecular signature of the stem cells inrelation to the self-regeneration and differentiation abilities ofthe cells The active JAK-STAT pathway belongs to the basicattributes defining the so-called ldquostemnessrdquo phenomenon Inthe embryonic stem cells the JAK-STAT signaling pathwaypromotes proliferation in the form of self-replicationThe activating factor LIF (Leukemia Inhibitory Factor)provokes phosphorylation and dimerisation of STAT3which after translocation to the cell nucleus stimulates theexpression of gene group responsible for self-replication ofthe embryonic stem cell However the JAK-STAT signalingpathway is not the only factor responsible for proliferationregulation of the embryonic stem cells Internal processesregulating the multiplication processes independent of theSTAT3 activity such as the Nanog and Oct-4 activity arealso important [15 17 26 27] In our study material theNanog and Oct-4 genes were not active in both types ofnonhematopoietic stem cells which is in agreement withother publications devoted to this subject [28 29] TheSTAT1 transcription factor overactive in our study in CD34minuscells of umbilical cord blood is responsible among othersfor the expression of interferon-activated genes involved incell defense against pathogenic factors [30] Moreover itis believed that STAT1 is a medium in expression of genegroup which is important for cell viability in response tostimulating factors In 2006 Kimrsquos team described STAT1as a highly overactive gene in human mesenchymal stemcells [31] In comparison the transcription factor STAT5which functions in the form of two isoforms STAT5a andSTAT5b is involved in the cellular proproliferation responsesand antiapoptosis processes [32 33] STAT5 is activatedby various cytokine receptors In our study the isoformSTAT5A showed increased activity It is believed that theSTAT5 factor activates specifically the cellular cycle genesthat is cyclins [34 35] In human stem cells the STAT factorsactivate the expression of cyclin Dwhich codes the regulatingsubunit of the CYCDCDK4 complex driving the G1S phasetransition which in turn activates the cell cycle progression[32 36] STAT5a and STAT5b also play an important rolein expression of genes promoting hematopoietic stem cellssurvival [37] Gene expression profiling over a range ofSTAT5 activities in human CB cells revealed subsets of genesthat are associated with the self-renewal and long-termexpansion phenotype Taking into account the fact thatin STAT5minusminus mice myelopoiesis appears to be relativelyunaffected it can be assumed that STAT5 can play a keyrole in assessing the quality of cells [38] Additionally notonly is the JAK-STAT signaling pathway a significant signaltransduction component which controls cell proliferationand purpose but it also regulates cell migration which isimportant both in the primary processes of gastrulationand in the tissue regeneration For instance the JAK-STATsignaling pathway is connected with cardioprotection [39]as well as with processes of regeneration and remodeling ofskeleton [40 41] The active JAK-STAT signaling pathwayhelps to regulate cell organization within cell grouping

[42] The above results may suggest a greater self-renewalpotential of CD34minus stem cells of umbilical cord bloodlong-term expansion phenotype and their higher ability interms of tissue regeneration

The Wnt signaling pathway is perceived as a signalingcascade involved in the control of fundamental processesof nondifferentiated mesenchymal stem cells such as cellreproduction and directed differentiation Both methods ofgene activity classification that is GO and KEGG showeda similar activity of the majority of Wnt signaling pathwaygenes (Table 4) In addition the analysis of separate genestaking part in the Wnt signaling pathway and coding 19glycosylated molecules of extracellular signaling of the Wntfamily cellular surface receptors including Frizzled receptorsintracellular signaling molecules and genes involved in cellcycle and taking part in regulation of proliferation growthand modification of proteins showed that the expressionprofilewas very similar except for single genes of an increasedand decreased expression in CD34minus stem cells of umbilicalcord blood in our study that is MMP7 PRKCB PSEN1RAC2 and CCND3 respectively as well as DKK1 WNT5AWNT5B FZD7 and TCF7L1 The Wnt5a ligand is involvedin the noncanonical Wnt signaling pathway transmissionand it is assumed that it may inhibit the canonical Wntpathway in mouse embryonic cells similarly to Dkk1 Therole of the noncanonical Wnt signaling pathway which inour results seemed to prevail in CD34minus stem cells of bonemarrow is not clear It is believed that the noncanonical Wntsignaling pathway controls the reorganization of cytoskeletonmuscle proteins tissue polarity and cell movement [43] Incontrast the canonical signaling pathway which activatestarget gene transcription through the increase of beta-cateninconcentration seems to be more active in CD34minus stemcells from umbilical cord cells Its role is to control thetissue-specific cell purpose during embryogenesis as well asregulation of proliferation in adult tissues [44] Additionallyhigh activity of WNT5A gene is noticeable in the humanMSCs differentiation into osteoblasts as well as in early stagesof adipogenesis [45 46] According to the publication ofYang et al the Wnt5a and Wnt5b glycoproteins are alsoresponsible for coordination of chondrocyte proliferation anddifferentiation [47] Wnt5a is believed to be an importantfactor taking part in the positive autocrinic regulation ofHSC repopulation ability [48] In their publication Prockopet al suggest that a high WNT5A expression in MSCs ischaracteristic for the end of the exponential and stationarygrowth phases in cell cultures in in vitro conditions [49]Slight changes in theWnt gene activity especially inWNT5Aactivity may result from the role played by MSCs in theirenvironmental niche Bone marrow MSCs seem to be moreinvolved in the renewal processes of the progenitor cells poolwhich reside in bone marrow The researches show that theexpression of Wnt ligands and Fz receptors in MSCs showssome correlation of WntFz pairs such as Wnt5aFz5 [50]The above discussed study results showed that there wasa coexpression of WNT5AFZD7 Their higher expressionwas characteristic for the bone marrow cells while thepreviously published data suggested differences in WntFzactivity depending on the origin-mouse versus humanMSCs

10 Stem Cells International

[51] Currently available data suggest differences which maybe characteristic not only for proliferation and differentiationprograms but also for the phenotype of MSCs from umbilicalcord blood and bone marrow which is in agreement withthe results published by Baksh et al [7] The remaining genestake part in theWnt signaling cascade of increased activity inCD34minus from umbilical cord blood such as MMP7 belongingto metalloproteinases which play a role in the extracellulararray remodeling and wound healing [5]

The Notch signaling pathway belongs to the processes ofintercellular communication which regulate gene expressionpatterns defining a dualistic character of stem cells reflectedby the asymmetric cell division [52] Additionally the Notchsignaling cascade controls various cells biological processesnot only proliferation and differentiation but also apoptosisand regeneration The signaling activity of Notch pathwaygenes determined in the study of 38 genes showed theirsimilar expression as defined by the KEGG classification(Table 4) A similar activity level was noted for all four Notch1ndash4 receptors as well as for JAG1 and JAG2 ligands The dif-ferences were related to two genes DTX4 and PSEN1 whichwere overactive in CD34minus cells from umbilical cord bloodThe function of the PSEN1 gene is mainly developmentalprocesses regarding cell differentiation during neurogenesisAnd the lack of this gene leads to a premature directeddifferentiation of neural progenitor cells [53] The activity ofthe Notch gene cascade plays an important role at all levelsof stem cell development starting from the embryonic stemcell through fetal cells to adult organism stem cells Notchis significantly involved in the asymmetric segregation offactors determining cell purpose during replication divisionsof the stem cell poolThe significance of receptors and ligandsis best described in the hematopoietic stem cells Expressionof the constitutively active Notch1 gene in hematopoieticstem cells induces the immortalized cell lineages which canreproduce the myeloid and lymphoid cell lineage in a long-term repopulation in animal studies [54 55] Apart from thatit delays differentiation of human hematopoietic progenitorstem cells in vitro [56] The studies suggest that Notch1 is animportant factor inHSC self-renewal and can also participatein the inhibition of stem cell differentiation The scientificfindings reveal that a suppression of the Notch signalingpathway via deletion of key genes in HSC did not cause cellsto lose their ability of long-term hematopoiesis renewal inanimal models [57] Therefore the role of ligands and Notchreceptors in HSC has not been precisely defined yet andcurrent publications do not provide precise data regardingthe role played by particular Notch signaling pathway genesin mesenchymal stem cells The only phenomenon whichappears to be superior and characteristic for all stem cellsincluding MSCs is the significance of the Notch cascadein asymmetrical cell divisions This is a significant com-ponent of stem cells population self-renewal as well asthe functionality of the cascade in the stem niche Thisphenomenon constitutes an integral part of the hypothesisabout stem cell population aging However further inves-tigation will be required to determine if the differentiallyexpressed genes described in the studies above could serve asstemness genes

Remaining signaling pathways which revealed increasedactivity in the studied CD34minus cells of umbilical cord bloodincludedMAPK (Mitogen-Activated Protein Kinase Figure 2)which regulates a wide range of cell biological activities suchas gene expression mitosis proliferation differentiationmigration and apoptosis processes [58]TheMAPKpathwaystake part in regulation of early embryonic development andin defining the role of embryonic stem cells from the earlydevelopmental phases until the moment of formation ofdifferentiated adult cells However this precise role played bythe MAPK pathways in various stem cell types has not yetbeen defined

Transmission via calcium ions was also singled out inthe group of the overactive signaling pathways of the studiedCD34minus stem cells of umbilical cord blood versus CD34minus stemcells of bone marrow (Figure 2) The ions are a vital andcommonly present mediator in the process of extracellularsignals transmission and conversion into a cellular responseVariations of calcium ions concentrations regulate geneexpression through an impact on the activity of numeroussignaling pathways Intracellular increase of calcium ionsconcentration also regulates cell activities including adhe-sion motility gene expression and proliferation [59] Thesignificance of transmission via calcium ions and the ways oftheir concentration changes in mesenchymal stem cells stillhave not been studied Kawano et al were the first to reportthat in the nondifferentiated MSCs there are spontaneouschanges of calcium ions concentrations despite the lack ofany stimulating factors [60] As reported the calcium iontransmission through the activation of different gene groupsmay direct the cells onto specific developmental pathways[61]

The integrin-mediated signaling pathway takes part inextracellular array remodeling which means that it con-trols cell behavior and tissue organization Its increasedactivity was observed in our study in CD34minus stem cells ofumbilical cord blood (Figure 2) In recent years the role ofintegrins and selectins has been stressed in the adhesion andtransendothelial migration which has a significant meaningin the processes of repopulation of tissues damaged bydiseases and their regeneration via the circulating MSCs Forinstance Ip et al showed that blocking of beta 1-integrinwhich constitutes a component of the adhesive moleculeVLA-4 in MSC weakened the ability of mesenchymal stemcells to engraft into the infarction-changedmyocardium [62]The above results may suggest a better migration abilityof CD34minus stem cells of umbilical cord blood into the areaof pathologic tissue MSCs own cytokinetic activity thathas similar significance in terms of tissue regenerationThe study of cytokinetic activity of CD34minus stem cells ofumbilical cord blood showed increased activity of signalingpathways activated via cytokines (Figure 2 Table 2) On theother hand the TLR signaling pathway (toll-like receptorsignaling) which in our own results revealed a significantincreased activity in CD34minus stem cells of umbilical cordblood (Figure 2) increases the immunosuppressive charac-teristics of MSCs Weakening of the immunological responsegenerated by the TLR pathway depends on the produc-tion of immunosuppressive kynurenines by the indoleamine

Stem Cells International 11

23-dioxygenase enzyme which degrades tryptophan Induc-tion of indoleamine 23-dioxygenase by TLR entails autocrinesignaling loop of interferon-beta which plays a role in MSCsimmunomodulative effects [63]

According to the GO and KEGG classifications carriedout for cluster 13 with the gene pool of a decreased expressiona statistically significant decrease of gene activity was shownfor the TGF-beta and BMP pathways (Figure 3 Table 3) Itis believed that the family of TGF-beta members whichalso includes BMP plays an important role in initiating theprocess of a directed MSCs differentiation TGF-beta sig-naling transmission runs through specific serine-threoninekinase receptors and their nuclear receptors called the Smadproteins TGF-beta signaling transmission promotes theprocess of early differentiation stages into the chondrogenicand osteogenic lineages [64 65] However cell differentiatinginto the adipogenic lineage results in inhibition of the TGF-beta signaling pathway This was confirmed in experimentalstudies developed by Ng et al where the inhibition of TGF-beta transmission resulted in an increase of adipogenesisand a decrease of differentiating ability towards chondrocytes[66] The BMP signaling pathway strongly promotes differ-entiation towards osteoblasts but it also leads to inhibitionof myogenesis and adipogenesis Such a gene expressionprofile may suggest that the MSCs biological characteristicsare strongly influenced by the significance of stem cell nicheTherefore the mesenchymal stem cells from bone marrowmay be more involved in the promotion of the phenomenawhich maintain the stability of bone marrow environment

5 Conclusions

Multipotentiality of the mesenchymal stem cells their highproliferation ability and not too-demanding culturing con-ditions reveal their potential for wide application in variousmedical areas These are the reasons for the common pursuitto understand their biology based onmore andmore sublimebiomolecular procedures Scientists have been searchingfor an unequivocal marker which would characterize themesenchymal stem cells and simultaneously would enable aseparation of a homogenous MSC population Understand-ing of molecular mechanisms controlling self-replication anddifferentiation is becoming a fundamental issue for clinicalapplication of MSC In this respect the activity of signalingpathways is important yet the interpretation of the pathwaysespecially their interdependence constitutes a great challengefor the scientific world Moreover the answer to the questionregarding MSC innidation and its survival ability in hosttissues after transplantation is yet to be found Explanationof the abovementioned issues will require a more globalunderstanding of biological activity of these cells especiallyon the basis of the significance of the mesenchymal nicheenvironment of stem cells and its impact on the gene andepigenetic activity It should be assumed that continuedprogress in the understanding of MSCs will finally lead totheir common therapeutic and preventive application andconsequently to the improvement of the quality and lengthof human life

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] R J Deans andA BMoseley ldquoMesenchymal stem cells biologyand potential clinical usesrdquo Experimental Hematology vol 28no 8 pp 875ndash884 2000

[2] M F Pittenger A M Mackay S C Beck et al ldquoMultilineagepotential of adult human mesenchymal stem cellsrdquo Science vol284 no 5411 pp 143ndash147 1999

[3] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006

[4] Y-J Chang D T-B Shih C-P Tseng T-B Hsieh D-C Leeand S-M Hwang ldquoDisparate mesenchyme-lineage tendenciesin mesenchymal stem cells from human bone marrow andumbilical cord bloodrdquo Stem Cells vol 24 no 3 pp 679ndash6852006

[5] R A Panepucci J L C Siufi W A Silva Jr et al ldquoComparisonof gene expression of umbilical cord vein and bone marrowndashderived mesenchymal stem cellsrdquo STEM CELLS vol 22 no 7pp 1263ndash1278 2004

[6] C K Rebelatto A M Aguiar M P Moretao et al ldquoDissimilardifferentiation of mesenchymal stem cells from bone marrowumbilical cord blood and adipose tissuerdquo Experimental Biologyand Medicine vol 233 no 7 pp 901ndash913 2008

[7] D Baksh R Yao and R S Tuan ldquoComparison of proliferativeand multilineage differentiation potential of human mesenchy-mal stem cells derived from umbilical cord and bone marrowrdquoStem Cells vol 25 no 6 pp 1384ndash1392 2007

[8] D L Clarke C B Johansson J Wilbertz et al ldquoGeneralizedpotential of adult neural stem cellsrdquo Science vol 288 no 5471pp 1660ndash1663 2000

[9] D J Anderson F H Gage and I L Weissman ldquoCan stem cellscross lineage boundariesrdquo Nature Medicine vol 7 no 4 pp393ndash395 2001

[10] D L Stemple and D J Anderson ldquoIsolation of a stem cell forneurons and glia from the mammalian neural crestrdquo Cell vol71 no 6 pp 973ndash985 1992

[11] R L Rietze H Valcanis G F Brooker T Thomas A K Vossand P F Bartlett ldquoPurification of a pluripotent neural stem cellfrom the adult mouse brainrdquoNature vol 412 no 6848 pp 736ndash739 2001

[12] R M Seaberg S R Smukler T J Kieffer et al ldquoClonalidentification of multipotent precursors from adult mousepancreas that generate neural and pancreatic lineagesrdquo NatureBiotechnology vol 22 no 9 pp 1115ndash1124 2004

[13] A P Beltrami L Barlucchi D Torella et al ldquoAdult cardiac stemcells are multipotent and support myocardial regenerationrdquoCell vol 114 no 6 pp 763ndash776 2003

[14] L Yang S Li H Hatch et al ldquoIn vitro trans-differentiation ofadult hepatic stem cells into pancreatic endocrine hormone-producing cellsrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 99 no 12 pp 8078ndash80832002

[15] I Chambers D Colby M Robertson et al ldquoFunctional expres-sion cloning of Nanog a pluripotency sustaining factor inembryonic stem cellsrdquo Cell vol 113 no 5 pp 643ndash655 2003

12 Stem Cells International

[16] M Dai P Wang A D Boyd et al ldquoEvolving genetranscriptdefinitions significantly alter the interpretation of GeneChipdatardquo Nucleic Acids Research vol 33 pp 175ndash179 2005

[17] RA Irizarry BHobbs F Collin et al ldquoExploration normaliza-tion and summaries of high density oligonucleotide array probelevel datardquo Biostatistics vol 4 no 2 pp 249ndash264 2003

[18] R B Dean and W J Dixon ldquoSimplified statistics for smallnumbers of observationsrdquo Analytical Chemistry vol 23 no 4pp 636ndash638 1951

[19] G J McLachlan and D Peel ldquoOn computational aspectsof clustering via mixtures of normal and t-componentsrdquo inProceedings of the American Statistical Association (BayesianStatistical Science Section) American Statistical AssociationIndianapolis Ind USA 2000

[20] J Polanska P Widlak J Rzeszowska-Wolny M Kimmel andA Polanski ldquoGaussian mixture decomposition of time-courseDNA microarray datardquo in Mathematical Modeling of BiologicalSystems Volume I pp 351ndash359 Birkhauser Boston Mass USA2007

[21] A J Friedenstein U F Gorskaja and N N Kulagina ldquoFibrob-last precursors in normal and irradiated mouse hematopoieticorgansrdquo Experimental Hematology vol 4 no 5 pp 267ndash2741976

[22] S Wakitani T Saito and A I Caplan ldquoMyogenic cells derivedfrom rat bone marrow mesenchymal stem cells exposed to 5-azacytidinerdquoMuscle ampNerve vol 18 no 12 pp 1417ndash1426 1995

[23] D Woodbury E J Schwarz D J Prockop and I B BlackldquoAdult rat and human bone marrow stromal cells differentiateinto neuronsrdquo Journal of Neuroscience Research vol 61 no 4pp 364ndash370 2000

[24] M Reyes T Lund T Lenvik D Aguiar L Koodie and CM Verfaillie ldquoPurification and ex vivo expansion of postnatalhumanmarrowmesodermal progenitor cellsrdquoBlood vol 98 no9 pp 2615ndash2625 2001

[25] M Ramalho-Santos S Yoon Y Matsuzaki R C Mulliganand D A Melton ldquolsquoStemnessrsquo transcriptional profiling ofembryonic and adult stem cellsrdquo Science vol 298 no 5593 pp597ndash600 2002

[26] KMitsui Y Tokuzawa H Itoh et al ldquoThe homeoprotein nanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003

[27] S Okumura-Nakanishi M Saito H Niwa and F IshikawaldquoOct-34 and Sox2 regulate Oct-34 gene in embryonic stemcellsrdquo The Journal of Biological Chemistry vol 280 no 7 pp5307ndash5317 2005

[28] F Ulloa-Montoya B L Kidder K A Pauwelyn et al ldquoCompar-ative transcriptome analysis of embryonic and adult stem cellswith extended and limited differentiation capacityrdquo GenomeBiology vol 8 no 8 article R163 2007

[29] L Song N EWebb Y Song and R S Tuan ldquoIdentification andfunctional analysis of candidate genes regulating mesenchymalstem cell self-renewal and multipotencyrdquo STEMCELLS vol 24no 7 pp 1707ndash1718 2006

[30] A H H Van Boxel-Dezaire M R S Rani and G R StarkldquoComplexmodulation of cell type-specific signaling in responseto type I interferonsrdquo Immunity vol 25 no 3 pp 361ndash372 2006

[31] C G Kim J J Lee D Y Jung et al ldquoProfiling of differentiallyexpressed genes in human stem cells by cDNA microarrayrdquoMolecules and Cells vol 21 no 3 pp 343ndash355 2006

[32] T Bowman R Garcia J Turkson and R Jove ldquoSTATs inoncogenesisrdquo Oncogene vol 19 no 21 pp 2474ndash2488 2000

[33] A Stephanou andD S Latchman ldquoOpposing actions of STAT-1and STAT-3rdquo Growth Factors vol 23 no 3 pp 177ndash182 2005

[34] A Martino J H Holmes J D Lord J J Moon and B HNelson ldquoStat5 and Sp1 regulate transcription of the cyclin D2gene in response to IL-2rdquo The Journal of Immunology vol 166no 3 pp 1723ndash1729 2001

[35] I Matsumura T Kitamura H Wakao et al ldquoTranscriptionalregulation of the cyclin D1 promoter by STAT5 its involvementin cytokine-dependent growth of hematopoietic cellsrdquo TheEMBO Journal vol 18 no 5 pp 1367ndash1377 1999

[36] V Calo M Migliavacca V Bazan et al ldquoSTAT proteins fromnormal control of cellular events to tumorigenesisrdquo Journal ofCellular Physiology vol 197 no 2 pp 157ndash168 2003

[37] M Socolovsky A E J Fallon S Wang C Brugnara and HF Lodish ldquoFetal anemia and apoptosis of red cell progenitorsin Stat5aminusminus5bminusminus mice a direct role for stat5 in Bcl-X(L)inductionrdquo Cell vol 98 no 2 pp 181ndash191 1999

[38] G Li Z Wang Y Zhang et al ldquoSTAT5 requires the N-domainto maintain hematopoietic stem cell repopulating function andappropriate lymphoid-myeloid lineage outputrdquo ExperimentalHematology vol 35 no 11 pp 1684ndash1694 2007

[39] S Negoro K Kunisada E Tone et al ldquoActivation of JAKSTATpathway transduces cytoprotective signal in rat acute myocar-dial infarctionrdquo Cardiovascular Research vol 47 no 4 pp 797ndash805 2000

[40] H C Blair L J Robinson and M Zaidi ldquoOsteoclast signallingpathwaysrdquo Biochemical and Biophysical Research Communica-tions vol 328 no 3 pp 728ndash738 2005

[41] C J Malemud ldquoProtein kinases in chondrocyte signaling andosteoarthritisrdquo Clinical Orthopaedics and Related Research no427 supplement pp S145ndashS151 2004

[42] E M Pinheiro and D J Montell ldquoRequirement for Par-6 andBazooka inDrosophila border cell migrationrdquoDevelopment vol131 no 21 pp 5243ndash5251 2004

[43] D Strutt ldquoFrizzled signalling and cell polarisation inDrosophilaand vertebratesrdquo Development vol 130 no 19 pp 4501ndash45132003

[44] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[45] J Guo J Jin and L F Cooper ldquoDissection of sets of genes thatcontrol the character of wnt5a-deficient mouse calvarial cellsrdquoBone vol 43 no 5 pp 961ndash971 2008

[46] M Nishizuka A Koyanagi S Osada and M Imagawa ldquoWnt4and Wnt5a promote adipocyte differentiationrdquo FEBS Lettersvol 582 no 21-22 pp 3201ndash3205 2008

[47] Y Yang L Topol H Lee and J Wu ldquoWnt5a andWnt5b exhibitdistinct activities in coordinating chondrocyte proliferation anddifferentiationrdquo Development vol 130 no 5 pp 1003ndash10152003

[48] D J Van Den Berg A K Sharma E Bruno and R Hoff-man ldquoRole of members of the Wnt gene family in humanhematopoiesisrdquo Blood vol 92 no 9 pp 3189ndash3202 1998

[49] D J Prockop C A Gregory and J L Spees ldquoOne strategy forcell and gene therapy harnessing the power of adult stemcells torepair tissuesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 1 pp 11917ndash119232003

[50] X He J-P Saint-Jeannet Y Wang J Nathans I Dawid and HVarmus ldquoA member of the Frizzled protein family mediatingaxis induction byWnt-5Ardquo Science vol 275 no 5306 pp 1652ndash1654 1997

Stem Cells International 13

[51] D G Phinney G Kopen R L Isaacson and D J ProckopldquoPlastic adherent stromal cells from the bone marrow ofcommonly used strains of inbred mice variations in yieldgrowth and differentiationrdquo Journal of Cellular Biochemistryvol 72 no 4 pp 570ndash585 1999

[52] N Sasaki T Sasamura H O Ishikawa et al ldquoPolarized exo-cytosis and transcytosis of Notch during its apical localizationin Drosophila epithelial cellsrdquo Genes to Cells vol 12 no 1 pp89ndash103 2007

[53] M Handler X Yang and J Shen ldquoPresenilin-1 regulatesneuronal differentiation during neurogenesisrdquo Developmentvol 127 no 12 pp 2593ndash2606 2000

[54] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006

[55] A W Duncan F M Rattis L N DiMascio et al ldquoIntegrationof Notch andWnt signaling in hematopoietic stem cell mainte-nancerdquo Nature Immunology vol 6 no 3 pp 314ndash322 2005

[56] N Carlesso J C Aster J Sklar and D T Scadden ldquoNotch1-induced delay of human hematopoietic progenitor cell differen-tiation is associated with altered cell cycle kineticsrdquo Blood vol93 no 3 pp 838ndash848 1999

[57] I Maillard U Koch A Dumortier et al ldquoCanonical notch sig-naling is dispensable for the maintenance of adult hematopoi-etic stem cellsrdquo Cell Stem Cell vol 2 no 4 pp 356ndash366 2008

[58] G Pearson F Robinson T B Gibson et al ldquoMitogen-activatedprotein (MAP) kinase pathways regulation and physiologicalfunctionsrdquo Endocrine Reviews vol 22 no 2 pp 153ndash183 2001

[59] R E Dolmetsch R S Lewis C C Goodnow and J I HealyldquoDifferential activation of transcription factors induced by Ca2+response amplitude and durationrdquoNature vol 386 no 6627 pp855ndash858 1997

[60] S Kawano S Shoji S Ichinose K Yamagata M Tagami andM Hiraoka ldquoCharacterization of Ca2+ signaling pathways inhuman mesenchymal stem cellsrdquo Cell Calcium vol 32 no 4pp 165ndash174 2002

[61] B Ye ldquoCa2+ oscillations and its transporters in mesenchymalstem cellsrdquo Physiological Research vol 59 no 3 pp 323ndash3292010

[62] J E Ip Y Wu J Huang L Zhang R E Pratt and V J DzauldquoMesenchymal stem cells use integrin 1205731 not CXC chemokinereceptor 4 for myocardial migration and engraftmentrdquoMolecu-lar Biology of the Cell vol 18 no 8 pp 2873ndash2882 2007

[63] C A Opitz U M Litzenburger C Lutz et al ldquoToll-like recep-tor engagement enhances the immunosuppressive propertiesof human bone marrow-derived mesenchymal stem cells byinducing indoleamine-23-dioxygenase-1 via Interferon-120573 andprotein kinase Rrdquo Stem Cells vol 27 no 4 pp 909ndash919 2009

[64] B Kulterer G Friedl A Jandrositz et al ldquoGene expressionprofiling of human mesenchymal stem cells derived from bonemarrow during expansion and osteoblast differentiationrdquo BMCGenomics vol 8 article 70 2007

[65] W G Li and X X Xu ldquoThe expression of N-cadherinfibronectin during chondrogenic differentiation of MSCinduced by TGF-1205731rdquo Chinese Journal of Traumatology vol 8no 6 pp 349ndash351 2005

[66] F Ng S Boucher S Koh et al ldquoPDGFTGF-120573 and FGF signal-ing is important for differentiation and growth of mesenchymalstem cells (mscs) transcriptional profiling can identify markersand signaling pathways important in differentiation of MSCsinto adipogenic chondrogenic and osteogenic lineagesrdquo Bloodvol 112 no 2 pp 295ndash307 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

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Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

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Advances in

Virolog y

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Nucleic AcidsJournal of

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Enzyme Research

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International Journal of

Microbiology

Stem Cells International 3

PrepPlus (AampABiotechnology) based on themodifiedChom-czynski and Sacchirsquos method At the initial stage the cellswere lysed while the endogenous RNases were inactivatedwith fenozol After adding chloroform the mixture wascentrifuged and the collected RNA was precipitated withisopropanol Next the mixture was centrifuged for 20minat 13 000 rmin the supernatant was decanted and the RNAsediment was washed twice with 75 ethanol After dryingthe sediment was stored at minus70∘C for the analysis

25 Qualitative Assessment of RNA Extracts RNA extractsunderwent qualitative assessment via the agarose gel elec-trophoresis in 08 agarose gel with ethidium bromide(05mgmL) in the SUBMINI apparatus 5120583L of extract wasmixed with 3 120583L of gel-loading dye (005 wv bromophenolblue 60 glycerol) and heated at 65∘C for 4min After theseparation the electropherograms were assessed with the UVtransilluminator (120582 = 260 nm)

26 Quantitative Assessment of RNA Extracts RNA extractsunderwent quantitative assessment via spectrophotomet-ric measurement of RNA concentration (Gene Quant IIPharmacia) The spectrophotometric assessment of extractscovered the absorbance measurement at the wave lengthsof 230 260 280 and 320mn and then the A

260

A280

ratio and protein content were determined for each sampleAbsorbance value at the 260 nm wave length was used tocalculate RNA concentration based on the assumption thatresults of cuvette measurements using the 1 cm optical pathequaling 1 OD

260

correspondwith the concentration of 40mgRNA in 1 cm3 of extract

27 Gene Expression Analysis with the OligonucleotideMicroarrays Technique

271 General Method Assumptions The material for thestudy consisted of total RNA extracted from the cell cultureswith the TRIzol reagent (Invitrogen) The isolated RNA waspurified with the RNeasy Total RNA Mini Kit and digestedwith DNase I (Qiagen) in order to eliminate any possiblecontamination with DNA Approximately 8 120583g of total RNAwas used for the double-stranded cDNA synthesis Biotin-labeled cRNA was synthesized and biotinylated cRNA wasthen fragmented and hybridized with the Test3 microarrayand HG U133A (Affymetrix) and labeled twice with thestreptavidin-phycoerythrin conjugate and biotinylated strep-tavidin antibodies

The fluorescence intensity was analyzedwith theGeneAr-ray Scanner G2500A The quantity and quality of total RNAcDNA and cRNA were assessed spectrophotometrically andby means of the electrophoresis technique in 1 agarose gel

272 Purification of Total RNA Extract with RNasy Mini KitAfter a prior concentration measurement 100120583L of the RNAextract was obtained with RNase-free water 350 120583L of RLTbuffer with 120573-mercaptoethanol and 250120583L of 96 ethanolwere added mixed by pipetting and then applied to thecolumn and centrifuged for 15 seconds at 12 000 rmin The

filtrate was discarded and the column was moved to a newtube and 350 120583L of RW1 was added and then centrifugedfor 15 seconds at 10ndash12 000 rmin For digestion 70 120583L ofRDD buffer was added to a tube with 10120583L of DNase Thismixture was applied on the column membrane and left todigest for 15min at room temperature Then 350 120583L of RW1solution was added the old flow-through was discarded andthe column was centrifuged for 15 sec at 12 000 rmin Theflow-throughwas again discarded and 500120583L of RPE solutionwas added to the column The extracts were centrifuged for1min at 14 000 rmin then the column was turned upsidedown and centrifuged again for the same time and rate Aftercentrifugation the column was placed in a new tube and30 120583L of H

2

O was added and was left for 10min Then thecolumn was centrifuged and 20120583L of H

2

O was added andleft for a few minutes and then centrifuged again After finalcleanup the RNA concentration in extracts was measured

28 cDNA Synthesis

281 First Strand cDNA Synthesis In order to obtain the firststrand of cDNA 2 120583L 100 pM of T7 starter-oligo(dT)

24

(51015840-GCCAGTGAATTGTAATACGACTCACTATAG GGAG-GCGG-31015840) was added to 11 120583L of RNA extract the mixturewas incubated at 70∘C for 10minutes and placed on iceThen4 120583L of 5x First Strand Buffer 2120583L of 01M DTT and 1 120583L of10mM dNTPs (Invitrogen) were added After 2 minutes ofpreincubation at 45∘C 1 120583L (200U) of reverse transcriptaseSupercscript II (Life Technologies) was added to the reactionmixture and incubation continued for another hour

282 Second Strand cDNA Synthesis In order to obtain thesecond strand of cDNA the following were added to themixture obtained after the first strand synthesis 30 120583L of5x Second Strand Buffer 91 120583L of RNase-free water 3 120583L of10mM dNTPs 4 120583L (40U) of E coli DNA Polymerase I1 120583L (10U) of E coli DNA Ligase and 1 120583L (2U) of Rnase H(Invitrogen) The mixture was incubated for 2 hours at 16∘C

Next 25 120583L (10U) of T4 DNA Polymerase I was addedto the mixture and the mixture was incubated for another5 minutes at 16∘C The composition of the reaction mixtureused for the second strand cDNA synthesis is presentedin Table 2 The reaction was stopped by adding 10 120583L of05M EDTA and the double-stranded cDNA was extractedusing the phenolchloroformisoamyl alcohol mixture Theaqueous phase was divided bymeans of Phase Lock Gel Light15mL tubes (Eppendorf) 05 volume of 75M ammoniumacetate (Sigma) and 25 volume of 96 ethyl alcohol wereadded to the aqueous phase and it was left for 12 h at minus20∘C

29 Purification of the Obtained cDNA After freezing for theentire night at minus20∘C 03 120583L of Palet Point (Merc) was addedto the reaction mixture and it was centrifuged for 20min at12 000 rmin at room temperature supernatant was discardedand the sediment was rinsed with 2x 70 ethanol (500120583L)and centrifuged for 5min at 12 000 rmin the sediment wasdried thoroughly and dissolved in 12 120583L of RNase-free water

4 Stem Cells International

210 Biotinylated cRNA Synthesis The template for thebiotinylated cRNA synthesis consisted of 10 120583L ds cDNAto which the following were added 12 120583L of RNase-freewater 4 120583L of 2x hybridization buffer 4 120583L of biotin-labeledribonucleotides 4 120583L of DTT 4 120583L RNase inhibitor and2 120583L of T7 polymerase (BioArray High Yield RNA TranscriptLabeling Kit Enzo) The whole was left at 37∘C for 5 h andmixed every 30 minutes

211 Biotinylated cRNA Purification After a 5-hour incuba-tion cRNA was cleaned up using 60 120583L of H

2

O 350 120583L ofRLT buffer and 250 120583L of 96 ethanol added to the reactionmixture The mixture was applied onto the RNAeasy columnTotal RNA Mini Kit (Qiagen) and was centrifuged twice for15 seconds at 10 000 rmin In the last stage 2x 20120583L of H

2

Owas added to the column membrane it was centrifuged at12 000 rmin and then the cRNA concentration was mea-sured

212 Biotinylated cRNA Fragmentation For fragmentationpurposes 16 120583g of cRNA and 8 120583L of fragmentation buffer(Affymetrix) were used and the mixture was supplementedwith up to 40 120583L of RNase-free water After completion of thisstage the mixture was centrifuged and left for 35min at 94∘Cand then placed on iceThe fragmentation result was checkedon 1 agarose gel

213 Assessment of cDNA Biotinylated cRNA and cRNASynthesis after Fragmentation via Electrophoresis in AgaroseGel Evidence of proper cDNA biotinylated cRNA andcRNA synthesis after fragmentation was assessed throughelectrophoresis in agarose gel with ethidium bromide Theresulting separation electrophoregram of the abovemen-tioned products verified that samples were acceptable to usein the following analysis stage

214 Hybridization of the Fragmented BiotinylatedcRNA with Microarray

2141 Preparation of Hybridization Cocktail In order toprepare the hybridization cocktail 375 120583L of fragmentedbiotinylated cRNA was used and the following were added5 120583L of control oligonucleotide B2 15 120583L of eukaryotichybridization control (Affymetrix) 3120583L of Herring Sperm(Invitrogen) 3 120583L of acetylated BSA (Invitrogen) 150 120583L of2x hybridization buffer and 865 120583L of RNase-free water Thecocktail was heated for 5min at 99∘C and for 5min at 45∘Cand centrifuged for 5min at 14 000 rmin

2142 Application of Cocktail on Microarray HG-U133Amicroarray (Affymetrix)was heated to room temperature andthen 100 120583L of 1x hybridization buffer was applied The chipwas placed in hybridization oven for 10min Next the bufferwas removed from the chip and 200120583L of hybridizationcocktail was applied The microarray was placed in thehybridization oven for 16 h at 45∘C at 55 rmin

2143 Washing and Scanning of Microarrays After a 16-hour hybridization the HG U133A microarray was washedwith buffer and stained with solutions (SAPE solutionstreptavidin antibody solution) and then scanned with theGeneArray Scanner G2500A (Affymetrix)

215 Statistical Analysis Data regarding expression levelsobtained directly from the HGU133A array were reannotatedwith CDF control files developed by Dai et al [16] Thenext step consisted of microarray normalization with theRMA algorithm [17] Three repetitions were replaced withan average value with a prior detection of outliers by meansof the Dean and Dixon method [18] Determination of mea-surement noise threshold was conducted through empiricaldecomposition of probability density function of expressionlevels into normal components [19] The component of thelowest mean value unchanged across all the microarrays wasassumed to be the noise model The threshold value of thefilter was constructed at level 28 (in the field of logarithmsto base 2) The mixture model of normal distributions con-structed for the average SRL (Signal Log Ratio) of CD34minus cellsfromumbilical cord blood versusCD34minus of bonemarrowwasused to define the affinity of certain genes to subgroups of anincreased decreased or unchanged value of expression levelMoreover thismixturemodel was used to determine the FDR(False Discovery Rate) value for each of them

In order to define qualitatively the processes in stemcells from umbilical cord blood in comparison with stemcells from bonemarrow an unsupervised gene clustering wasconducted Again the GaussianMixture Model for empiricaldecomposition of expression levels constituted the algorithmenabling such a division However in this analysis additionallimitations based on the gene composition of each compo-nent that were the same across the experimental conditionswere added to the algorithm [20] Clustersclasses obtainedin this manner were compared to cellular processes in whichthey are involved The categories of cellular processes weredefined by gene ontology terms that are commonly appliedfor this purpose A number of conditioning hypergeometrictests were conducted verifying the one-sided hypothesisabout overrepresentation For each ontological term thatwas found to be significantly overrepresented statisticallyin a given gene cluster the OR (Odds Ratio) value wasdetermined

Gene pathways whose components are genes belongingto specific clusters underwent an additional analysis whichwas independent of a direct description of cellular processesFor this purpose we used data from the KEGG databaseFor each pathway the hypothesis of overrepresentation of aspecified cluster of genes as compared with the whole scopeof the analyzed genes was verified Precise 119901 values weredetermined

Statistically significant values were those of FDR lt 010 or119901 lt 005

3 Results

The first stage of the study consisted of data reannotation forexpression levels through the CDF control files developed

Stem Cells International 5

by Dai et al [16] For many years the platform for geneexpression profile was the Affymetrix GeneChip databaseHowever their probe section was created on the basis ofprior gene and transcriptome annotations which significantlydiffer from the modern expertise A comparative analysisbetween many popular Gene Chips and redefined probesbased on updated gene data with consideration to geneticpolymorphism showed a 30ndash50 discrepancy [16] thereforeit was decided to reannotate data as the first step in ourstudyThe assessment of noise threshold was then completedcreating a mixture model of normal distributions for themean SLR of CD34minus cells from umbilical cord blood versusCD34minus from bone marrow

The next step comprised microarrays normalizationthrough the RMA algorithm [17] Three repetitions werereplaced with a mean value with a prior detection of outliersby means of the Dean and Dixon method [18] Determina-tion of the measurement noise threshold was conducted byempirical decomposition of probability density function ofexpression levels into normal components [19] The noisemodel was assumed to be the component of the lowest meanvalue which was unchanged across all the microarrays Thethreshold value of the filter was constructed at level 28 (inthe field of logarithms to base 2)

As a result of the abovementioned procedures a data setwas obtained which described 11 979 genes which underwentfurther analysis The first stage consisted of creating the mix-ture model of normal distributions constructed for a meanSLR (Signal Log Ratio) of CD34minus stem cells from umbilicalcord blood versus CD34minus stem cells from bone marrowwhich allowed defining the affinity criteria of specific genes tosubgroups of an increased decreased and unchanged valueof expression levels This also enabled us to determine a FDRvalue (False Discovery Rate) for each of them (Figure 1)

This method is based on decomposition that is thehistogram decomposition into (K)mdashGaussrsquos components aswell as modeling the probability density function distribu-tions (pdf) for given gene expression levels The method wasused for analysis of a histogram reflecting the frequency ofgenes of a specific expression level in the tested preparationThis initial analysis showed considerable similarity of geneexpression of CD34minus cells from umbilical cord blood ascompared with CD34minus cells from bone marrow

31 Unsupervised Gene Clustering During the next stage ofdata analysis a qualitative determination of the processes inCD34minus stem cells from umbilical cord blood in comparisonwith CD34minus stem cells from bone marrow was conductedusing unsupervised gene clustering Again the GaussianMixture Model for empirical decomposition of expressionlevels constituted the algorithm enabling such a divisionHowever in this analysis additional limitations based onthe gene composition of each component that were thesame across the experimental conditions were added to thealgorithm [20] As a result 15 gene clusters were obtainedtwo of which (numbers 9 and 13 in this study) containeddiscriminatory genes exhibiting a statistically significantincrease (cluster number 9) and decrease of gene expression(cluster number 13) (Table 1)

1600

1400

1200

1000

800

600

400

200

00 2 4 6 8

Num

ber o

f obs

erva

tions

Gene expressionminus10 minus8 minus6 minus4 minus2

SLR CD34H adnotacje Dai ver12

Figure 1Mixturemodel of normal distributions (GaussianMixtureModel) for a mean SLR (Signal Log Ratio) of CD34minus cells fromumbilical cord blood versus CD34minus cells from bone marrow Log(CD34-P)( CD34-Sz)

The remaining clusters differing in terms of the numberof genes within the cluster aggregated the genes of similarexpression values Differences betweenmean gene expressionlevels of CD34minus stem cells from bone marrow and CD34minusstem cells from umbilical cord blood in clusters numbered1ndash8 10ndash12 14 and 15 are small or nonexistent Thus all theseclusters were classified as the groups of genes exhibiting thesame expression levels

Cluster number 9 included 493 genes which show anincreased activity in CD34minus stem cells from umbilical cordblood of the mean SRL (Signal Log Ratio) equaling 306 Incontrast cluster number 13 included a lower number of genes(119899 = 387) of decreased expression in CD34minus stem cells fromumbilical cord blood for which SRL equals minus343

At the next stage gene clustersgroups obtained in thismanner were compared to biological processes in whichthey are involved as defined by gene ontology (GO) termsthat are commonly applied for this purpose A number ofconditioning hypergeometric tests were conducted verifyingthe one-sided hypothesis about overrepresentation For eachontological term that was found to be significantly overrep-resented statistically in a given gene cluster the OR (OddsRatio) value was determined All the clusters were classifiedthrough the GO classification system that is the clusterswith genes discriminating both cell populations as well as theclusters grouping genes of a similar expression

Four hundred ninety-three genes of cluster 9 classifiedvia the GO system determined the terms of biological pro-cesses (119901 lt 005) overrepresented in CD34minus stem cells fromumbilical cord blood The classification system determined256 GO terms of biological processes out of which GO termsof 119901 value below 004 underwent further analysis As a resultof this analysis the majority of GO terms represented only byone or two genes were rejected The GO terms for biologicalprocesses in cluster 9 were assigned to one of six func-tional groups including metabolism biological processes

6 Stem Cells International

Table 1 15 clusters obtained as a result of the unsupervised gene clusteringMean CD34minus BM (mean of CD34minus stem cells from bonemarrow)Mean CD34minus UCB (mean of CD34minus stem cells from umbilical cord blood)119873 (a number of genes in a cluster) SRL (Signal Log Ratio)

Cluster MeanCD34minus BM

MeanCD34minusUCB

SDCD34minusBM

SDCD34minusUCB

119873

119901 value MeanSLR

minus95CISLR

+95CISLR

Conclusion

1497 491 008 009

1102

000000023 minus006 minus008 minus004

No change2

1157 1149 035 047

54

026488907 minus008 minus022 006

No change3

802 805 030 032

592

063190010 001 minus005 007

No change4

445 440 006 005

999

000000001 minus006 minus008 minus004

No change5

702 714 024 021

898

000000009 011 007 016

No change6

554 550 012 013

1133

000000889 minus006 minus009 minus003

No change7

353 346 004 005

960

451861119864 minus 13 minus007 minus009 minus005

No change8

476 483 071 088

961

003102164 011 001 020

No change9 564 715 158 270 493 p lt 00000001 306 295 316 Up10

311 309 004 004

872

000325256 minus003 minus005 minus001

No change11

276 278 002 002

647

000002947 003 001 004

No change12

398 390 006 007

1133

544009119864 minus 15 minus008 minus010 minus006

No change13 739 486 285 101 387 p lt 00000001 minus343 minus357 minus329 Down14

621 621 026 021

1353

044902052 minus001 minus005 002

No change15

930 936 064 084

395

015369595 008 minus003 020

No change

0 001 002 003 004

Signal transduction (GO0007165)

Activation of MAPK activity (GO0000187)

Protein kinase cascade (GO0007243)

Regulation of MAP kinase activity (GO0043405)

Integrin-mediated signaling pathway (GO0007229)

Positive regulation of tyrosine phosphorylation ofSTAT protein (GO0042531)

Positive regulation of calcium-mediated signaling(GO0050850)

JAK-STAT cascade (GO0007259)

Cytokine-mediated signaling pathway (GO0019221)

Positive regulation of cell proliferation (GO0008284)

Calcium-mediated signaling (GO0019722)

Regulation of JAK-STAT cascade (GO0046425)

p value

Signaling pathway

Figure 2 Functional annotations 12 GO classification terms for biological processes in the group signaling pathways in cluster 9 dependingon the 119901 value (119901 lt 004)

regulation cellular homeostasis cellular defensive responseand response to stimuli cytokine production and signalingpathways The GO terms regarding signaling pathways arepresented in Figure 2

Thenext stage consisted of a similar analysis of genes fromcluster 13 GO system classification for 387 genes of cluster 13determined 119 terms of biological processes (119901 lt 005) of

a decreased activity in CD34minus cells fromumbilical cord bloodas compared with CD34minus cells from bone marrow The GOterms of 119901 value below 004 underwent further analysis As aresult of this analysis the majority of GO terms representedonly by one or two genes were rejected The GO terms forbiological processes in cluster 13 were assigned to one ofthree functional groups including metabolism biological

Stem Cells International 7

0000 0010 0020 0030

Negative regulation of signal transduction (GO0009968)

Vascular endothelial growth factor receptor signalingpathway (GO0048010)

Transmembrane receptor protein tyrosine kinase signalingpathway (GO0007169)

Transmembrane receptor protein serinethreonine kinasesignaling pathway (GO0007178)

Regulation of transforming growth factor beta receptorsignaling pathway (GO0017015)

Cell surface receptor linked signal transduction(GO0007166)

BMP signaling pathway (GO0030509)

p value

Signaling pathway

Figure 3 Functional annotations 7 GO classification terms for biological processes in the group signaling pathways in cluster 13 dependingon the 119901 value (119901 lt 004)

Table 2 Gene pathways based on the KEGG classification of an increased expression in CD34minus stem cells from umbilical cord blood

KEGGID 119901 value Odds ratio Exp count Count Size Term4060 0001 2947 14 33 223 Cytokine-cytokine receptor interaction4620 0001 3672 6 17 92 Toll-like receptor signaling pathway5120 0001 3748 4 12 63 Epithelial cell signaling inHelicobacter pylori infection4630 0012 2067 8 15 130 JAK-STAT signaling pathway

processes regulation and signaling pathways In CD34minus cellsfrom umbilical cord blood as compared with CD34minus cellsfrom bone marrow a decreased gene activity was observedof the BMP signaling pathway TGF beta transmembranereceptor of protein serinethreonine kinase transmembranereceptor of tyrosine kinase and VEGF (Figure 3)

Gene pathways whose components are genes belongingto specific clusters underwent an additional analysis whichwas independent of a direct description of cellular processesFor this purpose data from the KEGG database (KyotoEncyclopedia of Genes and Genomes) were used For eachpathway the hypothesis of overrepresentation of specificclusters of genes as compared with the whole scope ofanalyzed genes was verified and precise 119901 values weredetermined For the purpose of this study gene pathwaysfor clusters 9 and 13 were analyzed because they containedgenes discriminating both gene populations On the basisof KEGG database cytokine-cytokine receptor interactionpathway toll-like receptor signaling pathway epithelial cellsignaling in Helicobacter pylori infection and JAK-STATsignaling pathway are overrepresented in CD34minus cells fromumbilical cord blood (Table 2)

In cluster 13 on the basis of KEGG classification theCD34minus cells from umbilical cord blood show a decreasedactivity of focal adhesion signaling pathway cell junctionsand TGF-beta signaling pathway (Table 3)

At the next stage the genes of the remaining clustersnumbered 1ndash8 10ndash12 and 14 of a similar gene expression were

analyzed The analysis was based on the GO classification foreach of the abovementioned clusters The criteria used forchoosing the clusters included the biological processes con-nected with cellular cycle as well as the processes connectedwith signaling pathways activity A similar gene expression inCD34minus cells from umbilical cord blood and CD34minus cells frombone marrow was characteristic for such biological processesas cell division mitosis maintaining telomere endings RNAandDNA processing and a similar activity of Notch andWntsignaling pathways genes

An independent analysis was also carried out for genes ofa similar activity in clusters 1ndash8 10ndash12 and 14 on the basis ofthe KEGG database CD34minus cells from umbilical cord bloodand CD34minus cells from bone marrow showed similar activityof Wnt and Notch signaling pathways genes p53 signalingpathway genes cell cycle PPAR and mTOR (Table 4)

CD34minus cells from umbilical cord blood and CD34minus cellsfrom bone marrow showed similar gene activity of Wntand Notch signaling pathways both on the basis of the GOclassification system and the KEGG database Therefore atthe last stage of the study the activity of single genes of Wntand Notch as well as JAK-STAT signaling pathways chosenfrom 11976 sampled genes was analyzed

In accordance with the KEGG database the JAK-STATsignaling pathway is defined by 155 genes out of whichthere were 128 active genes in the studied material Genesexhibiting increased activity in CD34minus cells from umbilicalcord blood included SPRY2 CISH CSF2 GRB2 IFNGR1

8 Stem Cells International

Table 3 Gene pathways based on the KEGG classification of a decreased expression in CD34minus stem cells from umbilical cord blood

KEGGID 119901 value Odds ratio Exp count Count Size Term4510 0001 7517 7 33 188 Focal adhesion1430 0001 8157 4 22 109 Cell junctions4350 0001 4038 3 10 82 TGF-beta signaling pathway

Table 4 Gene pathways based on the KEGG classification of a similar expression in CD34minus stem cells from umbilical cord blood and CD34minuscells from bone marrow for clusters 1ndash8 10ndash12 and 14-15

KEGGID 119901 value Odds ratio Exp count Count Size Term04310 0018 1880 10 17 131 Wnt signaling pathway04330 0025 2722 3 7 39 Notch signaling pathway04115 0038 2157 5 9 61 p53 signaling pathway04110 0009 2028 10 18 100 Cell cycle03320 0020 2791 3 7 60 PPAR signaling pathway04150 0035 2220 5 9 46 mTOR signaling pathway

IL2RB IL2RG IL7R IL10RA IL15RA PIM1 PTPN6 STAT1STAT5A and CCND3 Genes exhibiting decreased activityincluded PIAS3 IL11RA and CBLB

On the basis of the KEGG database the Wnt signalingpathway is defined by 151 genes out of which there were132 active genes in the studied material Genes exhibit-ing increased activity in CD34minus cells from umbilical cordblood include MMP7 PRKCB PSEN1 RAC2 and CCND3Genes exhibiting decreased activity include DKK1 WNT5AWNT5B FZD7 and TCF7L1

On the basis of the KEGG database the Notch signalingpathway is defined by 47 genes out of which there were38 active genes in the studied material Genes exhibitingincreased activity in CD34minus cells from umbilical cord bloodincluded DTX4 and PSEN1

4 Discussion

Nonhematopoietic stem cells were first identified by Frieden-stein et al who described the progenitor cells of rat bonemarrow [21] Currently the nonhematopoietic stem cellswhich are frequently referred to although not quite correctlyas the mesenchymal stem cells are isolated from varioustissues including the umbilical cord blood placenta andumbilical cord A constantly increasing number of studieson MSCs have proved that in appropriate environmentalconditions they are capable of differentiating into special-ized mesodermal endodermal and even ectodermal celllineages [2 22ndash24] In natural conditions they play an impor-tant role in cell regeneration processes and in repairingthe tissues damaged by degenerating processes Anotherintriguing characteristic of MSCs is their ability to avoidimmunological identification and response therefore theyare referred to as the hypoimmunogenic cells Despite greatattention paid to mesenchymal stem cells their biology isnot yet fully understood Introducing multipotential stemcells into clinical application requires a full understandingof the mechanisms which control key properties of thecell such as cell self-replication versus differentiation and

mobilization versus tissue homing The understanding ofthese mechanisms must be based on strict definitions ofhuman multipotential stem cells at the molecular level Inaddition understanding of molecular mechanisms of self-replicating pluripotentiality plasticity and differentiation ofstem cells not only is a necessary condition to overcomelimitations of stem cells application as a therapeutic factorbut also is key to understand a fundamental mystery of thedevelopment of human organism

This paper presents a comparison of gene activity of thenonhematopoietic cells of umbilical cord blood and bonemarrow using oligonucleotidemicroarrays Gene activity wastested in stem cells CD34 and included 11 979 genes obtainedafter completing procedures to increase the purity of cellsamples Cell populations included in the study were alsolimited based on the noise threshold revealed in the finalcomparative analysis The gene expression profiles of thecompared populations of CD34minus stem cells from umbilicalcord blood and CD34minus stem cells from bone marrow werevery similar (Figure 1) For a better illustration of the biolog-ical molecular activity and the activity of signaling pathwaysin the studied cell populations a transcription profile analysiswith the GO and KEGG databases was completed In termsof fundamental signaling pathways for biological activityof stem cells like JAK-STAT Wnt and Notch we recordeddiscrete differences among expressed genes from umbilicalcord blood cells as compared to bone marrow cells

The analysis regarding signaling pathways showedincreased activity of the JAK-STAT pathway in CD34minusstem cells from umbilical cord blood both on the basis ofthe gene ontology classification and the KEGG database(Figure 2 and Table 2) The increased activity regarded 15genes which take part in the JAK-STAT cascade that isSPRY2 CISH CSF2 GRB2 IFNGR1 IL2RB IL2RG IL7RIL10RA IL15RA PIM1 PTPN6 and CCND3 including thegenes which code transcription factors STAT1 and STAT5AIn previous publications the significance of the JAK-STATsignaling pathway is best described in the embryonic andhematopoietic stem cells Ramalho-Santos et al searchedfor a transcription profile describing both the embryonic

Stem Cells International 9

stem cells and the stem cells of an adult organism on thebasis of the study of embryonic cells neural stem cellsand hematopoietic stem cells activity [25] These studiesattempted to create a molecular signature of the stem cells inrelation to the self-regeneration and differentiation abilities ofthe cells The active JAK-STAT pathway belongs to the basicattributes defining the so-called ldquostemnessrdquo phenomenon Inthe embryonic stem cells the JAK-STAT signaling pathwaypromotes proliferation in the form of self-replicationThe activating factor LIF (Leukemia Inhibitory Factor)provokes phosphorylation and dimerisation of STAT3which after translocation to the cell nucleus stimulates theexpression of gene group responsible for self-replication ofthe embryonic stem cell However the JAK-STAT signalingpathway is not the only factor responsible for proliferationregulation of the embryonic stem cells Internal processesregulating the multiplication processes independent of theSTAT3 activity such as the Nanog and Oct-4 activity arealso important [15 17 26 27] In our study material theNanog and Oct-4 genes were not active in both types ofnonhematopoietic stem cells which is in agreement withother publications devoted to this subject [28 29] TheSTAT1 transcription factor overactive in our study in CD34minuscells of umbilical cord blood is responsible among othersfor the expression of interferon-activated genes involved incell defense against pathogenic factors [30] Moreover itis believed that STAT1 is a medium in expression of genegroup which is important for cell viability in response tostimulating factors In 2006 Kimrsquos team described STAT1as a highly overactive gene in human mesenchymal stemcells [31] In comparison the transcription factor STAT5which functions in the form of two isoforms STAT5a andSTAT5b is involved in the cellular proproliferation responsesand antiapoptosis processes [32 33] STAT5 is activatedby various cytokine receptors In our study the isoformSTAT5A showed increased activity It is believed that theSTAT5 factor activates specifically the cellular cycle genesthat is cyclins [34 35] In human stem cells the STAT factorsactivate the expression of cyclin Dwhich codes the regulatingsubunit of the CYCDCDK4 complex driving the G1S phasetransition which in turn activates the cell cycle progression[32 36] STAT5a and STAT5b also play an important rolein expression of genes promoting hematopoietic stem cellssurvival [37] Gene expression profiling over a range ofSTAT5 activities in human CB cells revealed subsets of genesthat are associated with the self-renewal and long-termexpansion phenotype Taking into account the fact thatin STAT5minusminus mice myelopoiesis appears to be relativelyunaffected it can be assumed that STAT5 can play a keyrole in assessing the quality of cells [38] Additionally notonly is the JAK-STAT signaling pathway a significant signaltransduction component which controls cell proliferationand purpose but it also regulates cell migration which isimportant both in the primary processes of gastrulationand in the tissue regeneration For instance the JAK-STATsignaling pathway is connected with cardioprotection [39]as well as with processes of regeneration and remodeling ofskeleton [40 41] The active JAK-STAT signaling pathwayhelps to regulate cell organization within cell grouping

[42] The above results may suggest a greater self-renewalpotential of CD34minus stem cells of umbilical cord bloodlong-term expansion phenotype and their higher ability interms of tissue regeneration

The Wnt signaling pathway is perceived as a signalingcascade involved in the control of fundamental processesof nondifferentiated mesenchymal stem cells such as cellreproduction and directed differentiation Both methods ofgene activity classification that is GO and KEGG showeda similar activity of the majority of Wnt signaling pathwaygenes (Table 4) In addition the analysis of separate genestaking part in the Wnt signaling pathway and coding 19glycosylated molecules of extracellular signaling of the Wntfamily cellular surface receptors including Frizzled receptorsintracellular signaling molecules and genes involved in cellcycle and taking part in regulation of proliferation growthand modification of proteins showed that the expressionprofilewas very similar except for single genes of an increasedand decreased expression in CD34minus stem cells of umbilicalcord blood in our study that is MMP7 PRKCB PSEN1RAC2 and CCND3 respectively as well as DKK1 WNT5AWNT5B FZD7 and TCF7L1 The Wnt5a ligand is involvedin the noncanonical Wnt signaling pathway transmissionand it is assumed that it may inhibit the canonical Wntpathway in mouse embryonic cells similarly to Dkk1 Therole of the noncanonical Wnt signaling pathway which inour results seemed to prevail in CD34minus stem cells of bonemarrow is not clear It is believed that the noncanonical Wntsignaling pathway controls the reorganization of cytoskeletonmuscle proteins tissue polarity and cell movement [43] Incontrast the canonical signaling pathway which activatestarget gene transcription through the increase of beta-cateninconcentration seems to be more active in CD34minus stemcells from umbilical cord cells Its role is to control thetissue-specific cell purpose during embryogenesis as well asregulation of proliferation in adult tissues [44] Additionallyhigh activity of WNT5A gene is noticeable in the humanMSCs differentiation into osteoblasts as well as in early stagesof adipogenesis [45 46] According to the publication ofYang et al the Wnt5a and Wnt5b glycoproteins are alsoresponsible for coordination of chondrocyte proliferation anddifferentiation [47] Wnt5a is believed to be an importantfactor taking part in the positive autocrinic regulation ofHSC repopulation ability [48] In their publication Prockopet al suggest that a high WNT5A expression in MSCs ischaracteristic for the end of the exponential and stationarygrowth phases in cell cultures in in vitro conditions [49]Slight changes in theWnt gene activity especially inWNT5Aactivity may result from the role played by MSCs in theirenvironmental niche Bone marrow MSCs seem to be moreinvolved in the renewal processes of the progenitor cells poolwhich reside in bone marrow The researches show that theexpression of Wnt ligands and Fz receptors in MSCs showssome correlation of WntFz pairs such as Wnt5aFz5 [50]The above discussed study results showed that there wasa coexpression of WNT5AFZD7 Their higher expressionwas characteristic for the bone marrow cells while thepreviously published data suggested differences in WntFzactivity depending on the origin-mouse versus humanMSCs

10 Stem Cells International

[51] Currently available data suggest differences which maybe characteristic not only for proliferation and differentiationprograms but also for the phenotype of MSCs from umbilicalcord blood and bone marrow which is in agreement withthe results published by Baksh et al [7] The remaining genestake part in theWnt signaling cascade of increased activity inCD34minus from umbilical cord blood such as MMP7 belongingto metalloproteinases which play a role in the extracellulararray remodeling and wound healing [5]

The Notch signaling pathway belongs to the processes ofintercellular communication which regulate gene expressionpatterns defining a dualistic character of stem cells reflectedby the asymmetric cell division [52] Additionally the Notchsignaling cascade controls various cells biological processesnot only proliferation and differentiation but also apoptosisand regeneration The signaling activity of Notch pathwaygenes determined in the study of 38 genes showed theirsimilar expression as defined by the KEGG classification(Table 4) A similar activity level was noted for all four Notch1ndash4 receptors as well as for JAG1 and JAG2 ligands The dif-ferences were related to two genes DTX4 and PSEN1 whichwere overactive in CD34minus cells from umbilical cord bloodThe function of the PSEN1 gene is mainly developmentalprocesses regarding cell differentiation during neurogenesisAnd the lack of this gene leads to a premature directeddifferentiation of neural progenitor cells [53] The activity ofthe Notch gene cascade plays an important role at all levelsof stem cell development starting from the embryonic stemcell through fetal cells to adult organism stem cells Notchis significantly involved in the asymmetric segregation offactors determining cell purpose during replication divisionsof the stem cell poolThe significance of receptors and ligandsis best described in the hematopoietic stem cells Expressionof the constitutively active Notch1 gene in hematopoieticstem cells induces the immortalized cell lineages which canreproduce the myeloid and lymphoid cell lineage in a long-term repopulation in animal studies [54 55] Apart from thatit delays differentiation of human hematopoietic progenitorstem cells in vitro [56] The studies suggest that Notch1 is animportant factor inHSC self-renewal and can also participatein the inhibition of stem cell differentiation The scientificfindings reveal that a suppression of the Notch signalingpathway via deletion of key genes in HSC did not cause cellsto lose their ability of long-term hematopoiesis renewal inanimal models [57] Therefore the role of ligands and Notchreceptors in HSC has not been precisely defined yet andcurrent publications do not provide precise data regardingthe role played by particular Notch signaling pathway genesin mesenchymal stem cells The only phenomenon whichappears to be superior and characteristic for all stem cellsincluding MSCs is the significance of the Notch cascadein asymmetrical cell divisions This is a significant com-ponent of stem cells population self-renewal as well asthe functionality of the cascade in the stem niche Thisphenomenon constitutes an integral part of the hypothesisabout stem cell population aging However further inves-tigation will be required to determine if the differentiallyexpressed genes described in the studies above could serve asstemness genes

Remaining signaling pathways which revealed increasedactivity in the studied CD34minus cells of umbilical cord bloodincludedMAPK (Mitogen-Activated Protein Kinase Figure 2)which regulates a wide range of cell biological activities suchas gene expression mitosis proliferation differentiationmigration and apoptosis processes [58]TheMAPKpathwaystake part in regulation of early embryonic development andin defining the role of embryonic stem cells from the earlydevelopmental phases until the moment of formation ofdifferentiated adult cells However this precise role played bythe MAPK pathways in various stem cell types has not yetbeen defined

Transmission via calcium ions was also singled out inthe group of the overactive signaling pathways of the studiedCD34minus stem cells of umbilical cord blood versus CD34minus stemcells of bone marrow (Figure 2) The ions are a vital andcommonly present mediator in the process of extracellularsignals transmission and conversion into a cellular responseVariations of calcium ions concentrations regulate geneexpression through an impact on the activity of numeroussignaling pathways Intracellular increase of calcium ionsconcentration also regulates cell activities including adhe-sion motility gene expression and proliferation [59] Thesignificance of transmission via calcium ions and the ways oftheir concentration changes in mesenchymal stem cells stillhave not been studied Kawano et al were the first to reportthat in the nondifferentiated MSCs there are spontaneouschanges of calcium ions concentrations despite the lack ofany stimulating factors [60] As reported the calcium iontransmission through the activation of different gene groupsmay direct the cells onto specific developmental pathways[61]

The integrin-mediated signaling pathway takes part inextracellular array remodeling which means that it con-trols cell behavior and tissue organization Its increasedactivity was observed in our study in CD34minus stem cells ofumbilical cord blood (Figure 2) In recent years the role ofintegrins and selectins has been stressed in the adhesion andtransendothelial migration which has a significant meaningin the processes of repopulation of tissues damaged bydiseases and their regeneration via the circulating MSCs Forinstance Ip et al showed that blocking of beta 1-integrinwhich constitutes a component of the adhesive moleculeVLA-4 in MSC weakened the ability of mesenchymal stemcells to engraft into the infarction-changedmyocardium [62]The above results may suggest a better migration abilityof CD34minus stem cells of umbilical cord blood into the areaof pathologic tissue MSCs own cytokinetic activity thathas similar significance in terms of tissue regenerationThe study of cytokinetic activity of CD34minus stem cells ofumbilical cord blood showed increased activity of signalingpathways activated via cytokines (Figure 2 Table 2) On theother hand the TLR signaling pathway (toll-like receptorsignaling) which in our own results revealed a significantincreased activity in CD34minus stem cells of umbilical cordblood (Figure 2) increases the immunosuppressive charac-teristics of MSCs Weakening of the immunological responsegenerated by the TLR pathway depends on the produc-tion of immunosuppressive kynurenines by the indoleamine

Stem Cells International 11

23-dioxygenase enzyme which degrades tryptophan Induc-tion of indoleamine 23-dioxygenase by TLR entails autocrinesignaling loop of interferon-beta which plays a role in MSCsimmunomodulative effects [63]

According to the GO and KEGG classifications carriedout for cluster 13 with the gene pool of a decreased expressiona statistically significant decrease of gene activity was shownfor the TGF-beta and BMP pathways (Figure 3 Table 3) Itis believed that the family of TGF-beta members whichalso includes BMP plays an important role in initiating theprocess of a directed MSCs differentiation TGF-beta sig-naling transmission runs through specific serine-threoninekinase receptors and their nuclear receptors called the Smadproteins TGF-beta signaling transmission promotes theprocess of early differentiation stages into the chondrogenicand osteogenic lineages [64 65] However cell differentiatinginto the adipogenic lineage results in inhibition of the TGF-beta signaling pathway This was confirmed in experimentalstudies developed by Ng et al where the inhibition of TGF-beta transmission resulted in an increase of adipogenesisand a decrease of differentiating ability towards chondrocytes[66] The BMP signaling pathway strongly promotes differ-entiation towards osteoblasts but it also leads to inhibitionof myogenesis and adipogenesis Such a gene expressionprofile may suggest that the MSCs biological characteristicsare strongly influenced by the significance of stem cell nicheTherefore the mesenchymal stem cells from bone marrowmay be more involved in the promotion of the phenomenawhich maintain the stability of bone marrow environment

5 Conclusions

Multipotentiality of the mesenchymal stem cells their highproliferation ability and not too-demanding culturing con-ditions reveal their potential for wide application in variousmedical areas These are the reasons for the common pursuitto understand their biology based onmore andmore sublimebiomolecular procedures Scientists have been searchingfor an unequivocal marker which would characterize themesenchymal stem cells and simultaneously would enable aseparation of a homogenous MSC population Understand-ing of molecular mechanisms controlling self-replication anddifferentiation is becoming a fundamental issue for clinicalapplication of MSC In this respect the activity of signalingpathways is important yet the interpretation of the pathwaysespecially their interdependence constitutes a great challengefor the scientific world Moreover the answer to the questionregarding MSC innidation and its survival ability in hosttissues after transplantation is yet to be found Explanationof the abovementioned issues will require a more globalunderstanding of biological activity of these cells especiallyon the basis of the significance of the mesenchymal nicheenvironment of stem cells and its impact on the gene andepigenetic activity It should be assumed that continuedprogress in the understanding of MSCs will finally lead totheir common therapeutic and preventive application andconsequently to the improvement of the quality and lengthof human life

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] R J Deans andA BMoseley ldquoMesenchymal stem cells biologyand potential clinical usesrdquo Experimental Hematology vol 28no 8 pp 875ndash884 2000

[2] M F Pittenger A M Mackay S C Beck et al ldquoMultilineagepotential of adult human mesenchymal stem cellsrdquo Science vol284 no 5411 pp 143ndash147 1999

[3] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006

[4] Y-J Chang D T-B Shih C-P Tseng T-B Hsieh D-C Leeand S-M Hwang ldquoDisparate mesenchyme-lineage tendenciesin mesenchymal stem cells from human bone marrow andumbilical cord bloodrdquo Stem Cells vol 24 no 3 pp 679ndash6852006

[5] R A Panepucci J L C Siufi W A Silva Jr et al ldquoComparisonof gene expression of umbilical cord vein and bone marrowndashderived mesenchymal stem cellsrdquo STEM CELLS vol 22 no 7pp 1263ndash1278 2004

[6] C K Rebelatto A M Aguiar M P Moretao et al ldquoDissimilardifferentiation of mesenchymal stem cells from bone marrowumbilical cord blood and adipose tissuerdquo Experimental Biologyand Medicine vol 233 no 7 pp 901ndash913 2008

[7] D Baksh R Yao and R S Tuan ldquoComparison of proliferativeand multilineage differentiation potential of human mesenchy-mal stem cells derived from umbilical cord and bone marrowrdquoStem Cells vol 25 no 6 pp 1384ndash1392 2007

[8] D L Clarke C B Johansson J Wilbertz et al ldquoGeneralizedpotential of adult neural stem cellsrdquo Science vol 288 no 5471pp 1660ndash1663 2000

[9] D J Anderson F H Gage and I L Weissman ldquoCan stem cellscross lineage boundariesrdquo Nature Medicine vol 7 no 4 pp393ndash395 2001

[10] D L Stemple and D J Anderson ldquoIsolation of a stem cell forneurons and glia from the mammalian neural crestrdquo Cell vol71 no 6 pp 973ndash985 1992

[11] R L Rietze H Valcanis G F Brooker T Thomas A K Vossand P F Bartlett ldquoPurification of a pluripotent neural stem cellfrom the adult mouse brainrdquoNature vol 412 no 6848 pp 736ndash739 2001

[12] R M Seaberg S R Smukler T J Kieffer et al ldquoClonalidentification of multipotent precursors from adult mousepancreas that generate neural and pancreatic lineagesrdquo NatureBiotechnology vol 22 no 9 pp 1115ndash1124 2004

[13] A P Beltrami L Barlucchi D Torella et al ldquoAdult cardiac stemcells are multipotent and support myocardial regenerationrdquoCell vol 114 no 6 pp 763ndash776 2003

[14] L Yang S Li H Hatch et al ldquoIn vitro trans-differentiation ofadult hepatic stem cells into pancreatic endocrine hormone-producing cellsrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 99 no 12 pp 8078ndash80832002

[15] I Chambers D Colby M Robertson et al ldquoFunctional expres-sion cloning of Nanog a pluripotency sustaining factor inembryonic stem cellsrdquo Cell vol 113 no 5 pp 643ndash655 2003

12 Stem Cells International

[16] M Dai P Wang A D Boyd et al ldquoEvolving genetranscriptdefinitions significantly alter the interpretation of GeneChipdatardquo Nucleic Acids Research vol 33 pp 175ndash179 2005

[17] RA Irizarry BHobbs F Collin et al ldquoExploration normaliza-tion and summaries of high density oligonucleotide array probelevel datardquo Biostatistics vol 4 no 2 pp 249ndash264 2003

[18] R B Dean and W J Dixon ldquoSimplified statistics for smallnumbers of observationsrdquo Analytical Chemistry vol 23 no 4pp 636ndash638 1951

[19] G J McLachlan and D Peel ldquoOn computational aspectsof clustering via mixtures of normal and t-componentsrdquo inProceedings of the American Statistical Association (BayesianStatistical Science Section) American Statistical AssociationIndianapolis Ind USA 2000

[20] J Polanska P Widlak J Rzeszowska-Wolny M Kimmel andA Polanski ldquoGaussian mixture decomposition of time-courseDNA microarray datardquo in Mathematical Modeling of BiologicalSystems Volume I pp 351ndash359 Birkhauser Boston Mass USA2007

[21] A J Friedenstein U F Gorskaja and N N Kulagina ldquoFibrob-last precursors in normal and irradiated mouse hematopoieticorgansrdquo Experimental Hematology vol 4 no 5 pp 267ndash2741976

[22] S Wakitani T Saito and A I Caplan ldquoMyogenic cells derivedfrom rat bone marrow mesenchymal stem cells exposed to 5-azacytidinerdquoMuscle ampNerve vol 18 no 12 pp 1417ndash1426 1995

[23] D Woodbury E J Schwarz D J Prockop and I B BlackldquoAdult rat and human bone marrow stromal cells differentiateinto neuronsrdquo Journal of Neuroscience Research vol 61 no 4pp 364ndash370 2000

[24] M Reyes T Lund T Lenvik D Aguiar L Koodie and CM Verfaillie ldquoPurification and ex vivo expansion of postnatalhumanmarrowmesodermal progenitor cellsrdquoBlood vol 98 no9 pp 2615ndash2625 2001

[25] M Ramalho-Santos S Yoon Y Matsuzaki R C Mulliganand D A Melton ldquolsquoStemnessrsquo transcriptional profiling ofembryonic and adult stem cellsrdquo Science vol 298 no 5593 pp597ndash600 2002

[26] KMitsui Y Tokuzawa H Itoh et al ldquoThe homeoprotein nanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003

[27] S Okumura-Nakanishi M Saito H Niwa and F IshikawaldquoOct-34 and Sox2 regulate Oct-34 gene in embryonic stemcellsrdquo The Journal of Biological Chemistry vol 280 no 7 pp5307ndash5317 2005

[28] F Ulloa-Montoya B L Kidder K A Pauwelyn et al ldquoCompar-ative transcriptome analysis of embryonic and adult stem cellswith extended and limited differentiation capacityrdquo GenomeBiology vol 8 no 8 article R163 2007

[29] L Song N EWebb Y Song and R S Tuan ldquoIdentification andfunctional analysis of candidate genes regulating mesenchymalstem cell self-renewal and multipotencyrdquo STEMCELLS vol 24no 7 pp 1707ndash1718 2006

[30] A H H Van Boxel-Dezaire M R S Rani and G R StarkldquoComplexmodulation of cell type-specific signaling in responseto type I interferonsrdquo Immunity vol 25 no 3 pp 361ndash372 2006

[31] C G Kim J J Lee D Y Jung et al ldquoProfiling of differentiallyexpressed genes in human stem cells by cDNA microarrayrdquoMolecules and Cells vol 21 no 3 pp 343ndash355 2006

[32] T Bowman R Garcia J Turkson and R Jove ldquoSTATs inoncogenesisrdquo Oncogene vol 19 no 21 pp 2474ndash2488 2000

[33] A Stephanou andD S Latchman ldquoOpposing actions of STAT-1and STAT-3rdquo Growth Factors vol 23 no 3 pp 177ndash182 2005

[34] A Martino J H Holmes J D Lord J J Moon and B HNelson ldquoStat5 and Sp1 regulate transcription of the cyclin D2gene in response to IL-2rdquo The Journal of Immunology vol 166no 3 pp 1723ndash1729 2001

[35] I Matsumura T Kitamura H Wakao et al ldquoTranscriptionalregulation of the cyclin D1 promoter by STAT5 its involvementin cytokine-dependent growth of hematopoietic cellsrdquo TheEMBO Journal vol 18 no 5 pp 1367ndash1377 1999

[36] V Calo M Migliavacca V Bazan et al ldquoSTAT proteins fromnormal control of cellular events to tumorigenesisrdquo Journal ofCellular Physiology vol 197 no 2 pp 157ndash168 2003

[37] M Socolovsky A E J Fallon S Wang C Brugnara and HF Lodish ldquoFetal anemia and apoptosis of red cell progenitorsin Stat5aminusminus5bminusminus mice a direct role for stat5 in Bcl-X(L)inductionrdquo Cell vol 98 no 2 pp 181ndash191 1999

[38] G Li Z Wang Y Zhang et al ldquoSTAT5 requires the N-domainto maintain hematopoietic stem cell repopulating function andappropriate lymphoid-myeloid lineage outputrdquo ExperimentalHematology vol 35 no 11 pp 1684ndash1694 2007

[39] S Negoro K Kunisada E Tone et al ldquoActivation of JAKSTATpathway transduces cytoprotective signal in rat acute myocar-dial infarctionrdquo Cardiovascular Research vol 47 no 4 pp 797ndash805 2000

[40] H C Blair L J Robinson and M Zaidi ldquoOsteoclast signallingpathwaysrdquo Biochemical and Biophysical Research Communica-tions vol 328 no 3 pp 728ndash738 2005

[41] C J Malemud ldquoProtein kinases in chondrocyte signaling andosteoarthritisrdquo Clinical Orthopaedics and Related Research no427 supplement pp S145ndashS151 2004

[42] E M Pinheiro and D J Montell ldquoRequirement for Par-6 andBazooka inDrosophila border cell migrationrdquoDevelopment vol131 no 21 pp 5243ndash5251 2004

[43] D Strutt ldquoFrizzled signalling and cell polarisation inDrosophilaand vertebratesrdquo Development vol 130 no 19 pp 4501ndash45132003

[44] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[45] J Guo J Jin and L F Cooper ldquoDissection of sets of genes thatcontrol the character of wnt5a-deficient mouse calvarial cellsrdquoBone vol 43 no 5 pp 961ndash971 2008

[46] M Nishizuka A Koyanagi S Osada and M Imagawa ldquoWnt4and Wnt5a promote adipocyte differentiationrdquo FEBS Lettersvol 582 no 21-22 pp 3201ndash3205 2008

[47] Y Yang L Topol H Lee and J Wu ldquoWnt5a andWnt5b exhibitdistinct activities in coordinating chondrocyte proliferation anddifferentiationrdquo Development vol 130 no 5 pp 1003ndash10152003

[48] D J Van Den Berg A K Sharma E Bruno and R Hoff-man ldquoRole of members of the Wnt gene family in humanhematopoiesisrdquo Blood vol 92 no 9 pp 3189ndash3202 1998

[49] D J Prockop C A Gregory and J L Spees ldquoOne strategy forcell and gene therapy harnessing the power of adult stemcells torepair tissuesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 1 pp 11917ndash119232003

[50] X He J-P Saint-Jeannet Y Wang J Nathans I Dawid and HVarmus ldquoA member of the Frizzled protein family mediatingaxis induction byWnt-5Ardquo Science vol 275 no 5306 pp 1652ndash1654 1997

Stem Cells International 13

[51] D G Phinney G Kopen R L Isaacson and D J ProckopldquoPlastic adherent stromal cells from the bone marrow ofcommonly used strains of inbred mice variations in yieldgrowth and differentiationrdquo Journal of Cellular Biochemistryvol 72 no 4 pp 570ndash585 1999

[52] N Sasaki T Sasamura H O Ishikawa et al ldquoPolarized exo-cytosis and transcytosis of Notch during its apical localizationin Drosophila epithelial cellsrdquo Genes to Cells vol 12 no 1 pp89ndash103 2007

[53] M Handler X Yang and J Shen ldquoPresenilin-1 regulatesneuronal differentiation during neurogenesisrdquo Developmentvol 127 no 12 pp 2593ndash2606 2000

[54] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006

[55] A W Duncan F M Rattis L N DiMascio et al ldquoIntegrationof Notch andWnt signaling in hematopoietic stem cell mainte-nancerdquo Nature Immunology vol 6 no 3 pp 314ndash322 2005

[56] N Carlesso J C Aster J Sklar and D T Scadden ldquoNotch1-induced delay of human hematopoietic progenitor cell differen-tiation is associated with altered cell cycle kineticsrdquo Blood vol93 no 3 pp 838ndash848 1999

[57] I Maillard U Koch A Dumortier et al ldquoCanonical notch sig-naling is dispensable for the maintenance of adult hematopoi-etic stem cellsrdquo Cell Stem Cell vol 2 no 4 pp 356ndash366 2008

[58] G Pearson F Robinson T B Gibson et al ldquoMitogen-activatedprotein (MAP) kinase pathways regulation and physiologicalfunctionsrdquo Endocrine Reviews vol 22 no 2 pp 153ndash183 2001

[59] R E Dolmetsch R S Lewis C C Goodnow and J I HealyldquoDifferential activation of transcription factors induced by Ca2+response amplitude and durationrdquoNature vol 386 no 6627 pp855ndash858 1997

[60] S Kawano S Shoji S Ichinose K Yamagata M Tagami andM Hiraoka ldquoCharacterization of Ca2+ signaling pathways inhuman mesenchymal stem cellsrdquo Cell Calcium vol 32 no 4pp 165ndash174 2002

[61] B Ye ldquoCa2+ oscillations and its transporters in mesenchymalstem cellsrdquo Physiological Research vol 59 no 3 pp 323ndash3292010

[62] J E Ip Y Wu J Huang L Zhang R E Pratt and V J DzauldquoMesenchymal stem cells use integrin 1205731 not CXC chemokinereceptor 4 for myocardial migration and engraftmentrdquoMolecu-lar Biology of the Cell vol 18 no 8 pp 2873ndash2882 2007

[63] C A Opitz U M Litzenburger C Lutz et al ldquoToll-like recep-tor engagement enhances the immunosuppressive propertiesof human bone marrow-derived mesenchymal stem cells byinducing indoleamine-23-dioxygenase-1 via Interferon-120573 andprotein kinase Rrdquo Stem Cells vol 27 no 4 pp 909ndash919 2009

[64] B Kulterer G Friedl A Jandrositz et al ldquoGene expressionprofiling of human mesenchymal stem cells derived from bonemarrow during expansion and osteoblast differentiationrdquo BMCGenomics vol 8 article 70 2007

[65] W G Li and X X Xu ldquoThe expression of N-cadherinfibronectin during chondrogenic differentiation of MSCinduced by TGF-1205731rdquo Chinese Journal of Traumatology vol 8no 6 pp 349ndash351 2005

[66] F Ng S Boucher S Koh et al ldquoPDGFTGF-120573 and FGF signal-ing is important for differentiation and growth of mesenchymalstem cells (mscs) transcriptional profiling can identify markersand signaling pathways important in differentiation of MSCsinto adipogenic chondrogenic and osteogenic lineagesrdquo Bloodvol 112 no 2 pp 295ndash307 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 Stem Cells International

210 Biotinylated cRNA Synthesis The template for thebiotinylated cRNA synthesis consisted of 10 120583L ds cDNAto which the following were added 12 120583L of RNase-freewater 4 120583L of 2x hybridization buffer 4 120583L of biotin-labeledribonucleotides 4 120583L of DTT 4 120583L RNase inhibitor and2 120583L of T7 polymerase (BioArray High Yield RNA TranscriptLabeling Kit Enzo) The whole was left at 37∘C for 5 h andmixed every 30 minutes

211 Biotinylated cRNA Purification After a 5-hour incuba-tion cRNA was cleaned up using 60 120583L of H

2

O 350 120583L ofRLT buffer and 250 120583L of 96 ethanol added to the reactionmixture The mixture was applied onto the RNAeasy columnTotal RNA Mini Kit (Qiagen) and was centrifuged twice for15 seconds at 10 000 rmin In the last stage 2x 20120583L of H

2

Owas added to the column membrane it was centrifuged at12 000 rmin and then the cRNA concentration was mea-sured

212 Biotinylated cRNA Fragmentation For fragmentationpurposes 16 120583g of cRNA and 8 120583L of fragmentation buffer(Affymetrix) were used and the mixture was supplementedwith up to 40 120583L of RNase-free water After completion of thisstage the mixture was centrifuged and left for 35min at 94∘Cand then placed on iceThe fragmentation result was checkedon 1 agarose gel

213 Assessment of cDNA Biotinylated cRNA and cRNASynthesis after Fragmentation via Electrophoresis in AgaroseGel Evidence of proper cDNA biotinylated cRNA andcRNA synthesis after fragmentation was assessed throughelectrophoresis in agarose gel with ethidium bromide Theresulting separation electrophoregram of the abovemen-tioned products verified that samples were acceptable to usein the following analysis stage

214 Hybridization of the Fragmented BiotinylatedcRNA with Microarray

2141 Preparation of Hybridization Cocktail In order toprepare the hybridization cocktail 375 120583L of fragmentedbiotinylated cRNA was used and the following were added5 120583L of control oligonucleotide B2 15 120583L of eukaryotichybridization control (Affymetrix) 3120583L of Herring Sperm(Invitrogen) 3 120583L of acetylated BSA (Invitrogen) 150 120583L of2x hybridization buffer and 865 120583L of RNase-free water Thecocktail was heated for 5min at 99∘C and for 5min at 45∘Cand centrifuged for 5min at 14 000 rmin

2142 Application of Cocktail on Microarray HG-U133Amicroarray (Affymetrix)was heated to room temperature andthen 100 120583L of 1x hybridization buffer was applied The chipwas placed in hybridization oven for 10min Next the bufferwas removed from the chip and 200120583L of hybridizationcocktail was applied The microarray was placed in thehybridization oven for 16 h at 45∘C at 55 rmin

2143 Washing and Scanning of Microarrays After a 16-hour hybridization the HG U133A microarray was washedwith buffer and stained with solutions (SAPE solutionstreptavidin antibody solution) and then scanned with theGeneArray Scanner G2500A (Affymetrix)

215 Statistical Analysis Data regarding expression levelsobtained directly from the HGU133A array were reannotatedwith CDF control files developed by Dai et al [16] Thenext step consisted of microarray normalization with theRMA algorithm [17] Three repetitions were replaced withan average value with a prior detection of outliers by meansof the Dean and Dixon method [18] Determination of mea-surement noise threshold was conducted through empiricaldecomposition of probability density function of expressionlevels into normal components [19] The component of thelowest mean value unchanged across all the microarrays wasassumed to be the noise model The threshold value of thefilter was constructed at level 28 (in the field of logarithmsto base 2) The mixture model of normal distributions con-structed for the average SRL (Signal Log Ratio) of CD34minus cellsfromumbilical cord blood versusCD34minus of bonemarrowwasused to define the affinity of certain genes to subgroups of anincreased decreased or unchanged value of expression levelMoreover thismixturemodel was used to determine the FDR(False Discovery Rate) value for each of them

In order to define qualitatively the processes in stemcells from umbilical cord blood in comparison with stemcells from bonemarrow an unsupervised gene clustering wasconducted Again the GaussianMixture Model for empiricaldecomposition of expression levels constituted the algorithmenabling such a division However in this analysis additionallimitations based on the gene composition of each compo-nent that were the same across the experimental conditionswere added to the algorithm [20] Clustersclasses obtainedin this manner were compared to cellular processes in whichthey are involved The categories of cellular processes weredefined by gene ontology terms that are commonly appliedfor this purpose A number of conditioning hypergeometrictests were conducted verifying the one-sided hypothesisabout overrepresentation For each ontological term thatwas found to be significantly overrepresented statisticallyin a given gene cluster the OR (Odds Ratio) value wasdetermined

Gene pathways whose components are genes belongingto specific clusters underwent an additional analysis whichwas independent of a direct description of cellular processesFor this purpose we used data from the KEGG databaseFor each pathway the hypothesis of overrepresentation of aspecified cluster of genes as compared with the whole scopeof the analyzed genes was verified Precise 119901 values weredetermined

Statistically significant values were those of FDR lt 010 or119901 lt 005

3 Results

The first stage of the study consisted of data reannotation forexpression levels through the CDF control files developed

Stem Cells International 5

by Dai et al [16] For many years the platform for geneexpression profile was the Affymetrix GeneChip databaseHowever their probe section was created on the basis ofprior gene and transcriptome annotations which significantlydiffer from the modern expertise A comparative analysisbetween many popular Gene Chips and redefined probesbased on updated gene data with consideration to geneticpolymorphism showed a 30ndash50 discrepancy [16] thereforeit was decided to reannotate data as the first step in ourstudyThe assessment of noise threshold was then completedcreating a mixture model of normal distributions for themean SLR of CD34minus cells from umbilical cord blood versusCD34minus from bone marrow

The next step comprised microarrays normalizationthrough the RMA algorithm [17] Three repetitions werereplaced with a mean value with a prior detection of outliersby means of the Dean and Dixon method [18] Determina-tion of the measurement noise threshold was conducted byempirical decomposition of probability density function ofexpression levels into normal components [19] The noisemodel was assumed to be the component of the lowest meanvalue which was unchanged across all the microarrays Thethreshold value of the filter was constructed at level 28 (inthe field of logarithms to base 2)

As a result of the abovementioned procedures a data setwas obtained which described 11 979 genes which underwentfurther analysis The first stage consisted of creating the mix-ture model of normal distributions constructed for a meanSLR (Signal Log Ratio) of CD34minus stem cells from umbilicalcord blood versus CD34minus stem cells from bone marrowwhich allowed defining the affinity criteria of specific genes tosubgroups of an increased decreased and unchanged valueof expression levels This also enabled us to determine a FDRvalue (False Discovery Rate) for each of them (Figure 1)

This method is based on decomposition that is thehistogram decomposition into (K)mdashGaussrsquos components aswell as modeling the probability density function distribu-tions (pdf) for given gene expression levels The method wasused for analysis of a histogram reflecting the frequency ofgenes of a specific expression level in the tested preparationThis initial analysis showed considerable similarity of geneexpression of CD34minus cells from umbilical cord blood ascompared with CD34minus cells from bone marrow

31 Unsupervised Gene Clustering During the next stage ofdata analysis a qualitative determination of the processes inCD34minus stem cells from umbilical cord blood in comparisonwith CD34minus stem cells from bone marrow was conductedusing unsupervised gene clustering Again the GaussianMixture Model for empirical decomposition of expressionlevels constituted the algorithm enabling such a divisionHowever in this analysis additional limitations based onthe gene composition of each component that were thesame across the experimental conditions were added to thealgorithm [20] As a result 15 gene clusters were obtainedtwo of which (numbers 9 and 13 in this study) containeddiscriminatory genes exhibiting a statistically significantincrease (cluster number 9) and decrease of gene expression(cluster number 13) (Table 1)

1600

1400

1200

1000

800

600

400

200

00 2 4 6 8

Num

ber o

f obs

erva

tions

Gene expressionminus10 minus8 minus6 minus4 minus2

SLR CD34H adnotacje Dai ver12

Figure 1Mixturemodel of normal distributions (GaussianMixtureModel) for a mean SLR (Signal Log Ratio) of CD34minus cells fromumbilical cord blood versus CD34minus cells from bone marrow Log(CD34-P)( CD34-Sz)

The remaining clusters differing in terms of the numberof genes within the cluster aggregated the genes of similarexpression values Differences betweenmean gene expressionlevels of CD34minus stem cells from bone marrow and CD34minusstem cells from umbilical cord blood in clusters numbered1ndash8 10ndash12 14 and 15 are small or nonexistent Thus all theseclusters were classified as the groups of genes exhibiting thesame expression levels

Cluster number 9 included 493 genes which show anincreased activity in CD34minus stem cells from umbilical cordblood of the mean SRL (Signal Log Ratio) equaling 306 Incontrast cluster number 13 included a lower number of genes(119899 = 387) of decreased expression in CD34minus stem cells fromumbilical cord blood for which SRL equals minus343

At the next stage gene clustersgroups obtained in thismanner were compared to biological processes in whichthey are involved as defined by gene ontology (GO) termsthat are commonly applied for this purpose A number ofconditioning hypergeometric tests were conducted verifyingthe one-sided hypothesis about overrepresentation For eachontological term that was found to be significantly overrep-resented statistically in a given gene cluster the OR (OddsRatio) value was determined All the clusters were classifiedthrough the GO classification system that is the clusterswith genes discriminating both cell populations as well as theclusters grouping genes of a similar expression

Four hundred ninety-three genes of cluster 9 classifiedvia the GO system determined the terms of biological pro-cesses (119901 lt 005) overrepresented in CD34minus stem cells fromumbilical cord blood The classification system determined256 GO terms of biological processes out of which GO termsof 119901 value below 004 underwent further analysis As a resultof this analysis the majority of GO terms represented only byone or two genes were rejected The GO terms for biologicalprocesses in cluster 9 were assigned to one of six func-tional groups including metabolism biological processes

6 Stem Cells International

Table 1 15 clusters obtained as a result of the unsupervised gene clusteringMean CD34minus BM (mean of CD34minus stem cells from bonemarrow)Mean CD34minus UCB (mean of CD34minus stem cells from umbilical cord blood)119873 (a number of genes in a cluster) SRL (Signal Log Ratio)

Cluster MeanCD34minus BM

MeanCD34minusUCB

SDCD34minusBM

SDCD34minusUCB

119873

119901 value MeanSLR

minus95CISLR

+95CISLR

Conclusion

1497 491 008 009

1102

000000023 minus006 minus008 minus004

No change2

1157 1149 035 047

54

026488907 minus008 minus022 006

No change3

802 805 030 032

592

063190010 001 minus005 007

No change4

445 440 006 005

999

000000001 minus006 minus008 minus004

No change5

702 714 024 021

898

000000009 011 007 016

No change6

554 550 012 013

1133

000000889 minus006 minus009 minus003

No change7

353 346 004 005

960

451861119864 minus 13 minus007 minus009 minus005

No change8

476 483 071 088

961

003102164 011 001 020

No change9 564 715 158 270 493 p lt 00000001 306 295 316 Up10

311 309 004 004

872

000325256 minus003 minus005 minus001

No change11

276 278 002 002

647

000002947 003 001 004

No change12

398 390 006 007

1133

544009119864 minus 15 minus008 minus010 minus006

No change13 739 486 285 101 387 p lt 00000001 minus343 minus357 minus329 Down14

621 621 026 021

1353

044902052 minus001 minus005 002

No change15

930 936 064 084

395

015369595 008 minus003 020

No change

0 001 002 003 004

Signal transduction (GO0007165)

Activation of MAPK activity (GO0000187)

Protein kinase cascade (GO0007243)

Regulation of MAP kinase activity (GO0043405)

Integrin-mediated signaling pathway (GO0007229)

Positive regulation of tyrosine phosphorylation ofSTAT protein (GO0042531)

Positive regulation of calcium-mediated signaling(GO0050850)

JAK-STAT cascade (GO0007259)

Cytokine-mediated signaling pathway (GO0019221)

Positive regulation of cell proliferation (GO0008284)

Calcium-mediated signaling (GO0019722)

Regulation of JAK-STAT cascade (GO0046425)

p value

Signaling pathway

Figure 2 Functional annotations 12 GO classification terms for biological processes in the group signaling pathways in cluster 9 dependingon the 119901 value (119901 lt 004)

regulation cellular homeostasis cellular defensive responseand response to stimuli cytokine production and signalingpathways The GO terms regarding signaling pathways arepresented in Figure 2

Thenext stage consisted of a similar analysis of genes fromcluster 13 GO system classification for 387 genes of cluster 13determined 119 terms of biological processes (119901 lt 005) of

a decreased activity in CD34minus cells fromumbilical cord bloodas compared with CD34minus cells from bone marrow The GOterms of 119901 value below 004 underwent further analysis As aresult of this analysis the majority of GO terms representedonly by one or two genes were rejected The GO terms forbiological processes in cluster 13 were assigned to one ofthree functional groups including metabolism biological

Stem Cells International 7

0000 0010 0020 0030

Negative regulation of signal transduction (GO0009968)

Vascular endothelial growth factor receptor signalingpathway (GO0048010)

Transmembrane receptor protein tyrosine kinase signalingpathway (GO0007169)

Transmembrane receptor protein serinethreonine kinasesignaling pathway (GO0007178)

Regulation of transforming growth factor beta receptorsignaling pathway (GO0017015)

Cell surface receptor linked signal transduction(GO0007166)

BMP signaling pathway (GO0030509)

p value

Signaling pathway

Figure 3 Functional annotations 7 GO classification terms for biological processes in the group signaling pathways in cluster 13 dependingon the 119901 value (119901 lt 004)

Table 2 Gene pathways based on the KEGG classification of an increased expression in CD34minus stem cells from umbilical cord blood

KEGGID 119901 value Odds ratio Exp count Count Size Term4060 0001 2947 14 33 223 Cytokine-cytokine receptor interaction4620 0001 3672 6 17 92 Toll-like receptor signaling pathway5120 0001 3748 4 12 63 Epithelial cell signaling inHelicobacter pylori infection4630 0012 2067 8 15 130 JAK-STAT signaling pathway

processes regulation and signaling pathways In CD34minus cellsfrom umbilical cord blood as compared with CD34minus cellsfrom bone marrow a decreased gene activity was observedof the BMP signaling pathway TGF beta transmembranereceptor of protein serinethreonine kinase transmembranereceptor of tyrosine kinase and VEGF (Figure 3)

Gene pathways whose components are genes belongingto specific clusters underwent an additional analysis whichwas independent of a direct description of cellular processesFor this purpose data from the KEGG database (KyotoEncyclopedia of Genes and Genomes) were used For eachpathway the hypothesis of overrepresentation of specificclusters of genes as compared with the whole scope ofanalyzed genes was verified and precise 119901 values weredetermined For the purpose of this study gene pathwaysfor clusters 9 and 13 were analyzed because they containedgenes discriminating both gene populations On the basisof KEGG database cytokine-cytokine receptor interactionpathway toll-like receptor signaling pathway epithelial cellsignaling in Helicobacter pylori infection and JAK-STATsignaling pathway are overrepresented in CD34minus cells fromumbilical cord blood (Table 2)

In cluster 13 on the basis of KEGG classification theCD34minus cells from umbilical cord blood show a decreasedactivity of focal adhesion signaling pathway cell junctionsand TGF-beta signaling pathway (Table 3)

At the next stage the genes of the remaining clustersnumbered 1ndash8 10ndash12 and 14 of a similar gene expression were

analyzed The analysis was based on the GO classification foreach of the abovementioned clusters The criteria used forchoosing the clusters included the biological processes con-nected with cellular cycle as well as the processes connectedwith signaling pathways activity A similar gene expression inCD34minus cells from umbilical cord blood and CD34minus cells frombone marrow was characteristic for such biological processesas cell division mitosis maintaining telomere endings RNAandDNA processing and a similar activity of Notch andWntsignaling pathways genes

An independent analysis was also carried out for genes ofa similar activity in clusters 1ndash8 10ndash12 and 14 on the basis ofthe KEGG database CD34minus cells from umbilical cord bloodand CD34minus cells from bone marrow showed similar activityof Wnt and Notch signaling pathways genes p53 signalingpathway genes cell cycle PPAR and mTOR (Table 4)

CD34minus cells from umbilical cord blood and CD34minus cellsfrom bone marrow showed similar gene activity of Wntand Notch signaling pathways both on the basis of the GOclassification system and the KEGG database Therefore atthe last stage of the study the activity of single genes of Wntand Notch as well as JAK-STAT signaling pathways chosenfrom 11976 sampled genes was analyzed

In accordance with the KEGG database the JAK-STATsignaling pathway is defined by 155 genes out of whichthere were 128 active genes in the studied material Genesexhibiting increased activity in CD34minus cells from umbilicalcord blood included SPRY2 CISH CSF2 GRB2 IFNGR1

8 Stem Cells International

Table 3 Gene pathways based on the KEGG classification of a decreased expression in CD34minus stem cells from umbilical cord blood

KEGGID 119901 value Odds ratio Exp count Count Size Term4510 0001 7517 7 33 188 Focal adhesion1430 0001 8157 4 22 109 Cell junctions4350 0001 4038 3 10 82 TGF-beta signaling pathway

Table 4 Gene pathways based on the KEGG classification of a similar expression in CD34minus stem cells from umbilical cord blood and CD34minuscells from bone marrow for clusters 1ndash8 10ndash12 and 14-15

KEGGID 119901 value Odds ratio Exp count Count Size Term04310 0018 1880 10 17 131 Wnt signaling pathway04330 0025 2722 3 7 39 Notch signaling pathway04115 0038 2157 5 9 61 p53 signaling pathway04110 0009 2028 10 18 100 Cell cycle03320 0020 2791 3 7 60 PPAR signaling pathway04150 0035 2220 5 9 46 mTOR signaling pathway

IL2RB IL2RG IL7R IL10RA IL15RA PIM1 PTPN6 STAT1STAT5A and CCND3 Genes exhibiting decreased activityincluded PIAS3 IL11RA and CBLB

On the basis of the KEGG database the Wnt signalingpathway is defined by 151 genes out of which there were132 active genes in the studied material Genes exhibit-ing increased activity in CD34minus cells from umbilical cordblood include MMP7 PRKCB PSEN1 RAC2 and CCND3Genes exhibiting decreased activity include DKK1 WNT5AWNT5B FZD7 and TCF7L1

On the basis of the KEGG database the Notch signalingpathway is defined by 47 genes out of which there were38 active genes in the studied material Genes exhibitingincreased activity in CD34minus cells from umbilical cord bloodincluded DTX4 and PSEN1

4 Discussion

Nonhematopoietic stem cells were first identified by Frieden-stein et al who described the progenitor cells of rat bonemarrow [21] Currently the nonhematopoietic stem cellswhich are frequently referred to although not quite correctlyas the mesenchymal stem cells are isolated from varioustissues including the umbilical cord blood placenta andumbilical cord A constantly increasing number of studieson MSCs have proved that in appropriate environmentalconditions they are capable of differentiating into special-ized mesodermal endodermal and even ectodermal celllineages [2 22ndash24] In natural conditions they play an impor-tant role in cell regeneration processes and in repairingthe tissues damaged by degenerating processes Anotherintriguing characteristic of MSCs is their ability to avoidimmunological identification and response therefore theyare referred to as the hypoimmunogenic cells Despite greatattention paid to mesenchymal stem cells their biology isnot yet fully understood Introducing multipotential stemcells into clinical application requires a full understandingof the mechanisms which control key properties of thecell such as cell self-replication versus differentiation and

mobilization versus tissue homing The understanding ofthese mechanisms must be based on strict definitions ofhuman multipotential stem cells at the molecular level Inaddition understanding of molecular mechanisms of self-replicating pluripotentiality plasticity and differentiation ofstem cells not only is a necessary condition to overcomelimitations of stem cells application as a therapeutic factorbut also is key to understand a fundamental mystery of thedevelopment of human organism

This paper presents a comparison of gene activity of thenonhematopoietic cells of umbilical cord blood and bonemarrow using oligonucleotidemicroarrays Gene activity wastested in stem cells CD34 and included 11 979 genes obtainedafter completing procedures to increase the purity of cellsamples Cell populations included in the study were alsolimited based on the noise threshold revealed in the finalcomparative analysis The gene expression profiles of thecompared populations of CD34minus stem cells from umbilicalcord blood and CD34minus stem cells from bone marrow werevery similar (Figure 1) For a better illustration of the biolog-ical molecular activity and the activity of signaling pathwaysin the studied cell populations a transcription profile analysiswith the GO and KEGG databases was completed In termsof fundamental signaling pathways for biological activityof stem cells like JAK-STAT Wnt and Notch we recordeddiscrete differences among expressed genes from umbilicalcord blood cells as compared to bone marrow cells

The analysis regarding signaling pathways showedincreased activity of the JAK-STAT pathway in CD34minusstem cells from umbilical cord blood both on the basis ofthe gene ontology classification and the KEGG database(Figure 2 and Table 2) The increased activity regarded 15genes which take part in the JAK-STAT cascade that isSPRY2 CISH CSF2 GRB2 IFNGR1 IL2RB IL2RG IL7RIL10RA IL15RA PIM1 PTPN6 and CCND3 including thegenes which code transcription factors STAT1 and STAT5AIn previous publications the significance of the JAK-STATsignaling pathway is best described in the embryonic andhematopoietic stem cells Ramalho-Santos et al searchedfor a transcription profile describing both the embryonic

Stem Cells International 9

stem cells and the stem cells of an adult organism on thebasis of the study of embryonic cells neural stem cellsand hematopoietic stem cells activity [25] These studiesattempted to create a molecular signature of the stem cells inrelation to the self-regeneration and differentiation abilities ofthe cells The active JAK-STAT pathway belongs to the basicattributes defining the so-called ldquostemnessrdquo phenomenon Inthe embryonic stem cells the JAK-STAT signaling pathwaypromotes proliferation in the form of self-replicationThe activating factor LIF (Leukemia Inhibitory Factor)provokes phosphorylation and dimerisation of STAT3which after translocation to the cell nucleus stimulates theexpression of gene group responsible for self-replication ofthe embryonic stem cell However the JAK-STAT signalingpathway is not the only factor responsible for proliferationregulation of the embryonic stem cells Internal processesregulating the multiplication processes independent of theSTAT3 activity such as the Nanog and Oct-4 activity arealso important [15 17 26 27] In our study material theNanog and Oct-4 genes were not active in both types ofnonhematopoietic stem cells which is in agreement withother publications devoted to this subject [28 29] TheSTAT1 transcription factor overactive in our study in CD34minuscells of umbilical cord blood is responsible among othersfor the expression of interferon-activated genes involved incell defense against pathogenic factors [30] Moreover itis believed that STAT1 is a medium in expression of genegroup which is important for cell viability in response tostimulating factors In 2006 Kimrsquos team described STAT1as a highly overactive gene in human mesenchymal stemcells [31] In comparison the transcription factor STAT5which functions in the form of two isoforms STAT5a andSTAT5b is involved in the cellular proproliferation responsesand antiapoptosis processes [32 33] STAT5 is activatedby various cytokine receptors In our study the isoformSTAT5A showed increased activity It is believed that theSTAT5 factor activates specifically the cellular cycle genesthat is cyclins [34 35] In human stem cells the STAT factorsactivate the expression of cyclin Dwhich codes the regulatingsubunit of the CYCDCDK4 complex driving the G1S phasetransition which in turn activates the cell cycle progression[32 36] STAT5a and STAT5b also play an important rolein expression of genes promoting hematopoietic stem cellssurvival [37] Gene expression profiling over a range ofSTAT5 activities in human CB cells revealed subsets of genesthat are associated with the self-renewal and long-termexpansion phenotype Taking into account the fact thatin STAT5minusminus mice myelopoiesis appears to be relativelyunaffected it can be assumed that STAT5 can play a keyrole in assessing the quality of cells [38] Additionally notonly is the JAK-STAT signaling pathway a significant signaltransduction component which controls cell proliferationand purpose but it also regulates cell migration which isimportant both in the primary processes of gastrulationand in the tissue regeneration For instance the JAK-STATsignaling pathway is connected with cardioprotection [39]as well as with processes of regeneration and remodeling ofskeleton [40 41] The active JAK-STAT signaling pathwayhelps to regulate cell organization within cell grouping

[42] The above results may suggest a greater self-renewalpotential of CD34minus stem cells of umbilical cord bloodlong-term expansion phenotype and their higher ability interms of tissue regeneration

The Wnt signaling pathway is perceived as a signalingcascade involved in the control of fundamental processesof nondifferentiated mesenchymal stem cells such as cellreproduction and directed differentiation Both methods ofgene activity classification that is GO and KEGG showeda similar activity of the majority of Wnt signaling pathwaygenes (Table 4) In addition the analysis of separate genestaking part in the Wnt signaling pathway and coding 19glycosylated molecules of extracellular signaling of the Wntfamily cellular surface receptors including Frizzled receptorsintracellular signaling molecules and genes involved in cellcycle and taking part in regulation of proliferation growthand modification of proteins showed that the expressionprofilewas very similar except for single genes of an increasedand decreased expression in CD34minus stem cells of umbilicalcord blood in our study that is MMP7 PRKCB PSEN1RAC2 and CCND3 respectively as well as DKK1 WNT5AWNT5B FZD7 and TCF7L1 The Wnt5a ligand is involvedin the noncanonical Wnt signaling pathway transmissionand it is assumed that it may inhibit the canonical Wntpathway in mouse embryonic cells similarly to Dkk1 Therole of the noncanonical Wnt signaling pathway which inour results seemed to prevail in CD34minus stem cells of bonemarrow is not clear It is believed that the noncanonical Wntsignaling pathway controls the reorganization of cytoskeletonmuscle proteins tissue polarity and cell movement [43] Incontrast the canonical signaling pathway which activatestarget gene transcription through the increase of beta-cateninconcentration seems to be more active in CD34minus stemcells from umbilical cord cells Its role is to control thetissue-specific cell purpose during embryogenesis as well asregulation of proliferation in adult tissues [44] Additionallyhigh activity of WNT5A gene is noticeable in the humanMSCs differentiation into osteoblasts as well as in early stagesof adipogenesis [45 46] According to the publication ofYang et al the Wnt5a and Wnt5b glycoproteins are alsoresponsible for coordination of chondrocyte proliferation anddifferentiation [47] Wnt5a is believed to be an importantfactor taking part in the positive autocrinic regulation ofHSC repopulation ability [48] In their publication Prockopet al suggest that a high WNT5A expression in MSCs ischaracteristic for the end of the exponential and stationarygrowth phases in cell cultures in in vitro conditions [49]Slight changes in theWnt gene activity especially inWNT5Aactivity may result from the role played by MSCs in theirenvironmental niche Bone marrow MSCs seem to be moreinvolved in the renewal processes of the progenitor cells poolwhich reside in bone marrow The researches show that theexpression of Wnt ligands and Fz receptors in MSCs showssome correlation of WntFz pairs such as Wnt5aFz5 [50]The above discussed study results showed that there wasa coexpression of WNT5AFZD7 Their higher expressionwas characteristic for the bone marrow cells while thepreviously published data suggested differences in WntFzactivity depending on the origin-mouse versus humanMSCs

10 Stem Cells International

[51] Currently available data suggest differences which maybe characteristic not only for proliferation and differentiationprograms but also for the phenotype of MSCs from umbilicalcord blood and bone marrow which is in agreement withthe results published by Baksh et al [7] The remaining genestake part in theWnt signaling cascade of increased activity inCD34minus from umbilical cord blood such as MMP7 belongingto metalloproteinases which play a role in the extracellulararray remodeling and wound healing [5]

The Notch signaling pathway belongs to the processes ofintercellular communication which regulate gene expressionpatterns defining a dualistic character of stem cells reflectedby the asymmetric cell division [52] Additionally the Notchsignaling cascade controls various cells biological processesnot only proliferation and differentiation but also apoptosisand regeneration The signaling activity of Notch pathwaygenes determined in the study of 38 genes showed theirsimilar expression as defined by the KEGG classification(Table 4) A similar activity level was noted for all four Notch1ndash4 receptors as well as for JAG1 and JAG2 ligands The dif-ferences were related to two genes DTX4 and PSEN1 whichwere overactive in CD34minus cells from umbilical cord bloodThe function of the PSEN1 gene is mainly developmentalprocesses regarding cell differentiation during neurogenesisAnd the lack of this gene leads to a premature directeddifferentiation of neural progenitor cells [53] The activity ofthe Notch gene cascade plays an important role at all levelsof stem cell development starting from the embryonic stemcell through fetal cells to adult organism stem cells Notchis significantly involved in the asymmetric segregation offactors determining cell purpose during replication divisionsof the stem cell poolThe significance of receptors and ligandsis best described in the hematopoietic stem cells Expressionof the constitutively active Notch1 gene in hematopoieticstem cells induces the immortalized cell lineages which canreproduce the myeloid and lymphoid cell lineage in a long-term repopulation in animal studies [54 55] Apart from thatit delays differentiation of human hematopoietic progenitorstem cells in vitro [56] The studies suggest that Notch1 is animportant factor inHSC self-renewal and can also participatein the inhibition of stem cell differentiation The scientificfindings reveal that a suppression of the Notch signalingpathway via deletion of key genes in HSC did not cause cellsto lose their ability of long-term hematopoiesis renewal inanimal models [57] Therefore the role of ligands and Notchreceptors in HSC has not been precisely defined yet andcurrent publications do not provide precise data regardingthe role played by particular Notch signaling pathway genesin mesenchymal stem cells The only phenomenon whichappears to be superior and characteristic for all stem cellsincluding MSCs is the significance of the Notch cascadein asymmetrical cell divisions This is a significant com-ponent of stem cells population self-renewal as well asthe functionality of the cascade in the stem niche Thisphenomenon constitutes an integral part of the hypothesisabout stem cell population aging However further inves-tigation will be required to determine if the differentiallyexpressed genes described in the studies above could serve asstemness genes

Remaining signaling pathways which revealed increasedactivity in the studied CD34minus cells of umbilical cord bloodincludedMAPK (Mitogen-Activated Protein Kinase Figure 2)which regulates a wide range of cell biological activities suchas gene expression mitosis proliferation differentiationmigration and apoptosis processes [58]TheMAPKpathwaystake part in regulation of early embryonic development andin defining the role of embryonic stem cells from the earlydevelopmental phases until the moment of formation ofdifferentiated adult cells However this precise role played bythe MAPK pathways in various stem cell types has not yetbeen defined

Transmission via calcium ions was also singled out inthe group of the overactive signaling pathways of the studiedCD34minus stem cells of umbilical cord blood versus CD34minus stemcells of bone marrow (Figure 2) The ions are a vital andcommonly present mediator in the process of extracellularsignals transmission and conversion into a cellular responseVariations of calcium ions concentrations regulate geneexpression through an impact on the activity of numeroussignaling pathways Intracellular increase of calcium ionsconcentration also regulates cell activities including adhe-sion motility gene expression and proliferation [59] Thesignificance of transmission via calcium ions and the ways oftheir concentration changes in mesenchymal stem cells stillhave not been studied Kawano et al were the first to reportthat in the nondifferentiated MSCs there are spontaneouschanges of calcium ions concentrations despite the lack ofany stimulating factors [60] As reported the calcium iontransmission through the activation of different gene groupsmay direct the cells onto specific developmental pathways[61]

The integrin-mediated signaling pathway takes part inextracellular array remodeling which means that it con-trols cell behavior and tissue organization Its increasedactivity was observed in our study in CD34minus stem cells ofumbilical cord blood (Figure 2) In recent years the role ofintegrins and selectins has been stressed in the adhesion andtransendothelial migration which has a significant meaningin the processes of repopulation of tissues damaged bydiseases and their regeneration via the circulating MSCs Forinstance Ip et al showed that blocking of beta 1-integrinwhich constitutes a component of the adhesive moleculeVLA-4 in MSC weakened the ability of mesenchymal stemcells to engraft into the infarction-changedmyocardium [62]The above results may suggest a better migration abilityof CD34minus stem cells of umbilical cord blood into the areaof pathologic tissue MSCs own cytokinetic activity thathas similar significance in terms of tissue regenerationThe study of cytokinetic activity of CD34minus stem cells ofumbilical cord blood showed increased activity of signalingpathways activated via cytokines (Figure 2 Table 2) On theother hand the TLR signaling pathway (toll-like receptorsignaling) which in our own results revealed a significantincreased activity in CD34minus stem cells of umbilical cordblood (Figure 2) increases the immunosuppressive charac-teristics of MSCs Weakening of the immunological responsegenerated by the TLR pathway depends on the produc-tion of immunosuppressive kynurenines by the indoleamine

Stem Cells International 11

23-dioxygenase enzyme which degrades tryptophan Induc-tion of indoleamine 23-dioxygenase by TLR entails autocrinesignaling loop of interferon-beta which plays a role in MSCsimmunomodulative effects [63]

According to the GO and KEGG classifications carriedout for cluster 13 with the gene pool of a decreased expressiona statistically significant decrease of gene activity was shownfor the TGF-beta and BMP pathways (Figure 3 Table 3) Itis believed that the family of TGF-beta members whichalso includes BMP plays an important role in initiating theprocess of a directed MSCs differentiation TGF-beta sig-naling transmission runs through specific serine-threoninekinase receptors and their nuclear receptors called the Smadproteins TGF-beta signaling transmission promotes theprocess of early differentiation stages into the chondrogenicand osteogenic lineages [64 65] However cell differentiatinginto the adipogenic lineage results in inhibition of the TGF-beta signaling pathway This was confirmed in experimentalstudies developed by Ng et al where the inhibition of TGF-beta transmission resulted in an increase of adipogenesisand a decrease of differentiating ability towards chondrocytes[66] The BMP signaling pathway strongly promotes differ-entiation towards osteoblasts but it also leads to inhibitionof myogenesis and adipogenesis Such a gene expressionprofile may suggest that the MSCs biological characteristicsare strongly influenced by the significance of stem cell nicheTherefore the mesenchymal stem cells from bone marrowmay be more involved in the promotion of the phenomenawhich maintain the stability of bone marrow environment

5 Conclusions

Multipotentiality of the mesenchymal stem cells their highproliferation ability and not too-demanding culturing con-ditions reveal their potential for wide application in variousmedical areas These are the reasons for the common pursuitto understand their biology based onmore andmore sublimebiomolecular procedures Scientists have been searchingfor an unequivocal marker which would characterize themesenchymal stem cells and simultaneously would enable aseparation of a homogenous MSC population Understand-ing of molecular mechanisms controlling self-replication anddifferentiation is becoming a fundamental issue for clinicalapplication of MSC In this respect the activity of signalingpathways is important yet the interpretation of the pathwaysespecially their interdependence constitutes a great challengefor the scientific world Moreover the answer to the questionregarding MSC innidation and its survival ability in hosttissues after transplantation is yet to be found Explanationof the abovementioned issues will require a more globalunderstanding of biological activity of these cells especiallyon the basis of the significance of the mesenchymal nicheenvironment of stem cells and its impact on the gene andepigenetic activity It should be assumed that continuedprogress in the understanding of MSCs will finally lead totheir common therapeutic and preventive application andconsequently to the improvement of the quality and lengthof human life

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] R J Deans andA BMoseley ldquoMesenchymal stem cells biologyand potential clinical usesrdquo Experimental Hematology vol 28no 8 pp 875ndash884 2000

[2] M F Pittenger A M Mackay S C Beck et al ldquoMultilineagepotential of adult human mesenchymal stem cellsrdquo Science vol284 no 5411 pp 143ndash147 1999

[3] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006

[4] Y-J Chang D T-B Shih C-P Tseng T-B Hsieh D-C Leeand S-M Hwang ldquoDisparate mesenchyme-lineage tendenciesin mesenchymal stem cells from human bone marrow andumbilical cord bloodrdquo Stem Cells vol 24 no 3 pp 679ndash6852006

[5] R A Panepucci J L C Siufi W A Silva Jr et al ldquoComparisonof gene expression of umbilical cord vein and bone marrowndashderived mesenchymal stem cellsrdquo STEM CELLS vol 22 no 7pp 1263ndash1278 2004

[6] C K Rebelatto A M Aguiar M P Moretao et al ldquoDissimilardifferentiation of mesenchymal stem cells from bone marrowumbilical cord blood and adipose tissuerdquo Experimental Biologyand Medicine vol 233 no 7 pp 901ndash913 2008

[7] D Baksh R Yao and R S Tuan ldquoComparison of proliferativeand multilineage differentiation potential of human mesenchy-mal stem cells derived from umbilical cord and bone marrowrdquoStem Cells vol 25 no 6 pp 1384ndash1392 2007

[8] D L Clarke C B Johansson J Wilbertz et al ldquoGeneralizedpotential of adult neural stem cellsrdquo Science vol 288 no 5471pp 1660ndash1663 2000

[9] D J Anderson F H Gage and I L Weissman ldquoCan stem cellscross lineage boundariesrdquo Nature Medicine vol 7 no 4 pp393ndash395 2001

[10] D L Stemple and D J Anderson ldquoIsolation of a stem cell forneurons and glia from the mammalian neural crestrdquo Cell vol71 no 6 pp 973ndash985 1992

[11] R L Rietze H Valcanis G F Brooker T Thomas A K Vossand P F Bartlett ldquoPurification of a pluripotent neural stem cellfrom the adult mouse brainrdquoNature vol 412 no 6848 pp 736ndash739 2001

[12] R M Seaberg S R Smukler T J Kieffer et al ldquoClonalidentification of multipotent precursors from adult mousepancreas that generate neural and pancreatic lineagesrdquo NatureBiotechnology vol 22 no 9 pp 1115ndash1124 2004

[13] A P Beltrami L Barlucchi D Torella et al ldquoAdult cardiac stemcells are multipotent and support myocardial regenerationrdquoCell vol 114 no 6 pp 763ndash776 2003

[14] L Yang S Li H Hatch et al ldquoIn vitro trans-differentiation ofadult hepatic stem cells into pancreatic endocrine hormone-producing cellsrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 99 no 12 pp 8078ndash80832002

[15] I Chambers D Colby M Robertson et al ldquoFunctional expres-sion cloning of Nanog a pluripotency sustaining factor inembryonic stem cellsrdquo Cell vol 113 no 5 pp 643ndash655 2003

12 Stem Cells International

[16] M Dai P Wang A D Boyd et al ldquoEvolving genetranscriptdefinitions significantly alter the interpretation of GeneChipdatardquo Nucleic Acids Research vol 33 pp 175ndash179 2005

[17] RA Irizarry BHobbs F Collin et al ldquoExploration normaliza-tion and summaries of high density oligonucleotide array probelevel datardquo Biostatistics vol 4 no 2 pp 249ndash264 2003

[18] R B Dean and W J Dixon ldquoSimplified statistics for smallnumbers of observationsrdquo Analytical Chemistry vol 23 no 4pp 636ndash638 1951

[19] G J McLachlan and D Peel ldquoOn computational aspectsof clustering via mixtures of normal and t-componentsrdquo inProceedings of the American Statistical Association (BayesianStatistical Science Section) American Statistical AssociationIndianapolis Ind USA 2000

[20] J Polanska P Widlak J Rzeszowska-Wolny M Kimmel andA Polanski ldquoGaussian mixture decomposition of time-courseDNA microarray datardquo in Mathematical Modeling of BiologicalSystems Volume I pp 351ndash359 Birkhauser Boston Mass USA2007

[21] A J Friedenstein U F Gorskaja and N N Kulagina ldquoFibrob-last precursors in normal and irradiated mouse hematopoieticorgansrdquo Experimental Hematology vol 4 no 5 pp 267ndash2741976

[22] S Wakitani T Saito and A I Caplan ldquoMyogenic cells derivedfrom rat bone marrow mesenchymal stem cells exposed to 5-azacytidinerdquoMuscle ampNerve vol 18 no 12 pp 1417ndash1426 1995

[23] D Woodbury E J Schwarz D J Prockop and I B BlackldquoAdult rat and human bone marrow stromal cells differentiateinto neuronsrdquo Journal of Neuroscience Research vol 61 no 4pp 364ndash370 2000

[24] M Reyes T Lund T Lenvik D Aguiar L Koodie and CM Verfaillie ldquoPurification and ex vivo expansion of postnatalhumanmarrowmesodermal progenitor cellsrdquoBlood vol 98 no9 pp 2615ndash2625 2001

[25] M Ramalho-Santos S Yoon Y Matsuzaki R C Mulliganand D A Melton ldquolsquoStemnessrsquo transcriptional profiling ofembryonic and adult stem cellsrdquo Science vol 298 no 5593 pp597ndash600 2002

[26] KMitsui Y Tokuzawa H Itoh et al ldquoThe homeoprotein nanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003

[27] S Okumura-Nakanishi M Saito H Niwa and F IshikawaldquoOct-34 and Sox2 regulate Oct-34 gene in embryonic stemcellsrdquo The Journal of Biological Chemistry vol 280 no 7 pp5307ndash5317 2005

[28] F Ulloa-Montoya B L Kidder K A Pauwelyn et al ldquoCompar-ative transcriptome analysis of embryonic and adult stem cellswith extended and limited differentiation capacityrdquo GenomeBiology vol 8 no 8 article R163 2007

[29] L Song N EWebb Y Song and R S Tuan ldquoIdentification andfunctional analysis of candidate genes regulating mesenchymalstem cell self-renewal and multipotencyrdquo STEMCELLS vol 24no 7 pp 1707ndash1718 2006

[30] A H H Van Boxel-Dezaire M R S Rani and G R StarkldquoComplexmodulation of cell type-specific signaling in responseto type I interferonsrdquo Immunity vol 25 no 3 pp 361ndash372 2006

[31] C G Kim J J Lee D Y Jung et al ldquoProfiling of differentiallyexpressed genes in human stem cells by cDNA microarrayrdquoMolecules and Cells vol 21 no 3 pp 343ndash355 2006

[32] T Bowman R Garcia J Turkson and R Jove ldquoSTATs inoncogenesisrdquo Oncogene vol 19 no 21 pp 2474ndash2488 2000

[33] A Stephanou andD S Latchman ldquoOpposing actions of STAT-1and STAT-3rdquo Growth Factors vol 23 no 3 pp 177ndash182 2005

[34] A Martino J H Holmes J D Lord J J Moon and B HNelson ldquoStat5 and Sp1 regulate transcription of the cyclin D2gene in response to IL-2rdquo The Journal of Immunology vol 166no 3 pp 1723ndash1729 2001

[35] I Matsumura T Kitamura H Wakao et al ldquoTranscriptionalregulation of the cyclin D1 promoter by STAT5 its involvementin cytokine-dependent growth of hematopoietic cellsrdquo TheEMBO Journal vol 18 no 5 pp 1367ndash1377 1999

[36] V Calo M Migliavacca V Bazan et al ldquoSTAT proteins fromnormal control of cellular events to tumorigenesisrdquo Journal ofCellular Physiology vol 197 no 2 pp 157ndash168 2003

[37] M Socolovsky A E J Fallon S Wang C Brugnara and HF Lodish ldquoFetal anemia and apoptosis of red cell progenitorsin Stat5aminusminus5bminusminus mice a direct role for stat5 in Bcl-X(L)inductionrdquo Cell vol 98 no 2 pp 181ndash191 1999

[38] G Li Z Wang Y Zhang et al ldquoSTAT5 requires the N-domainto maintain hematopoietic stem cell repopulating function andappropriate lymphoid-myeloid lineage outputrdquo ExperimentalHematology vol 35 no 11 pp 1684ndash1694 2007

[39] S Negoro K Kunisada E Tone et al ldquoActivation of JAKSTATpathway transduces cytoprotective signal in rat acute myocar-dial infarctionrdquo Cardiovascular Research vol 47 no 4 pp 797ndash805 2000

[40] H C Blair L J Robinson and M Zaidi ldquoOsteoclast signallingpathwaysrdquo Biochemical and Biophysical Research Communica-tions vol 328 no 3 pp 728ndash738 2005

[41] C J Malemud ldquoProtein kinases in chondrocyte signaling andosteoarthritisrdquo Clinical Orthopaedics and Related Research no427 supplement pp S145ndashS151 2004

[42] E M Pinheiro and D J Montell ldquoRequirement for Par-6 andBazooka inDrosophila border cell migrationrdquoDevelopment vol131 no 21 pp 5243ndash5251 2004

[43] D Strutt ldquoFrizzled signalling and cell polarisation inDrosophilaand vertebratesrdquo Development vol 130 no 19 pp 4501ndash45132003

[44] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[45] J Guo J Jin and L F Cooper ldquoDissection of sets of genes thatcontrol the character of wnt5a-deficient mouse calvarial cellsrdquoBone vol 43 no 5 pp 961ndash971 2008

[46] M Nishizuka A Koyanagi S Osada and M Imagawa ldquoWnt4and Wnt5a promote adipocyte differentiationrdquo FEBS Lettersvol 582 no 21-22 pp 3201ndash3205 2008

[47] Y Yang L Topol H Lee and J Wu ldquoWnt5a andWnt5b exhibitdistinct activities in coordinating chondrocyte proliferation anddifferentiationrdquo Development vol 130 no 5 pp 1003ndash10152003

[48] D J Van Den Berg A K Sharma E Bruno and R Hoff-man ldquoRole of members of the Wnt gene family in humanhematopoiesisrdquo Blood vol 92 no 9 pp 3189ndash3202 1998

[49] D J Prockop C A Gregory and J L Spees ldquoOne strategy forcell and gene therapy harnessing the power of adult stemcells torepair tissuesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 1 pp 11917ndash119232003

[50] X He J-P Saint-Jeannet Y Wang J Nathans I Dawid and HVarmus ldquoA member of the Frizzled protein family mediatingaxis induction byWnt-5Ardquo Science vol 275 no 5306 pp 1652ndash1654 1997

Stem Cells International 13

[51] D G Phinney G Kopen R L Isaacson and D J ProckopldquoPlastic adherent stromal cells from the bone marrow ofcommonly used strains of inbred mice variations in yieldgrowth and differentiationrdquo Journal of Cellular Biochemistryvol 72 no 4 pp 570ndash585 1999

[52] N Sasaki T Sasamura H O Ishikawa et al ldquoPolarized exo-cytosis and transcytosis of Notch during its apical localizationin Drosophila epithelial cellsrdquo Genes to Cells vol 12 no 1 pp89ndash103 2007

[53] M Handler X Yang and J Shen ldquoPresenilin-1 regulatesneuronal differentiation during neurogenesisrdquo Developmentvol 127 no 12 pp 2593ndash2606 2000

[54] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006

[55] A W Duncan F M Rattis L N DiMascio et al ldquoIntegrationof Notch andWnt signaling in hematopoietic stem cell mainte-nancerdquo Nature Immunology vol 6 no 3 pp 314ndash322 2005

[56] N Carlesso J C Aster J Sklar and D T Scadden ldquoNotch1-induced delay of human hematopoietic progenitor cell differen-tiation is associated with altered cell cycle kineticsrdquo Blood vol93 no 3 pp 838ndash848 1999

[57] I Maillard U Koch A Dumortier et al ldquoCanonical notch sig-naling is dispensable for the maintenance of adult hematopoi-etic stem cellsrdquo Cell Stem Cell vol 2 no 4 pp 356ndash366 2008

[58] G Pearson F Robinson T B Gibson et al ldquoMitogen-activatedprotein (MAP) kinase pathways regulation and physiologicalfunctionsrdquo Endocrine Reviews vol 22 no 2 pp 153ndash183 2001

[59] R E Dolmetsch R S Lewis C C Goodnow and J I HealyldquoDifferential activation of transcription factors induced by Ca2+response amplitude and durationrdquoNature vol 386 no 6627 pp855ndash858 1997

[60] S Kawano S Shoji S Ichinose K Yamagata M Tagami andM Hiraoka ldquoCharacterization of Ca2+ signaling pathways inhuman mesenchymal stem cellsrdquo Cell Calcium vol 32 no 4pp 165ndash174 2002

[61] B Ye ldquoCa2+ oscillations and its transporters in mesenchymalstem cellsrdquo Physiological Research vol 59 no 3 pp 323ndash3292010

[62] J E Ip Y Wu J Huang L Zhang R E Pratt and V J DzauldquoMesenchymal stem cells use integrin 1205731 not CXC chemokinereceptor 4 for myocardial migration and engraftmentrdquoMolecu-lar Biology of the Cell vol 18 no 8 pp 2873ndash2882 2007

[63] C A Opitz U M Litzenburger C Lutz et al ldquoToll-like recep-tor engagement enhances the immunosuppressive propertiesof human bone marrow-derived mesenchymal stem cells byinducing indoleamine-23-dioxygenase-1 via Interferon-120573 andprotein kinase Rrdquo Stem Cells vol 27 no 4 pp 909ndash919 2009

[64] B Kulterer G Friedl A Jandrositz et al ldquoGene expressionprofiling of human mesenchymal stem cells derived from bonemarrow during expansion and osteoblast differentiationrdquo BMCGenomics vol 8 article 70 2007

[65] W G Li and X X Xu ldquoThe expression of N-cadherinfibronectin during chondrogenic differentiation of MSCinduced by TGF-1205731rdquo Chinese Journal of Traumatology vol 8no 6 pp 349ndash351 2005

[66] F Ng S Boucher S Koh et al ldquoPDGFTGF-120573 and FGF signal-ing is important for differentiation and growth of mesenchymalstem cells (mscs) transcriptional profiling can identify markersand signaling pathways important in differentiation of MSCsinto adipogenic chondrogenic and osteogenic lineagesrdquo Bloodvol 112 no 2 pp 295ndash307 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

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Signal TransductionJournal of

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Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Nucleic AcidsJournal of

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Enzyme Research

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International Journal of

Microbiology

Stem Cells International 5

by Dai et al [16] For many years the platform for geneexpression profile was the Affymetrix GeneChip databaseHowever their probe section was created on the basis ofprior gene and transcriptome annotations which significantlydiffer from the modern expertise A comparative analysisbetween many popular Gene Chips and redefined probesbased on updated gene data with consideration to geneticpolymorphism showed a 30ndash50 discrepancy [16] thereforeit was decided to reannotate data as the first step in ourstudyThe assessment of noise threshold was then completedcreating a mixture model of normal distributions for themean SLR of CD34minus cells from umbilical cord blood versusCD34minus from bone marrow

The next step comprised microarrays normalizationthrough the RMA algorithm [17] Three repetitions werereplaced with a mean value with a prior detection of outliersby means of the Dean and Dixon method [18] Determina-tion of the measurement noise threshold was conducted byempirical decomposition of probability density function ofexpression levels into normal components [19] The noisemodel was assumed to be the component of the lowest meanvalue which was unchanged across all the microarrays Thethreshold value of the filter was constructed at level 28 (inthe field of logarithms to base 2)

As a result of the abovementioned procedures a data setwas obtained which described 11 979 genes which underwentfurther analysis The first stage consisted of creating the mix-ture model of normal distributions constructed for a meanSLR (Signal Log Ratio) of CD34minus stem cells from umbilicalcord blood versus CD34minus stem cells from bone marrowwhich allowed defining the affinity criteria of specific genes tosubgroups of an increased decreased and unchanged valueof expression levels This also enabled us to determine a FDRvalue (False Discovery Rate) for each of them (Figure 1)

This method is based on decomposition that is thehistogram decomposition into (K)mdashGaussrsquos components aswell as modeling the probability density function distribu-tions (pdf) for given gene expression levels The method wasused for analysis of a histogram reflecting the frequency ofgenes of a specific expression level in the tested preparationThis initial analysis showed considerable similarity of geneexpression of CD34minus cells from umbilical cord blood ascompared with CD34minus cells from bone marrow

31 Unsupervised Gene Clustering During the next stage ofdata analysis a qualitative determination of the processes inCD34minus stem cells from umbilical cord blood in comparisonwith CD34minus stem cells from bone marrow was conductedusing unsupervised gene clustering Again the GaussianMixture Model for empirical decomposition of expressionlevels constituted the algorithm enabling such a divisionHowever in this analysis additional limitations based onthe gene composition of each component that were thesame across the experimental conditions were added to thealgorithm [20] As a result 15 gene clusters were obtainedtwo of which (numbers 9 and 13 in this study) containeddiscriminatory genes exhibiting a statistically significantincrease (cluster number 9) and decrease of gene expression(cluster number 13) (Table 1)

1600

1400

1200

1000

800

600

400

200

00 2 4 6 8

Num

ber o

f obs

erva

tions

Gene expressionminus10 minus8 minus6 minus4 minus2

SLR CD34H adnotacje Dai ver12

Figure 1Mixturemodel of normal distributions (GaussianMixtureModel) for a mean SLR (Signal Log Ratio) of CD34minus cells fromumbilical cord blood versus CD34minus cells from bone marrow Log(CD34-P)( CD34-Sz)

The remaining clusters differing in terms of the numberof genes within the cluster aggregated the genes of similarexpression values Differences betweenmean gene expressionlevels of CD34minus stem cells from bone marrow and CD34minusstem cells from umbilical cord blood in clusters numbered1ndash8 10ndash12 14 and 15 are small or nonexistent Thus all theseclusters were classified as the groups of genes exhibiting thesame expression levels

Cluster number 9 included 493 genes which show anincreased activity in CD34minus stem cells from umbilical cordblood of the mean SRL (Signal Log Ratio) equaling 306 Incontrast cluster number 13 included a lower number of genes(119899 = 387) of decreased expression in CD34minus stem cells fromumbilical cord blood for which SRL equals minus343

At the next stage gene clustersgroups obtained in thismanner were compared to biological processes in whichthey are involved as defined by gene ontology (GO) termsthat are commonly applied for this purpose A number ofconditioning hypergeometric tests were conducted verifyingthe one-sided hypothesis about overrepresentation For eachontological term that was found to be significantly overrep-resented statistically in a given gene cluster the OR (OddsRatio) value was determined All the clusters were classifiedthrough the GO classification system that is the clusterswith genes discriminating both cell populations as well as theclusters grouping genes of a similar expression

Four hundred ninety-three genes of cluster 9 classifiedvia the GO system determined the terms of biological pro-cesses (119901 lt 005) overrepresented in CD34minus stem cells fromumbilical cord blood The classification system determined256 GO terms of biological processes out of which GO termsof 119901 value below 004 underwent further analysis As a resultof this analysis the majority of GO terms represented only byone or two genes were rejected The GO terms for biologicalprocesses in cluster 9 were assigned to one of six func-tional groups including metabolism biological processes

6 Stem Cells International

Table 1 15 clusters obtained as a result of the unsupervised gene clusteringMean CD34minus BM (mean of CD34minus stem cells from bonemarrow)Mean CD34minus UCB (mean of CD34minus stem cells from umbilical cord blood)119873 (a number of genes in a cluster) SRL (Signal Log Ratio)

Cluster MeanCD34minus BM

MeanCD34minusUCB

SDCD34minusBM

SDCD34minusUCB

119873

119901 value MeanSLR

minus95CISLR

+95CISLR

Conclusion

1497 491 008 009

1102

000000023 minus006 minus008 minus004

No change2

1157 1149 035 047

54

026488907 minus008 minus022 006

No change3

802 805 030 032

592

063190010 001 minus005 007

No change4

445 440 006 005

999

000000001 minus006 minus008 minus004

No change5

702 714 024 021

898

000000009 011 007 016

No change6

554 550 012 013

1133

000000889 minus006 minus009 minus003

No change7

353 346 004 005

960

451861119864 minus 13 minus007 minus009 minus005

No change8

476 483 071 088

961

003102164 011 001 020

No change9 564 715 158 270 493 p lt 00000001 306 295 316 Up10

311 309 004 004

872

000325256 minus003 minus005 minus001

No change11

276 278 002 002

647

000002947 003 001 004

No change12

398 390 006 007

1133

544009119864 minus 15 minus008 minus010 minus006

No change13 739 486 285 101 387 p lt 00000001 minus343 minus357 minus329 Down14

621 621 026 021

1353

044902052 minus001 minus005 002

No change15

930 936 064 084

395

015369595 008 minus003 020

No change

0 001 002 003 004

Signal transduction (GO0007165)

Activation of MAPK activity (GO0000187)

Protein kinase cascade (GO0007243)

Regulation of MAP kinase activity (GO0043405)

Integrin-mediated signaling pathway (GO0007229)

Positive regulation of tyrosine phosphorylation ofSTAT protein (GO0042531)

Positive regulation of calcium-mediated signaling(GO0050850)

JAK-STAT cascade (GO0007259)

Cytokine-mediated signaling pathway (GO0019221)

Positive regulation of cell proliferation (GO0008284)

Calcium-mediated signaling (GO0019722)

Regulation of JAK-STAT cascade (GO0046425)

p value

Signaling pathway

Figure 2 Functional annotations 12 GO classification terms for biological processes in the group signaling pathways in cluster 9 dependingon the 119901 value (119901 lt 004)

regulation cellular homeostasis cellular defensive responseand response to stimuli cytokine production and signalingpathways The GO terms regarding signaling pathways arepresented in Figure 2

Thenext stage consisted of a similar analysis of genes fromcluster 13 GO system classification for 387 genes of cluster 13determined 119 terms of biological processes (119901 lt 005) of

a decreased activity in CD34minus cells fromumbilical cord bloodas compared with CD34minus cells from bone marrow The GOterms of 119901 value below 004 underwent further analysis As aresult of this analysis the majority of GO terms representedonly by one or two genes were rejected The GO terms forbiological processes in cluster 13 were assigned to one ofthree functional groups including metabolism biological

Stem Cells International 7

0000 0010 0020 0030

Negative regulation of signal transduction (GO0009968)

Vascular endothelial growth factor receptor signalingpathway (GO0048010)

Transmembrane receptor protein tyrosine kinase signalingpathway (GO0007169)

Transmembrane receptor protein serinethreonine kinasesignaling pathway (GO0007178)

Regulation of transforming growth factor beta receptorsignaling pathway (GO0017015)

Cell surface receptor linked signal transduction(GO0007166)

BMP signaling pathway (GO0030509)

p value

Signaling pathway

Figure 3 Functional annotations 7 GO classification terms for biological processes in the group signaling pathways in cluster 13 dependingon the 119901 value (119901 lt 004)

Table 2 Gene pathways based on the KEGG classification of an increased expression in CD34minus stem cells from umbilical cord blood

KEGGID 119901 value Odds ratio Exp count Count Size Term4060 0001 2947 14 33 223 Cytokine-cytokine receptor interaction4620 0001 3672 6 17 92 Toll-like receptor signaling pathway5120 0001 3748 4 12 63 Epithelial cell signaling inHelicobacter pylori infection4630 0012 2067 8 15 130 JAK-STAT signaling pathway

processes regulation and signaling pathways In CD34minus cellsfrom umbilical cord blood as compared with CD34minus cellsfrom bone marrow a decreased gene activity was observedof the BMP signaling pathway TGF beta transmembranereceptor of protein serinethreonine kinase transmembranereceptor of tyrosine kinase and VEGF (Figure 3)

Gene pathways whose components are genes belongingto specific clusters underwent an additional analysis whichwas independent of a direct description of cellular processesFor this purpose data from the KEGG database (KyotoEncyclopedia of Genes and Genomes) were used For eachpathway the hypothesis of overrepresentation of specificclusters of genes as compared with the whole scope ofanalyzed genes was verified and precise 119901 values weredetermined For the purpose of this study gene pathwaysfor clusters 9 and 13 were analyzed because they containedgenes discriminating both gene populations On the basisof KEGG database cytokine-cytokine receptor interactionpathway toll-like receptor signaling pathway epithelial cellsignaling in Helicobacter pylori infection and JAK-STATsignaling pathway are overrepresented in CD34minus cells fromumbilical cord blood (Table 2)

In cluster 13 on the basis of KEGG classification theCD34minus cells from umbilical cord blood show a decreasedactivity of focal adhesion signaling pathway cell junctionsand TGF-beta signaling pathway (Table 3)

At the next stage the genes of the remaining clustersnumbered 1ndash8 10ndash12 and 14 of a similar gene expression were

analyzed The analysis was based on the GO classification foreach of the abovementioned clusters The criteria used forchoosing the clusters included the biological processes con-nected with cellular cycle as well as the processes connectedwith signaling pathways activity A similar gene expression inCD34minus cells from umbilical cord blood and CD34minus cells frombone marrow was characteristic for such biological processesas cell division mitosis maintaining telomere endings RNAandDNA processing and a similar activity of Notch andWntsignaling pathways genes

An independent analysis was also carried out for genes ofa similar activity in clusters 1ndash8 10ndash12 and 14 on the basis ofthe KEGG database CD34minus cells from umbilical cord bloodand CD34minus cells from bone marrow showed similar activityof Wnt and Notch signaling pathways genes p53 signalingpathway genes cell cycle PPAR and mTOR (Table 4)

CD34minus cells from umbilical cord blood and CD34minus cellsfrom bone marrow showed similar gene activity of Wntand Notch signaling pathways both on the basis of the GOclassification system and the KEGG database Therefore atthe last stage of the study the activity of single genes of Wntand Notch as well as JAK-STAT signaling pathways chosenfrom 11976 sampled genes was analyzed

In accordance with the KEGG database the JAK-STATsignaling pathway is defined by 155 genes out of whichthere were 128 active genes in the studied material Genesexhibiting increased activity in CD34minus cells from umbilicalcord blood included SPRY2 CISH CSF2 GRB2 IFNGR1

8 Stem Cells International

Table 3 Gene pathways based on the KEGG classification of a decreased expression in CD34minus stem cells from umbilical cord blood

KEGGID 119901 value Odds ratio Exp count Count Size Term4510 0001 7517 7 33 188 Focal adhesion1430 0001 8157 4 22 109 Cell junctions4350 0001 4038 3 10 82 TGF-beta signaling pathway

Table 4 Gene pathways based on the KEGG classification of a similar expression in CD34minus stem cells from umbilical cord blood and CD34minuscells from bone marrow for clusters 1ndash8 10ndash12 and 14-15

KEGGID 119901 value Odds ratio Exp count Count Size Term04310 0018 1880 10 17 131 Wnt signaling pathway04330 0025 2722 3 7 39 Notch signaling pathway04115 0038 2157 5 9 61 p53 signaling pathway04110 0009 2028 10 18 100 Cell cycle03320 0020 2791 3 7 60 PPAR signaling pathway04150 0035 2220 5 9 46 mTOR signaling pathway

IL2RB IL2RG IL7R IL10RA IL15RA PIM1 PTPN6 STAT1STAT5A and CCND3 Genes exhibiting decreased activityincluded PIAS3 IL11RA and CBLB

On the basis of the KEGG database the Wnt signalingpathway is defined by 151 genes out of which there were132 active genes in the studied material Genes exhibit-ing increased activity in CD34minus cells from umbilical cordblood include MMP7 PRKCB PSEN1 RAC2 and CCND3Genes exhibiting decreased activity include DKK1 WNT5AWNT5B FZD7 and TCF7L1

On the basis of the KEGG database the Notch signalingpathway is defined by 47 genes out of which there were38 active genes in the studied material Genes exhibitingincreased activity in CD34minus cells from umbilical cord bloodincluded DTX4 and PSEN1

4 Discussion

Nonhematopoietic stem cells were first identified by Frieden-stein et al who described the progenitor cells of rat bonemarrow [21] Currently the nonhematopoietic stem cellswhich are frequently referred to although not quite correctlyas the mesenchymal stem cells are isolated from varioustissues including the umbilical cord blood placenta andumbilical cord A constantly increasing number of studieson MSCs have proved that in appropriate environmentalconditions they are capable of differentiating into special-ized mesodermal endodermal and even ectodermal celllineages [2 22ndash24] In natural conditions they play an impor-tant role in cell regeneration processes and in repairingthe tissues damaged by degenerating processes Anotherintriguing characteristic of MSCs is their ability to avoidimmunological identification and response therefore theyare referred to as the hypoimmunogenic cells Despite greatattention paid to mesenchymal stem cells their biology isnot yet fully understood Introducing multipotential stemcells into clinical application requires a full understandingof the mechanisms which control key properties of thecell such as cell self-replication versus differentiation and

mobilization versus tissue homing The understanding ofthese mechanisms must be based on strict definitions ofhuman multipotential stem cells at the molecular level Inaddition understanding of molecular mechanisms of self-replicating pluripotentiality plasticity and differentiation ofstem cells not only is a necessary condition to overcomelimitations of stem cells application as a therapeutic factorbut also is key to understand a fundamental mystery of thedevelopment of human organism

This paper presents a comparison of gene activity of thenonhematopoietic cells of umbilical cord blood and bonemarrow using oligonucleotidemicroarrays Gene activity wastested in stem cells CD34 and included 11 979 genes obtainedafter completing procedures to increase the purity of cellsamples Cell populations included in the study were alsolimited based on the noise threshold revealed in the finalcomparative analysis The gene expression profiles of thecompared populations of CD34minus stem cells from umbilicalcord blood and CD34minus stem cells from bone marrow werevery similar (Figure 1) For a better illustration of the biolog-ical molecular activity and the activity of signaling pathwaysin the studied cell populations a transcription profile analysiswith the GO and KEGG databases was completed In termsof fundamental signaling pathways for biological activityof stem cells like JAK-STAT Wnt and Notch we recordeddiscrete differences among expressed genes from umbilicalcord blood cells as compared to bone marrow cells

The analysis regarding signaling pathways showedincreased activity of the JAK-STAT pathway in CD34minusstem cells from umbilical cord blood both on the basis ofthe gene ontology classification and the KEGG database(Figure 2 and Table 2) The increased activity regarded 15genes which take part in the JAK-STAT cascade that isSPRY2 CISH CSF2 GRB2 IFNGR1 IL2RB IL2RG IL7RIL10RA IL15RA PIM1 PTPN6 and CCND3 including thegenes which code transcription factors STAT1 and STAT5AIn previous publications the significance of the JAK-STATsignaling pathway is best described in the embryonic andhematopoietic stem cells Ramalho-Santos et al searchedfor a transcription profile describing both the embryonic

Stem Cells International 9

stem cells and the stem cells of an adult organism on thebasis of the study of embryonic cells neural stem cellsand hematopoietic stem cells activity [25] These studiesattempted to create a molecular signature of the stem cells inrelation to the self-regeneration and differentiation abilities ofthe cells The active JAK-STAT pathway belongs to the basicattributes defining the so-called ldquostemnessrdquo phenomenon Inthe embryonic stem cells the JAK-STAT signaling pathwaypromotes proliferation in the form of self-replicationThe activating factor LIF (Leukemia Inhibitory Factor)provokes phosphorylation and dimerisation of STAT3which after translocation to the cell nucleus stimulates theexpression of gene group responsible for self-replication ofthe embryonic stem cell However the JAK-STAT signalingpathway is not the only factor responsible for proliferationregulation of the embryonic stem cells Internal processesregulating the multiplication processes independent of theSTAT3 activity such as the Nanog and Oct-4 activity arealso important [15 17 26 27] In our study material theNanog and Oct-4 genes were not active in both types ofnonhematopoietic stem cells which is in agreement withother publications devoted to this subject [28 29] TheSTAT1 transcription factor overactive in our study in CD34minuscells of umbilical cord blood is responsible among othersfor the expression of interferon-activated genes involved incell defense against pathogenic factors [30] Moreover itis believed that STAT1 is a medium in expression of genegroup which is important for cell viability in response tostimulating factors In 2006 Kimrsquos team described STAT1as a highly overactive gene in human mesenchymal stemcells [31] In comparison the transcription factor STAT5which functions in the form of two isoforms STAT5a andSTAT5b is involved in the cellular proproliferation responsesand antiapoptosis processes [32 33] STAT5 is activatedby various cytokine receptors In our study the isoformSTAT5A showed increased activity It is believed that theSTAT5 factor activates specifically the cellular cycle genesthat is cyclins [34 35] In human stem cells the STAT factorsactivate the expression of cyclin Dwhich codes the regulatingsubunit of the CYCDCDK4 complex driving the G1S phasetransition which in turn activates the cell cycle progression[32 36] STAT5a and STAT5b also play an important rolein expression of genes promoting hematopoietic stem cellssurvival [37] Gene expression profiling over a range ofSTAT5 activities in human CB cells revealed subsets of genesthat are associated with the self-renewal and long-termexpansion phenotype Taking into account the fact thatin STAT5minusminus mice myelopoiesis appears to be relativelyunaffected it can be assumed that STAT5 can play a keyrole in assessing the quality of cells [38] Additionally notonly is the JAK-STAT signaling pathway a significant signaltransduction component which controls cell proliferationand purpose but it also regulates cell migration which isimportant both in the primary processes of gastrulationand in the tissue regeneration For instance the JAK-STATsignaling pathway is connected with cardioprotection [39]as well as with processes of regeneration and remodeling ofskeleton [40 41] The active JAK-STAT signaling pathwayhelps to regulate cell organization within cell grouping

[42] The above results may suggest a greater self-renewalpotential of CD34minus stem cells of umbilical cord bloodlong-term expansion phenotype and their higher ability interms of tissue regeneration

The Wnt signaling pathway is perceived as a signalingcascade involved in the control of fundamental processesof nondifferentiated mesenchymal stem cells such as cellreproduction and directed differentiation Both methods ofgene activity classification that is GO and KEGG showeda similar activity of the majority of Wnt signaling pathwaygenes (Table 4) In addition the analysis of separate genestaking part in the Wnt signaling pathway and coding 19glycosylated molecules of extracellular signaling of the Wntfamily cellular surface receptors including Frizzled receptorsintracellular signaling molecules and genes involved in cellcycle and taking part in regulation of proliferation growthand modification of proteins showed that the expressionprofilewas very similar except for single genes of an increasedand decreased expression in CD34minus stem cells of umbilicalcord blood in our study that is MMP7 PRKCB PSEN1RAC2 and CCND3 respectively as well as DKK1 WNT5AWNT5B FZD7 and TCF7L1 The Wnt5a ligand is involvedin the noncanonical Wnt signaling pathway transmissionand it is assumed that it may inhibit the canonical Wntpathway in mouse embryonic cells similarly to Dkk1 Therole of the noncanonical Wnt signaling pathway which inour results seemed to prevail in CD34minus stem cells of bonemarrow is not clear It is believed that the noncanonical Wntsignaling pathway controls the reorganization of cytoskeletonmuscle proteins tissue polarity and cell movement [43] Incontrast the canonical signaling pathway which activatestarget gene transcription through the increase of beta-cateninconcentration seems to be more active in CD34minus stemcells from umbilical cord cells Its role is to control thetissue-specific cell purpose during embryogenesis as well asregulation of proliferation in adult tissues [44] Additionallyhigh activity of WNT5A gene is noticeable in the humanMSCs differentiation into osteoblasts as well as in early stagesof adipogenesis [45 46] According to the publication ofYang et al the Wnt5a and Wnt5b glycoproteins are alsoresponsible for coordination of chondrocyte proliferation anddifferentiation [47] Wnt5a is believed to be an importantfactor taking part in the positive autocrinic regulation ofHSC repopulation ability [48] In their publication Prockopet al suggest that a high WNT5A expression in MSCs ischaracteristic for the end of the exponential and stationarygrowth phases in cell cultures in in vitro conditions [49]Slight changes in theWnt gene activity especially inWNT5Aactivity may result from the role played by MSCs in theirenvironmental niche Bone marrow MSCs seem to be moreinvolved in the renewal processes of the progenitor cells poolwhich reside in bone marrow The researches show that theexpression of Wnt ligands and Fz receptors in MSCs showssome correlation of WntFz pairs such as Wnt5aFz5 [50]The above discussed study results showed that there wasa coexpression of WNT5AFZD7 Their higher expressionwas characteristic for the bone marrow cells while thepreviously published data suggested differences in WntFzactivity depending on the origin-mouse versus humanMSCs

10 Stem Cells International

[51] Currently available data suggest differences which maybe characteristic not only for proliferation and differentiationprograms but also for the phenotype of MSCs from umbilicalcord blood and bone marrow which is in agreement withthe results published by Baksh et al [7] The remaining genestake part in theWnt signaling cascade of increased activity inCD34minus from umbilical cord blood such as MMP7 belongingto metalloproteinases which play a role in the extracellulararray remodeling and wound healing [5]

The Notch signaling pathway belongs to the processes ofintercellular communication which regulate gene expressionpatterns defining a dualistic character of stem cells reflectedby the asymmetric cell division [52] Additionally the Notchsignaling cascade controls various cells biological processesnot only proliferation and differentiation but also apoptosisand regeneration The signaling activity of Notch pathwaygenes determined in the study of 38 genes showed theirsimilar expression as defined by the KEGG classification(Table 4) A similar activity level was noted for all four Notch1ndash4 receptors as well as for JAG1 and JAG2 ligands The dif-ferences were related to two genes DTX4 and PSEN1 whichwere overactive in CD34minus cells from umbilical cord bloodThe function of the PSEN1 gene is mainly developmentalprocesses regarding cell differentiation during neurogenesisAnd the lack of this gene leads to a premature directeddifferentiation of neural progenitor cells [53] The activity ofthe Notch gene cascade plays an important role at all levelsof stem cell development starting from the embryonic stemcell through fetal cells to adult organism stem cells Notchis significantly involved in the asymmetric segregation offactors determining cell purpose during replication divisionsof the stem cell poolThe significance of receptors and ligandsis best described in the hematopoietic stem cells Expressionof the constitutively active Notch1 gene in hematopoieticstem cells induces the immortalized cell lineages which canreproduce the myeloid and lymphoid cell lineage in a long-term repopulation in animal studies [54 55] Apart from thatit delays differentiation of human hematopoietic progenitorstem cells in vitro [56] The studies suggest that Notch1 is animportant factor inHSC self-renewal and can also participatein the inhibition of stem cell differentiation The scientificfindings reveal that a suppression of the Notch signalingpathway via deletion of key genes in HSC did not cause cellsto lose their ability of long-term hematopoiesis renewal inanimal models [57] Therefore the role of ligands and Notchreceptors in HSC has not been precisely defined yet andcurrent publications do not provide precise data regardingthe role played by particular Notch signaling pathway genesin mesenchymal stem cells The only phenomenon whichappears to be superior and characteristic for all stem cellsincluding MSCs is the significance of the Notch cascadein asymmetrical cell divisions This is a significant com-ponent of stem cells population self-renewal as well asthe functionality of the cascade in the stem niche Thisphenomenon constitutes an integral part of the hypothesisabout stem cell population aging However further inves-tigation will be required to determine if the differentiallyexpressed genes described in the studies above could serve asstemness genes

Remaining signaling pathways which revealed increasedactivity in the studied CD34minus cells of umbilical cord bloodincludedMAPK (Mitogen-Activated Protein Kinase Figure 2)which regulates a wide range of cell biological activities suchas gene expression mitosis proliferation differentiationmigration and apoptosis processes [58]TheMAPKpathwaystake part in regulation of early embryonic development andin defining the role of embryonic stem cells from the earlydevelopmental phases until the moment of formation ofdifferentiated adult cells However this precise role played bythe MAPK pathways in various stem cell types has not yetbeen defined

Transmission via calcium ions was also singled out inthe group of the overactive signaling pathways of the studiedCD34minus stem cells of umbilical cord blood versus CD34minus stemcells of bone marrow (Figure 2) The ions are a vital andcommonly present mediator in the process of extracellularsignals transmission and conversion into a cellular responseVariations of calcium ions concentrations regulate geneexpression through an impact on the activity of numeroussignaling pathways Intracellular increase of calcium ionsconcentration also regulates cell activities including adhe-sion motility gene expression and proliferation [59] Thesignificance of transmission via calcium ions and the ways oftheir concentration changes in mesenchymal stem cells stillhave not been studied Kawano et al were the first to reportthat in the nondifferentiated MSCs there are spontaneouschanges of calcium ions concentrations despite the lack ofany stimulating factors [60] As reported the calcium iontransmission through the activation of different gene groupsmay direct the cells onto specific developmental pathways[61]

The integrin-mediated signaling pathway takes part inextracellular array remodeling which means that it con-trols cell behavior and tissue organization Its increasedactivity was observed in our study in CD34minus stem cells ofumbilical cord blood (Figure 2) In recent years the role ofintegrins and selectins has been stressed in the adhesion andtransendothelial migration which has a significant meaningin the processes of repopulation of tissues damaged bydiseases and their regeneration via the circulating MSCs Forinstance Ip et al showed that blocking of beta 1-integrinwhich constitutes a component of the adhesive moleculeVLA-4 in MSC weakened the ability of mesenchymal stemcells to engraft into the infarction-changedmyocardium [62]The above results may suggest a better migration abilityof CD34minus stem cells of umbilical cord blood into the areaof pathologic tissue MSCs own cytokinetic activity thathas similar significance in terms of tissue regenerationThe study of cytokinetic activity of CD34minus stem cells ofumbilical cord blood showed increased activity of signalingpathways activated via cytokines (Figure 2 Table 2) On theother hand the TLR signaling pathway (toll-like receptorsignaling) which in our own results revealed a significantincreased activity in CD34minus stem cells of umbilical cordblood (Figure 2) increases the immunosuppressive charac-teristics of MSCs Weakening of the immunological responsegenerated by the TLR pathway depends on the produc-tion of immunosuppressive kynurenines by the indoleamine

Stem Cells International 11

23-dioxygenase enzyme which degrades tryptophan Induc-tion of indoleamine 23-dioxygenase by TLR entails autocrinesignaling loop of interferon-beta which plays a role in MSCsimmunomodulative effects [63]

According to the GO and KEGG classifications carriedout for cluster 13 with the gene pool of a decreased expressiona statistically significant decrease of gene activity was shownfor the TGF-beta and BMP pathways (Figure 3 Table 3) Itis believed that the family of TGF-beta members whichalso includes BMP plays an important role in initiating theprocess of a directed MSCs differentiation TGF-beta sig-naling transmission runs through specific serine-threoninekinase receptors and their nuclear receptors called the Smadproteins TGF-beta signaling transmission promotes theprocess of early differentiation stages into the chondrogenicand osteogenic lineages [64 65] However cell differentiatinginto the adipogenic lineage results in inhibition of the TGF-beta signaling pathway This was confirmed in experimentalstudies developed by Ng et al where the inhibition of TGF-beta transmission resulted in an increase of adipogenesisand a decrease of differentiating ability towards chondrocytes[66] The BMP signaling pathway strongly promotes differ-entiation towards osteoblasts but it also leads to inhibitionof myogenesis and adipogenesis Such a gene expressionprofile may suggest that the MSCs biological characteristicsare strongly influenced by the significance of stem cell nicheTherefore the mesenchymal stem cells from bone marrowmay be more involved in the promotion of the phenomenawhich maintain the stability of bone marrow environment

5 Conclusions

Multipotentiality of the mesenchymal stem cells their highproliferation ability and not too-demanding culturing con-ditions reveal their potential for wide application in variousmedical areas These are the reasons for the common pursuitto understand their biology based onmore andmore sublimebiomolecular procedures Scientists have been searchingfor an unequivocal marker which would characterize themesenchymal stem cells and simultaneously would enable aseparation of a homogenous MSC population Understand-ing of molecular mechanisms controlling self-replication anddifferentiation is becoming a fundamental issue for clinicalapplication of MSC In this respect the activity of signalingpathways is important yet the interpretation of the pathwaysespecially their interdependence constitutes a great challengefor the scientific world Moreover the answer to the questionregarding MSC innidation and its survival ability in hosttissues after transplantation is yet to be found Explanationof the abovementioned issues will require a more globalunderstanding of biological activity of these cells especiallyon the basis of the significance of the mesenchymal nicheenvironment of stem cells and its impact on the gene andepigenetic activity It should be assumed that continuedprogress in the understanding of MSCs will finally lead totheir common therapeutic and preventive application andconsequently to the improvement of the quality and lengthof human life

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] R J Deans andA BMoseley ldquoMesenchymal stem cells biologyand potential clinical usesrdquo Experimental Hematology vol 28no 8 pp 875ndash884 2000

[2] M F Pittenger A M Mackay S C Beck et al ldquoMultilineagepotential of adult human mesenchymal stem cellsrdquo Science vol284 no 5411 pp 143ndash147 1999

[3] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006

[4] Y-J Chang D T-B Shih C-P Tseng T-B Hsieh D-C Leeand S-M Hwang ldquoDisparate mesenchyme-lineage tendenciesin mesenchymal stem cells from human bone marrow andumbilical cord bloodrdquo Stem Cells vol 24 no 3 pp 679ndash6852006

[5] R A Panepucci J L C Siufi W A Silva Jr et al ldquoComparisonof gene expression of umbilical cord vein and bone marrowndashderived mesenchymal stem cellsrdquo STEM CELLS vol 22 no 7pp 1263ndash1278 2004

[6] C K Rebelatto A M Aguiar M P Moretao et al ldquoDissimilardifferentiation of mesenchymal stem cells from bone marrowumbilical cord blood and adipose tissuerdquo Experimental Biologyand Medicine vol 233 no 7 pp 901ndash913 2008

[7] D Baksh R Yao and R S Tuan ldquoComparison of proliferativeand multilineage differentiation potential of human mesenchy-mal stem cells derived from umbilical cord and bone marrowrdquoStem Cells vol 25 no 6 pp 1384ndash1392 2007

[8] D L Clarke C B Johansson J Wilbertz et al ldquoGeneralizedpotential of adult neural stem cellsrdquo Science vol 288 no 5471pp 1660ndash1663 2000

[9] D J Anderson F H Gage and I L Weissman ldquoCan stem cellscross lineage boundariesrdquo Nature Medicine vol 7 no 4 pp393ndash395 2001

[10] D L Stemple and D J Anderson ldquoIsolation of a stem cell forneurons and glia from the mammalian neural crestrdquo Cell vol71 no 6 pp 973ndash985 1992

[11] R L Rietze H Valcanis G F Brooker T Thomas A K Vossand P F Bartlett ldquoPurification of a pluripotent neural stem cellfrom the adult mouse brainrdquoNature vol 412 no 6848 pp 736ndash739 2001

[12] R M Seaberg S R Smukler T J Kieffer et al ldquoClonalidentification of multipotent precursors from adult mousepancreas that generate neural and pancreatic lineagesrdquo NatureBiotechnology vol 22 no 9 pp 1115ndash1124 2004

[13] A P Beltrami L Barlucchi D Torella et al ldquoAdult cardiac stemcells are multipotent and support myocardial regenerationrdquoCell vol 114 no 6 pp 763ndash776 2003

[14] L Yang S Li H Hatch et al ldquoIn vitro trans-differentiation ofadult hepatic stem cells into pancreatic endocrine hormone-producing cellsrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 99 no 12 pp 8078ndash80832002

[15] I Chambers D Colby M Robertson et al ldquoFunctional expres-sion cloning of Nanog a pluripotency sustaining factor inembryonic stem cellsrdquo Cell vol 113 no 5 pp 643ndash655 2003

12 Stem Cells International

[16] M Dai P Wang A D Boyd et al ldquoEvolving genetranscriptdefinitions significantly alter the interpretation of GeneChipdatardquo Nucleic Acids Research vol 33 pp 175ndash179 2005

[17] RA Irizarry BHobbs F Collin et al ldquoExploration normaliza-tion and summaries of high density oligonucleotide array probelevel datardquo Biostatistics vol 4 no 2 pp 249ndash264 2003

[18] R B Dean and W J Dixon ldquoSimplified statistics for smallnumbers of observationsrdquo Analytical Chemistry vol 23 no 4pp 636ndash638 1951

[19] G J McLachlan and D Peel ldquoOn computational aspectsof clustering via mixtures of normal and t-componentsrdquo inProceedings of the American Statistical Association (BayesianStatistical Science Section) American Statistical AssociationIndianapolis Ind USA 2000

[20] J Polanska P Widlak J Rzeszowska-Wolny M Kimmel andA Polanski ldquoGaussian mixture decomposition of time-courseDNA microarray datardquo in Mathematical Modeling of BiologicalSystems Volume I pp 351ndash359 Birkhauser Boston Mass USA2007

[21] A J Friedenstein U F Gorskaja and N N Kulagina ldquoFibrob-last precursors in normal and irradiated mouse hematopoieticorgansrdquo Experimental Hematology vol 4 no 5 pp 267ndash2741976

[22] S Wakitani T Saito and A I Caplan ldquoMyogenic cells derivedfrom rat bone marrow mesenchymal stem cells exposed to 5-azacytidinerdquoMuscle ampNerve vol 18 no 12 pp 1417ndash1426 1995

[23] D Woodbury E J Schwarz D J Prockop and I B BlackldquoAdult rat and human bone marrow stromal cells differentiateinto neuronsrdquo Journal of Neuroscience Research vol 61 no 4pp 364ndash370 2000

[24] M Reyes T Lund T Lenvik D Aguiar L Koodie and CM Verfaillie ldquoPurification and ex vivo expansion of postnatalhumanmarrowmesodermal progenitor cellsrdquoBlood vol 98 no9 pp 2615ndash2625 2001

[25] M Ramalho-Santos S Yoon Y Matsuzaki R C Mulliganand D A Melton ldquolsquoStemnessrsquo transcriptional profiling ofembryonic and adult stem cellsrdquo Science vol 298 no 5593 pp597ndash600 2002

[26] KMitsui Y Tokuzawa H Itoh et al ldquoThe homeoprotein nanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003

[27] S Okumura-Nakanishi M Saito H Niwa and F IshikawaldquoOct-34 and Sox2 regulate Oct-34 gene in embryonic stemcellsrdquo The Journal of Biological Chemistry vol 280 no 7 pp5307ndash5317 2005

[28] F Ulloa-Montoya B L Kidder K A Pauwelyn et al ldquoCompar-ative transcriptome analysis of embryonic and adult stem cellswith extended and limited differentiation capacityrdquo GenomeBiology vol 8 no 8 article R163 2007

[29] L Song N EWebb Y Song and R S Tuan ldquoIdentification andfunctional analysis of candidate genes regulating mesenchymalstem cell self-renewal and multipotencyrdquo STEMCELLS vol 24no 7 pp 1707ndash1718 2006

[30] A H H Van Boxel-Dezaire M R S Rani and G R StarkldquoComplexmodulation of cell type-specific signaling in responseto type I interferonsrdquo Immunity vol 25 no 3 pp 361ndash372 2006

[31] C G Kim J J Lee D Y Jung et al ldquoProfiling of differentiallyexpressed genes in human stem cells by cDNA microarrayrdquoMolecules and Cells vol 21 no 3 pp 343ndash355 2006

[32] T Bowman R Garcia J Turkson and R Jove ldquoSTATs inoncogenesisrdquo Oncogene vol 19 no 21 pp 2474ndash2488 2000

[33] A Stephanou andD S Latchman ldquoOpposing actions of STAT-1and STAT-3rdquo Growth Factors vol 23 no 3 pp 177ndash182 2005

[34] A Martino J H Holmes J D Lord J J Moon and B HNelson ldquoStat5 and Sp1 regulate transcription of the cyclin D2gene in response to IL-2rdquo The Journal of Immunology vol 166no 3 pp 1723ndash1729 2001

[35] I Matsumura T Kitamura H Wakao et al ldquoTranscriptionalregulation of the cyclin D1 promoter by STAT5 its involvementin cytokine-dependent growth of hematopoietic cellsrdquo TheEMBO Journal vol 18 no 5 pp 1367ndash1377 1999

[36] V Calo M Migliavacca V Bazan et al ldquoSTAT proteins fromnormal control of cellular events to tumorigenesisrdquo Journal ofCellular Physiology vol 197 no 2 pp 157ndash168 2003

[37] M Socolovsky A E J Fallon S Wang C Brugnara and HF Lodish ldquoFetal anemia and apoptosis of red cell progenitorsin Stat5aminusminus5bminusminus mice a direct role for stat5 in Bcl-X(L)inductionrdquo Cell vol 98 no 2 pp 181ndash191 1999

[38] G Li Z Wang Y Zhang et al ldquoSTAT5 requires the N-domainto maintain hematopoietic stem cell repopulating function andappropriate lymphoid-myeloid lineage outputrdquo ExperimentalHematology vol 35 no 11 pp 1684ndash1694 2007

[39] S Negoro K Kunisada E Tone et al ldquoActivation of JAKSTATpathway transduces cytoprotective signal in rat acute myocar-dial infarctionrdquo Cardiovascular Research vol 47 no 4 pp 797ndash805 2000

[40] H C Blair L J Robinson and M Zaidi ldquoOsteoclast signallingpathwaysrdquo Biochemical and Biophysical Research Communica-tions vol 328 no 3 pp 728ndash738 2005

[41] C J Malemud ldquoProtein kinases in chondrocyte signaling andosteoarthritisrdquo Clinical Orthopaedics and Related Research no427 supplement pp S145ndashS151 2004

[42] E M Pinheiro and D J Montell ldquoRequirement for Par-6 andBazooka inDrosophila border cell migrationrdquoDevelopment vol131 no 21 pp 5243ndash5251 2004

[43] D Strutt ldquoFrizzled signalling and cell polarisation inDrosophilaand vertebratesrdquo Development vol 130 no 19 pp 4501ndash45132003

[44] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[45] J Guo J Jin and L F Cooper ldquoDissection of sets of genes thatcontrol the character of wnt5a-deficient mouse calvarial cellsrdquoBone vol 43 no 5 pp 961ndash971 2008

[46] M Nishizuka A Koyanagi S Osada and M Imagawa ldquoWnt4and Wnt5a promote adipocyte differentiationrdquo FEBS Lettersvol 582 no 21-22 pp 3201ndash3205 2008

[47] Y Yang L Topol H Lee and J Wu ldquoWnt5a andWnt5b exhibitdistinct activities in coordinating chondrocyte proliferation anddifferentiationrdquo Development vol 130 no 5 pp 1003ndash10152003

[48] D J Van Den Berg A K Sharma E Bruno and R Hoff-man ldquoRole of members of the Wnt gene family in humanhematopoiesisrdquo Blood vol 92 no 9 pp 3189ndash3202 1998

[49] D J Prockop C A Gregory and J L Spees ldquoOne strategy forcell and gene therapy harnessing the power of adult stemcells torepair tissuesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 1 pp 11917ndash119232003

[50] X He J-P Saint-Jeannet Y Wang J Nathans I Dawid and HVarmus ldquoA member of the Frizzled protein family mediatingaxis induction byWnt-5Ardquo Science vol 275 no 5306 pp 1652ndash1654 1997

Stem Cells International 13

[51] D G Phinney G Kopen R L Isaacson and D J ProckopldquoPlastic adherent stromal cells from the bone marrow ofcommonly used strains of inbred mice variations in yieldgrowth and differentiationrdquo Journal of Cellular Biochemistryvol 72 no 4 pp 570ndash585 1999

[52] N Sasaki T Sasamura H O Ishikawa et al ldquoPolarized exo-cytosis and transcytosis of Notch during its apical localizationin Drosophila epithelial cellsrdquo Genes to Cells vol 12 no 1 pp89ndash103 2007

[53] M Handler X Yang and J Shen ldquoPresenilin-1 regulatesneuronal differentiation during neurogenesisrdquo Developmentvol 127 no 12 pp 2593ndash2606 2000

[54] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006

[55] A W Duncan F M Rattis L N DiMascio et al ldquoIntegrationof Notch andWnt signaling in hematopoietic stem cell mainte-nancerdquo Nature Immunology vol 6 no 3 pp 314ndash322 2005

[56] N Carlesso J C Aster J Sklar and D T Scadden ldquoNotch1-induced delay of human hematopoietic progenitor cell differen-tiation is associated with altered cell cycle kineticsrdquo Blood vol93 no 3 pp 838ndash848 1999

[57] I Maillard U Koch A Dumortier et al ldquoCanonical notch sig-naling is dispensable for the maintenance of adult hematopoi-etic stem cellsrdquo Cell Stem Cell vol 2 no 4 pp 356ndash366 2008

[58] G Pearson F Robinson T B Gibson et al ldquoMitogen-activatedprotein (MAP) kinase pathways regulation and physiologicalfunctionsrdquo Endocrine Reviews vol 22 no 2 pp 153ndash183 2001

[59] R E Dolmetsch R S Lewis C C Goodnow and J I HealyldquoDifferential activation of transcription factors induced by Ca2+response amplitude and durationrdquoNature vol 386 no 6627 pp855ndash858 1997

[60] S Kawano S Shoji S Ichinose K Yamagata M Tagami andM Hiraoka ldquoCharacterization of Ca2+ signaling pathways inhuman mesenchymal stem cellsrdquo Cell Calcium vol 32 no 4pp 165ndash174 2002

[61] B Ye ldquoCa2+ oscillations and its transporters in mesenchymalstem cellsrdquo Physiological Research vol 59 no 3 pp 323ndash3292010

[62] J E Ip Y Wu J Huang L Zhang R E Pratt and V J DzauldquoMesenchymal stem cells use integrin 1205731 not CXC chemokinereceptor 4 for myocardial migration and engraftmentrdquoMolecu-lar Biology of the Cell vol 18 no 8 pp 2873ndash2882 2007

[63] C A Opitz U M Litzenburger C Lutz et al ldquoToll-like recep-tor engagement enhances the immunosuppressive propertiesof human bone marrow-derived mesenchymal stem cells byinducing indoleamine-23-dioxygenase-1 via Interferon-120573 andprotein kinase Rrdquo Stem Cells vol 27 no 4 pp 909ndash919 2009

[64] B Kulterer G Friedl A Jandrositz et al ldquoGene expressionprofiling of human mesenchymal stem cells derived from bonemarrow during expansion and osteoblast differentiationrdquo BMCGenomics vol 8 article 70 2007

[65] W G Li and X X Xu ldquoThe expression of N-cadherinfibronectin during chondrogenic differentiation of MSCinduced by TGF-1205731rdquo Chinese Journal of Traumatology vol 8no 6 pp 349ndash351 2005

[66] F Ng S Boucher S Koh et al ldquoPDGFTGF-120573 and FGF signal-ing is important for differentiation and growth of mesenchymalstem cells (mscs) transcriptional profiling can identify markersand signaling pathways important in differentiation of MSCsinto adipogenic chondrogenic and osteogenic lineagesrdquo Bloodvol 112 no 2 pp 295ndash307 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 Stem Cells International

Table 1 15 clusters obtained as a result of the unsupervised gene clusteringMean CD34minus BM (mean of CD34minus stem cells from bonemarrow)Mean CD34minus UCB (mean of CD34minus stem cells from umbilical cord blood)119873 (a number of genes in a cluster) SRL (Signal Log Ratio)

Cluster MeanCD34minus BM

MeanCD34minusUCB

SDCD34minusBM

SDCD34minusUCB

119873

119901 value MeanSLR

minus95CISLR

+95CISLR

Conclusion

1497 491 008 009

1102

000000023 minus006 minus008 minus004

No change2

1157 1149 035 047

54

026488907 minus008 minus022 006

No change3

802 805 030 032

592

063190010 001 minus005 007

No change4

445 440 006 005

999

000000001 minus006 minus008 minus004

No change5

702 714 024 021

898

000000009 011 007 016

No change6

554 550 012 013

1133

000000889 minus006 minus009 minus003

No change7

353 346 004 005

960

451861119864 minus 13 minus007 minus009 minus005

No change8

476 483 071 088

961

003102164 011 001 020

No change9 564 715 158 270 493 p lt 00000001 306 295 316 Up10

311 309 004 004

872

000325256 minus003 minus005 minus001

No change11

276 278 002 002

647

000002947 003 001 004

No change12

398 390 006 007

1133

544009119864 minus 15 minus008 minus010 minus006

No change13 739 486 285 101 387 p lt 00000001 minus343 minus357 minus329 Down14

621 621 026 021

1353

044902052 minus001 minus005 002

No change15

930 936 064 084

395

015369595 008 minus003 020

No change

0 001 002 003 004

Signal transduction (GO0007165)

Activation of MAPK activity (GO0000187)

Protein kinase cascade (GO0007243)

Regulation of MAP kinase activity (GO0043405)

Integrin-mediated signaling pathway (GO0007229)

Positive regulation of tyrosine phosphorylation ofSTAT protein (GO0042531)

Positive regulation of calcium-mediated signaling(GO0050850)

JAK-STAT cascade (GO0007259)

Cytokine-mediated signaling pathway (GO0019221)

Positive regulation of cell proliferation (GO0008284)

Calcium-mediated signaling (GO0019722)

Regulation of JAK-STAT cascade (GO0046425)

p value

Signaling pathway

Figure 2 Functional annotations 12 GO classification terms for biological processes in the group signaling pathways in cluster 9 dependingon the 119901 value (119901 lt 004)

regulation cellular homeostasis cellular defensive responseand response to stimuli cytokine production and signalingpathways The GO terms regarding signaling pathways arepresented in Figure 2

Thenext stage consisted of a similar analysis of genes fromcluster 13 GO system classification for 387 genes of cluster 13determined 119 terms of biological processes (119901 lt 005) of

a decreased activity in CD34minus cells fromumbilical cord bloodas compared with CD34minus cells from bone marrow The GOterms of 119901 value below 004 underwent further analysis As aresult of this analysis the majority of GO terms representedonly by one or two genes were rejected The GO terms forbiological processes in cluster 13 were assigned to one ofthree functional groups including metabolism biological

Stem Cells International 7

0000 0010 0020 0030

Negative regulation of signal transduction (GO0009968)

Vascular endothelial growth factor receptor signalingpathway (GO0048010)

Transmembrane receptor protein tyrosine kinase signalingpathway (GO0007169)

Transmembrane receptor protein serinethreonine kinasesignaling pathway (GO0007178)

Regulation of transforming growth factor beta receptorsignaling pathway (GO0017015)

Cell surface receptor linked signal transduction(GO0007166)

BMP signaling pathway (GO0030509)

p value

Signaling pathway

Figure 3 Functional annotations 7 GO classification terms for biological processes in the group signaling pathways in cluster 13 dependingon the 119901 value (119901 lt 004)

Table 2 Gene pathways based on the KEGG classification of an increased expression in CD34minus stem cells from umbilical cord blood

KEGGID 119901 value Odds ratio Exp count Count Size Term4060 0001 2947 14 33 223 Cytokine-cytokine receptor interaction4620 0001 3672 6 17 92 Toll-like receptor signaling pathway5120 0001 3748 4 12 63 Epithelial cell signaling inHelicobacter pylori infection4630 0012 2067 8 15 130 JAK-STAT signaling pathway

processes regulation and signaling pathways In CD34minus cellsfrom umbilical cord blood as compared with CD34minus cellsfrom bone marrow a decreased gene activity was observedof the BMP signaling pathway TGF beta transmembranereceptor of protein serinethreonine kinase transmembranereceptor of tyrosine kinase and VEGF (Figure 3)

Gene pathways whose components are genes belongingto specific clusters underwent an additional analysis whichwas independent of a direct description of cellular processesFor this purpose data from the KEGG database (KyotoEncyclopedia of Genes and Genomes) were used For eachpathway the hypothesis of overrepresentation of specificclusters of genes as compared with the whole scope ofanalyzed genes was verified and precise 119901 values weredetermined For the purpose of this study gene pathwaysfor clusters 9 and 13 were analyzed because they containedgenes discriminating both gene populations On the basisof KEGG database cytokine-cytokine receptor interactionpathway toll-like receptor signaling pathway epithelial cellsignaling in Helicobacter pylori infection and JAK-STATsignaling pathway are overrepresented in CD34minus cells fromumbilical cord blood (Table 2)

In cluster 13 on the basis of KEGG classification theCD34minus cells from umbilical cord blood show a decreasedactivity of focal adhesion signaling pathway cell junctionsand TGF-beta signaling pathway (Table 3)

At the next stage the genes of the remaining clustersnumbered 1ndash8 10ndash12 and 14 of a similar gene expression were

analyzed The analysis was based on the GO classification foreach of the abovementioned clusters The criteria used forchoosing the clusters included the biological processes con-nected with cellular cycle as well as the processes connectedwith signaling pathways activity A similar gene expression inCD34minus cells from umbilical cord blood and CD34minus cells frombone marrow was characteristic for such biological processesas cell division mitosis maintaining telomere endings RNAandDNA processing and a similar activity of Notch andWntsignaling pathways genes

An independent analysis was also carried out for genes ofa similar activity in clusters 1ndash8 10ndash12 and 14 on the basis ofthe KEGG database CD34minus cells from umbilical cord bloodand CD34minus cells from bone marrow showed similar activityof Wnt and Notch signaling pathways genes p53 signalingpathway genes cell cycle PPAR and mTOR (Table 4)

CD34minus cells from umbilical cord blood and CD34minus cellsfrom bone marrow showed similar gene activity of Wntand Notch signaling pathways both on the basis of the GOclassification system and the KEGG database Therefore atthe last stage of the study the activity of single genes of Wntand Notch as well as JAK-STAT signaling pathways chosenfrom 11976 sampled genes was analyzed

In accordance with the KEGG database the JAK-STATsignaling pathway is defined by 155 genes out of whichthere were 128 active genes in the studied material Genesexhibiting increased activity in CD34minus cells from umbilicalcord blood included SPRY2 CISH CSF2 GRB2 IFNGR1

8 Stem Cells International

Table 3 Gene pathways based on the KEGG classification of a decreased expression in CD34minus stem cells from umbilical cord blood

KEGGID 119901 value Odds ratio Exp count Count Size Term4510 0001 7517 7 33 188 Focal adhesion1430 0001 8157 4 22 109 Cell junctions4350 0001 4038 3 10 82 TGF-beta signaling pathway

Table 4 Gene pathways based on the KEGG classification of a similar expression in CD34minus stem cells from umbilical cord blood and CD34minuscells from bone marrow for clusters 1ndash8 10ndash12 and 14-15

KEGGID 119901 value Odds ratio Exp count Count Size Term04310 0018 1880 10 17 131 Wnt signaling pathway04330 0025 2722 3 7 39 Notch signaling pathway04115 0038 2157 5 9 61 p53 signaling pathway04110 0009 2028 10 18 100 Cell cycle03320 0020 2791 3 7 60 PPAR signaling pathway04150 0035 2220 5 9 46 mTOR signaling pathway

IL2RB IL2RG IL7R IL10RA IL15RA PIM1 PTPN6 STAT1STAT5A and CCND3 Genes exhibiting decreased activityincluded PIAS3 IL11RA and CBLB

On the basis of the KEGG database the Wnt signalingpathway is defined by 151 genes out of which there were132 active genes in the studied material Genes exhibit-ing increased activity in CD34minus cells from umbilical cordblood include MMP7 PRKCB PSEN1 RAC2 and CCND3Genes exhibiting decreased activity include DKK1 WNT5AWNT5B FZD7 and TCF7L1

On the basis of the KEGG database the Notch signalingpathway is defined by 47 genes out of which there were38 active genes in the studied material Genes exhibitingincreased activity in CD34minus cells from umbilical cord bloodincluded DTX4 and PSEN1

4 Discussion

Nonhematopoietic stem cells were first identified by Frieden-stein et al who described the progenitor cells of rat bonemarrow [21] Currently the nonhematopoietic stem cellswhich are frequently referred to although not quite correctlyas the mesenchymal stem cells are isolated from varioustissues including the umbilical cord blood placenta andumbilical cord A constantly increasing number of studieson MSCs have proved that in appropriate environmentalconditions they are capable of differentiating into special-ized mesodermal endodermal and even ectodermal celllineages [2 22ndash24] In natural conditions they play an impor-tant role in cell regeneration processes and in repairingthe tissues damaged by degenerating processes Anotherintriguing characteristic of MSCs is their ability to avoidimmunological identification and response therefore theyare referred to as the hypoimmunogenic cells Despite greatattention paid to mesenchymal stem cells their biology isnot yet fully understood Introducing multipotential stemcells into clinical application requires a full understandingof the mechanisms which control key properties of thecell such as cell self-replication versus differentiation and

mobilization versus tissue homing The understanding ofthese mechanisms must be based on strict definitions ofhuman multipotential stem cells at the molecular level Inaddition understanding of molecular mechanisms of self-replicating pluripotentiality plasticity and differentiation ofstem cells not only is a necessary condition to overcomelimitations of stem cells application as a therapeutic factorbut also is key to understand a fundamental mystery of thedevelopment of human organism

This paper presents a comparison of gene activity of thenonhematopoietic cells of umbilical cord blood and bonemarrow using oligonucleotidemicroarrays Gene activity wastested in stem cells CD34 and included 11 979 genes obtainedafter completing procedures to increase the purity of cellsamples Cell populations included in the study were alsolimited based on the noise threshold revealed in the finalcomparative analysis The gene expression profiles of thecompared populations of CD34minus stem cells from umbilicalcord blood and CD34minus stem cells from bone marrow werevery similar (Figure 1) For a better illustration of the biolog-ical molecular activity and the activity of signaling pathwaysin the studied cell populations a transcription profile analysiswith the GO and KEGG databases was completed In termsof fundamental signaling pathways for biological activityof stem cells like JAK-STAT Wnt and Notch we recordeddiscrete differences among expressed genes from umbilicalcord blood cells as compared to bone marrow cells

The analysis regarding signaling pathways showedincreased activity of the JAK-STAT pathway in CD34minusstem cells from umbilical cord blood both on the basis ofthe gene ontology classification and the KEGG database(Figure 2 and Table 2) The increased activity regarded 15genes which take part in the JAK-STAT cascade that isSPRY2 CISH CSF2 GRB2 IFNGR1 IL2RB IL2RG IL7RIL10RA IL15RA PIM1 PTPN6 and CCND3 including thegenes which code transcription factors STAT1 and STAT5AIn previous publications the significance of the JAK-STATsignaling pathway is best described in the embryonic andhematopoietic stem cells Ramalho-Santos et al searchedfor a transcription profile describing both the embryonic

Stem Cells International 9

stem cells and the stem cells of an adult organism on thebasis of the study of embryonic cells neural stem cellsand hematopoietic stem cells activity [25] These studiesattempted to create a molecular signature of the stem cells inrelation to the self-regeneration and differentiation abilities ofthe cells The active JAK-STAT pathway belongs to the basicattributes defining the so-called ldquostemnessrdquo phenomenon Inthe embryonic stem cells the JAK-STAT signaling pathwaypromotes proliferation in the form of self-replicationThe activating factor LIF (Leukemia Inhibitory Factor)provokes phosphorylation and dimerisation of STAT3which after translocation to the cell nucleus stimulates theexpression of gene group responsible for self-replication ofthe embryonic stem cell However the JAK-STAT signalingpathway is not the only factor responsible for proliferationregulation of the embryonic stem cells Internal processesregulating the multiplication processes independent of theSTAT3 activity such as the Nanog and Oct-4 activity arealso important [15 17 26 27] In our study material theNanog and Oct-4 genes were not active in both types ofnonhematopoietic stem cells which is in agreement withother publications devoted to this subject [28 29] TheSTAT1 transcription factor overactive in our study in CD34minuscells of umbilical cord blood is responsible among othersfor the expression of interferon-activated genes involved incell defense against pathogenic factors [30] Moreover itis believed that STAT1 is a medium in expression of genegroup which is important for cell viability in response tostimulating factors In 2006 Kimrsquos team described STAT1as a highly overactive gene in human mesenchymal stemcells [31] In comparison the transcription factor STAT5which functions in the form of two isoforms STAT5a andSTAT5b is involved in the cellular proproliferation responsesand antiapoptosis processes [32 33] STAT5 is activatedby various cytokine receptors In our study the isoformSTAT5A showed increased activity It is believed that theSTAT5 factor activates specifically the cellular cycle genesthat is cyclins [34 35] In human stem cells the STAT factorsactivate the expression of cyclin Dwhich codes the regulatingsubunit of the CYCDCDK4 complex driving the G1S phasetransition which in turn activates the cell cycle progression[32 36] STAT5a and STAT5b also play an important rolein expression of genes promoting hematopoietic stem cellssurvival [37] Gene expression profiling over a range ofSTAT5 activities in human CB cells revealed subsets of genesthat are associated with the self-renewal and long-termexpansion phenotype Taking into account the fact thatin STAT5minusminus mice myelopoiesis appears to be relativelyunaffected it can be assumed that STAT5 can play a keyrole in assessing the quality of cells [38] Additionally notonly is the JAK-STAT signaling pathway a significant signaltransduction component which controls cell proliferationand purpose but it also regulates cell migration which isimportant both in the primary processes of gastrulationand in the tissue regeneration For instance the JAK-STATsignaling pathway is connected with cardioprotection [39]as well as with processes of regeneration and remodeling ofskeleton [40 41] The active JAK-STAT signaling pathwayhelps to regulate cell organization within cell grouping

[42] The above results may suggest a greater self-renewalpotential of CD34minus stem cells of umbilical cord bloodlong-term expansion phenotype and their higher ability interms of tissue regeneration

The Wnt signaling pathway is perceived as a signalingcascade involved in the control of fundamental processesof nondifferentiated mesenchymal stem cells such as cellreproduction and directed differentiation Both methods ofgene activity classification that is GO and KEGG showeda similar activity of the majority of Wnt signaling pathwaygenes (Table 4) In addition the analysis of separate genestaking part in the Wnt signaling pathway and coding 19glycosylated molecules of extracellular signaling of the Wntfamily cellular surface receptors including Frizzled receptorsintracellular signaling molecules and genes involved in cellcycle and taking part in regulation of proliferation growthand modification of proteins showed that the expressionprofilewas very similar except for single genes of an increasedand decreased expression in CD34minus stem cells of umbilicalcord blood in our study that is MMP7 PRKCB PSEN1RAC2 and CCND3 respectively as well as DKK1 WNT5AWNT5B FZD7 and TCF7L1 The Wnt5a ligand is involvedin the noncanonical Wnt signaling pathway transmissionand it is assumed that it may inhibit the canonical Wntpathway in mouse embryonic cells similarly to Dkk1 Therole of the noncanonical Wnt signaling pathway which inour results seemed to prevail in CD34minus stem cells of bonemarrow is not clear It is believed that the noncanonical Wntsignaling pathway controls the reorganization of cytoskeletonmuscle proteins tissue polarity and cell movement [43] Incontrast the canonical signaling pathway which activatestarget gene transcription through the increase of beta-cateninconcentration seems to be more active in CD34minus stemcells from umbilical cord cells Its role is to control thetissue-specific cell purpose during embryogenesis as well asregulation of proliferation in adult tissues [44] Additionallyhigh activity of WNT5A gene is noticeable in the humanMSCs differentiation into osteoblasts as well as in early stagesof adipogenesis [45 46] According to the publication ofYang et al the Wnt5a and Wnt5b glycoproteins are alsoresponsible for coordination of chondrocyte proliferation anddifferentiation [47] Wnt5a is believed to be an importantfactor taking part in the positive autocrinic regulation ofHSC repopulation ability [48] In their publication Prockopet al suggest that a high WNT5A expression in MSCs ischaracteristic for the end of the exponential and stationarygrowth phases in cell cultures in in vitro conditions [49]Slight changes in theWnt gene activity especially inWNT5Aactivity may result from the role played by MSCs in theirenvironmental niche Bone marrow MSCs seem to be moreinvolved in the renewal processes of the progenitor cells poolwhich reside in bone marrow The researches show that theexpression of Wnt ligands and Fz receptors in MSCs showssome correlation of WntFz pairs such as Wnt5aFz5 [50]The above discussed study results showed that there wasa coexpression of WNT5AFZD7 Their higher expressionwas characteristic for the bone marrow cells while thepreviously published data suggested differences in WntFzactivity depending on the origin-mouse versus humanMSCs

10 Stem Cells International

[51] Currently available data suggest differences which maybe characteristic not only for proliferation and differentiationprograms but also for the phenotype of MSCs from umbilicalcord blood and bone marrow which is in agreement withthe results published by Baksh et al [7] The remaining genestake part in theWnt signaling cascade of increased activity inCD34minus from umbilical cord blood such as MMP7 belongingto metalloproteinases which play a role in the extracellulararray remodeling and wound healing [5]

The Notch signaling pathway belongs to the processes ofintercellular communication which regulate gene expressionpatterns defining a dualistic character of stem cells reflectedby the asymmetric cell division [52] Additionally the Notchsignaling cascade controls various cells biological processesnot only proliferation and differentiation but also apoptosisand regeneration The signaling activity of Notch pathwaygenes determined in the study of 38 genes showed theirsimilar expression as defined by the KEGG classification(Table 4) A similar activity level was noted for all four Notch1ndash4 receptors as well as for JAG1 and JAG2 ligands The dif-ferences were related to two genes DTX4 and PSEN1 whichwere overactive in CD34minus cells from umbilical cord bloodThe function of the PSEN1 gene is mainly developmentalprocesses regarding cell differentiation during neurogenesisAnd the lack of this gene leads to a premature directeddifferentiation of neural progenitor cells [53] The activity ofthe Notch gene cascade plays an important role at all levelsof stem cell development starting from the embryonic stemcell through fetal cells to adult organism stem cells Notchis significantly involved in the asymmetric segregation offactors determining cell purpose during replication divisionsof the stem cell poolThe significance of receptors and ligandsis best described in the hematopoietic stem cells Expressionof the constitutively active Notch1 gene in hematopoieticstem cells induces the immortalized cell lineages which canreproduce the myeloid and lymphoid cell lineage in a long-term repopulation in animal studies [54 55] Apart from thatit delays differentiation of human hematopoietic progenitorstem cells in vitro [56] The studies suggest that Notch1 is animportant factor inHSC self-renewal and can also participatein the inhibition of stem cell differentiation The scientificfindings reveal that a suppression of the Notch signalingpathway via deletion of key genes in HSC did not cause cellsto lose their ability of long-term hematopoiesis renewal inanimal models [57] Therefore the role of ligands and Notchreceptors in HSC has not been precisely defined yet andcurrent publications do not provide precise data regardingthe role played by particular Notch signaling pathway genesin mesenchymal stem cells The only phenomenon whichappears to be superior and characteristic for all stem cellsincluding MSCs is the significance of the Notch cascadein asymmetrical cell divisions This is a significant com-ponent of stem cells population self-renewal as well asthe functionality of the cascade in the stem niche Thisphenomenon constitutes an integral part of the hypothesisabout stem cell population aging However further inves-tigation will be required to determine if the differentiallyexpressed genes described in the studies above could serve asstemness genes

Remaining signaling pathways which revealed increasedactivity in the studied CD34minus cells of umbilical cord bloodincludedMAPK (Mitogen-Activated Protein Kinase Figure 2)which regulates a wide range of cell biological activities suchas gene expression mitosis proliferation differentiationmigration and apoptosis processes [58]TheMAPKpathwaystake part in regulation of early embryonic development andin defining the role of embryonic stem cells from the earlydevelopmental phases until the moment of formation ofdifferentiated adult cells However this precise role played bythe MAPK pathways in various stem cell types has not yetbeen defined

Transmission via calcium ions was also singled out inthe group of the overactive signaling pathways of the studiedCD34minus stem cells of umbilical cord blood versus CD34minus stemcells of bone marrow (Figure 2) The ions are a vital andcommonly present mediator in the process of extracellularsignals transmission and conversion into a cellular responseVariations of calcium ions concentrations regulate geneexpression through an impact on the activity of numeroussignaling pathways Intracellular increase of calcium ionsconcentration also regulates cell activities including adhe-sion motility gene expression and proliferation [59] Thesignificance of transmission via calcium ions and the ways oftheir concentration changes in mesenchymal stem cells stillhave not been studied Kawano et al were the first to reportthat in the nondifferentiated MSCs there are spontaneouschanges of calcium ions concentrations despite the lack ofany stimulating factors [60] As reported the calcium iontransmission through the activation of different gene groupsmay direct the cells onto specific developmental pathways[61]

The integrin-mediated signaling pathway takes part inextracellular array remodeling which means that it con-trols cell behavior and tissue organization Its increasedactivity was observed in our study in CD34minus stem cells ofumbilical cord blood (Figure 2) In recent years the role ofintegrins and selectins has been stressed in the adhesion andtransendothelial migration which has a significant meaningin the processes of repopulation of tissues damaged bydiseases and their regeneration via the circulating MSCs Forinstance Ip et al showed that blocking of beta 1-integrinwhich constitutes a component of the adhesive moleculeVLA-4 in MSC weakened the ability of mesenchymal stemcells to engraft into the infarction-changedmyocardium [62]The above results may suggest a better migration abilityof CD34minus stem cells of umbilical cord blood into the areaof pathologic tissue MSCs own cytokinetic activity thathas similar significance in terms of tissue regenerationThe study of cytokinetic activity of CD34minus stem cells ofumbilical cord blood showed increased activity of signalingpathways activated via cytokines (Figure 2 Table 2) On theother hand the TLR signaling pathway (toll-like receptorsignaling) which in our own results revealed a significantincreased activity in CD34minus stem cells of umbilical cordblood (Figure 2) increases the immunosuppressive charac-teristics of MSCs Weakening of the immunological responsegenerated by the TLR pathway depends on the produc-tion of immunosuppressive kynurenines by the indoleamine

Stem Cells International 11

23-dioxygenase enzyme which degrades tryptophan Induc-tion of indoleamine 23-dioxygenase by TLR entails autocrinesignaling loop of interferon-beta which plays a role in MSCsimmunomodulative effects [63]

According to the GO and KEGG classifications carriedout for cluster 13 with the gene pool of a decreased expressiona statistically significant decrease of gene activity was shownfor the TGF-beta and BMP pathways (Figure 3 Table 3) Itis believed that the family of TGF-beta members whichalso includes BMP plays an important role in initiating theprocess of a directed MSCs differentiation TGF-beta sig-naling transmission runs through specific serine-threoninekinase receptors and their nuclear receptors called the Smadproteins TGF-beta signaling transmission promotes theprocess of early differentiation stages into the chondrogenicand osteogenic lineages [64 65] However cell differentiatinginto the adipogenic lineage results in inhibition of the TGF-beta signaling pathway This was confirmed in experimentalstudies developed by Ng et al where the inhibition of TGF-beta transmission resulted in an increase of adipogenesisand a decrease of differentiating ability towards chondrocytes[66] The BMP signaling pathway strongly promotes differ-entiation towards osteoblasts but it also leads to inhibitionof myogenesis and adipogenesis Such a gene expressionprofile may suggest that the MSCs biological characteristicsare strongly influenced by the significance of stem cell nicheTherefore the mesenchymal stem cells from bone marrowmay be more involved in the promotion of the phenomenawhich maintain the stability of bone marrow environment

5 Conclusions

Multipotentiality of the mesenchymal stem cells their highproliferation ability and not too-demanding culturing con-ditions reveal their potential for wide application in variousmedical areas These are the reasons for the common pursuitto understand their biology based onmore andmore sublimebiomolecular procedures Scientists have been searchingfor an unequivocal marker which would characterize themesenchymal stem cells and simultaneously would enable aseparation of a homogenous MSC population Understand-ing of molecular mechanisms controlling self-replication anddifferentiation is becoming a fundamental issue for clinicalapplication of MSC In this respect the activity of signalingpathways is important yet the interpretation of the pathwaysespecially their interdependence constitutes a great challengefor the scientific world Moreover the answer to the questionregarding MSC innidation and its survival ability in hosttissues after transplantation is yet to be found Explanationof the abovementioned issues will require a more globalunderstanding of biological activity of these cells especiallyon the basis of the significance of the mesenchymal nicheenvironment of stem cells and its impact on the gene andepigenetic activity It should be assumed that continuedprogress in the understanding of MSCs will finally lead totheir common therapeutic and preventive application andconsequently to the improvement of the quality and lengthof human life

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] R J Deans andA BMoseley ldquoMesenchymal stem cells biologyand potential clinical usesrdquo Experimental Hematology vol 28no 8 pp 875ndash884 2000

[2] M F Pittenger A M Mackay S C Beck et al ldquoMultilineagepotential of adult human mesenchymal stem cellsrdquo Science vol284 no 5411 pp 143ndash147 1999

[3] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006

[4] Y-J Chang D T-B Shih C-P Tseng T-B Hsieh D-C Leeand S-M Hwang ldquoDisparate mesenchyme-lineage tendenciesin mesenchymal stem cells from human bone marrow andumbilical cord bloodrdquo Stem Cells vol 24 no 3 pp 679ndash6852006

[5] R A Panepucci J L C Siufi W A Silva Jr et al ldquoComparisonof gene expression of umbilical cord vein and bone marrowndashderived mesenchymal stem cellsrdquo STEM CELLS vol 22 no 7pp 1263ndash1278 2004

[6] C K Rebelatto A M Aguiar M P Moretao et al ldquoDissimilardifferentiation of mesenchymal stem cells from bone marrowumbilical cord blood and adipose tissuerdquo Experimental Biologyand Medicine vol 233 no 7 pp 901ndash913 2008

[7] D Baksh R Yao and R S Tuan ldquoComparison of proliferativeand multilineage differentiation potential of human mesenchy-mal stem cells derived from umbilical cord and bone marrowrdquoStem Cells vol 25 no 6 pp 1384ndash1392 2007

[8] D L Clarke C B Johansson J Wilbertz et al ldquoGeneralizedpotential of adult neural stem cellsrdquo Science vol 288 no 5471pp 1660ndash1663 2000

[9] D J Anderson F H Gage and I L Weissman ldquoCan stem cellscross lineage boundariesrdquo Nature Medicine vol 7 no 4 pp393ndash395 2001

[10] D L Stemple and D J Anderson ldquoIsolation of a stem cell forneurons and glia from the mammalian neural crestrdquo Cell vol71 no 6 pp 973ndash985 1992

[11] R L Rietze H Valcanis G F Brooker T Thomas A K Vossand P F Bartlett ldquoPurification of a pluripotent neural stem cellfrom the adult mouse brainrdquoNature vol 412 no 6848 pp 736ndash739 2001

[12] R M Seaberg S R Smukler T J Kieffer et al ldquoClonalidentification of multipotent precursors from adult mousepancreas that generate neural and pancreatic lineagesrdquo NatureBiotechnology vol 22 no 9 pp 1115ndash1124 2004

[13] A P Beltrami L Barlucchi D Torella et al ldquoAdult cardiac stemcells are multipotent and support myocardial regenerationrdquoCell vol 114 no 6 pp 763ndash776 2003

[14] L Yang S Li H Hatch et al ldquoIn vitro trans-differentiation ofadult hepatic stem cells into pancreatic endocrine hormone-producing cellsrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 99 no 12 pp 8078ndash80832002

[15] I Chambers D Colby M Robertson et al ldquoFunctional expres-sion cloning of Nanog a pluripotency sustaining factor inembryonic stem cellsrdquo Cell vol 113 no 5 pp 643ndash655 2003

12 Stem Cells International

[16] M Dai P Wang A D Boyd et al ldquoEvolving genetranscriptdefinitions significantly alter the interpretation of GeneChipdatardquo Nucleic Acids Research vol 33 pp 175ndash179 2005

[17] RA Irizarry BHobbs F Collin et al ldquoExploration normaliza-tion and summaries of high density oligonucleotide array probelevel datardquo Biostatistics vol 4 no 2 pp 249ndash264 2003

[18] R B Dean and W J Dixon ldquoSimplified statistics for smallnumbers of observationsrdquo Analytical Chemistry vol 23 no 4pp 636ndash638 1951

[19] G J McLachlan and D Peel ldquoOn computational aspectsof clustering via mixtures of normal and t-componentsrdquo inProceedings of the American Statistical Association (BayesianStatistical Science Section) American Statistical AssociationIndianapolis Ind USA 2000

[20] J Polanska P Widlak J Rzeszowska-Wolny M Kimmel andA Polanski ldquoGaussian mixture decomposition of time-courseDNA microarray datardquo in Mathematical Modeling of BiologicalSystems Volume I pp 351ndash359 Birkhauser Boston Mass USA2007

[21] A J Friedenstein U F Gorskaja and N N Kulagina ldquoFibrob-last precursors in normal and irradiated mouse hematopoieticorgansrdquo Experimental Hematology vol 4 no 5 pp 267ndash2741976

[22] S Wakitani T Saito and A I Caplan ldquoMyogenic cells derivedfrom rat bone marrow mesenchymal stem cells exposed to 5-azacytidinerdquoMuscle ampNerve vol 18 no 12 pp 1417ndash1426 1995

[23] D Woodbury E J Schwarz D J Prockop and I B BlackldquoAdult rat and human bone marrow stromal cells differentiateinto neuronsrdquo Journal of Neuroscience Research vol 61 no 4pp 364ndash370 2000

[24] M Reyes T Lund T Lenvik D Aguiar L Koodie and CM Verfaillie ldquoPurification and ex vivo expansion of postnatalhumanmarrowmesodermal progenitor cellsrdquoBlood vol 98 no9 pp 2615ndash2625 2001

[25] M Ramalho-Santos S Yoon Y Matsuzaki R C Mulliganand D A Melton ldquolsquoStemnessrsquo transcriptional profiling ofembryonic and adult stem cellsrdquo Science vol 298 no 5593 pp597ndash600 2002

[26] KMitsui Y Tokuzawa H Itoh et al ldquoThe homeoprotein nanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003

[27] S Okumura-Nakanishi M Saito H Niwa and F IshikawaldquoOct-34 and Sox2 regulate Oct-34 gene in embryonic stemcellsrdquo The Journal of Biological Chemistry vol 280 no 7 pp5307ndash5317 2005

[28] F Ulloa-Montoya B L Kidder K A Pauwelyn et al ldquoCompar-ative transcriptome analysis of embryonic and adult stem cellswith extended and limited differentiation capacityrdquo GenomeBiology vol 8 no 8 article R163 2007

[29] L Song N EWebb Y Song and R S Tuan ldquoIdentification andfunctional analysis of candidate genes regulating mesenchymalstem cell self-renewal and multipotencyrdquo STEMCELLS vol 24no 7 pp 1707ndash1718 2006

[30] A H H Van Boxel-Dezaire M R S Rani and G R StarkldquoComplexmodulation of cell type-specific signaling in responseto type I interferonsrdquo Immunity vol 25 no 3 pp 361ndash372 2006

[31] C G Kim J J Lee D Y Jung et al ldquoProfiling of differentiallyexpressed genes in human stem cells by cDNA microarrayrdquoMolecules and Cells vol 21 no 3 pp 343ndash355 2006

[32] T Bowman R Garcia J Turkson and R Jove ldquoSTATs inoncogenesisrdquo Oncogene vol 19 no 21 pp 2474ndash2488 2000

[33] A Stephanou andD S Latchman ldquoOpposing actions of STAT-1and STAT-3rdquo Growth Factors vol 23 no 3 pp 177ndash182 2005

[34] A Martino J H Holmes J D Lord J J Moon and B HNelson ldquoStat5 and Sp1 regulate transcription of the cyclin D2gene in response to IL-2rdquo The Journal of Immunology vol 166no 3 pp 1723ndash1729 2001

[35] I Matsumura T Kitamura H Wakao et al ldquoTranscriptionalregulation of the cyclin D1 promoter by STAT5 its involvementin cytokine-dependent growth of hematopoietic cellsrdquo TheEMBO Journal vol 18 no 5 pp 1367ndash1377 1999

[36] V Calo M Migliavacca V Bazan et al ldquoSTAT proteins fromnormal control of cellular events to tumorigenesisrdquo Journal ofCellular Physiology vol 197 no 2 pp 157ndash168 2003

[37] M Socolovsky A E J Fallon S Wang C Brugnara and HF Lodish ldquoFetal anemia and apoptosis of red cell progenitorsin Stat5aminusminus5bminusminus mice a direct role for stat5 in Bcl-X(L)inductionrdquo Cell vol 98 no 2 pp 181ndash191 1999

[38] G Li Z Wang Y Zhang et al ldquoSTAT5 requires the N-domainto maintain hematopoietic stem cell repopulating function andappropriate lymphoid-myeloid lineage outputrdquo ExperimentalHematology vol 35 no 11 pp 1684ndash1694 2007

[39] S Negoro K Kunisada E Tone et al ldquoActivation of JAKSTATpathway transduces cytoprotective signal in rat acute myocar-dial infarctionrdquo Cardiovascular Research vol 47 no 4 pp 797ndash805 2000

[40] H C Blair L J Robinson and M Zaidi ldquoOsteoclast signallingpathwaysrdquo Biochemical and Biophysical Research Communica-tions vol 328 no 3 pp 728ndash738 2005

[41] C J Malemud ldquoProtein kinases in chondrocyte signaling andosteoarthritisrdquo Clinical Orthopaedics and Related Research no427 supplement pp S145ndashS151 2004

[42] E M Pinheiro and D J Montell ldquoRequirement for Par-6 andBazooka inDrosophila border cell migrationrdquoDevelopment vol131 no 21 pp 5243ndash5251 2004

[43] D Strutt ldquoFrizzled signalling and cell polarisation inDrosophilaand vertebratesrdquo Development vol 130 no 19 pp 4501ndash45132003

[44] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[45] J Guo J Jin and L F Cooper ldquoDissection of sets of genes thatcontrol the character of wnt5a-deficient mouse calvarial cellsrdquoBone vol 43 no 5 pp 961ndash971 2008

[46] M Nishizuka A Koyanagi S Osada and M Imagawa ldquoWnt4and Wnt5a promote adipocyte differentiationrdquo FEBS Lettersvol 582 no 21-22 pp 3201ndash3205 2008

[47] Y Yang L Topol H Lee and J Wu ldquoWnt5a andWnt5b exhibitdistinct activities in coordinating chondrocyte proliferation anddifferentiationrdquo Development vol 130 no 5 pp 1003ndash10152003

[48] D J Van Den Berg A K Sharma E Bruno and R Hoff-man ldquoRole of members of the Wnt gene family in humanhematopoiesisrdquo Blood vol 92 no 9 pp 3189ndash3202 1998

[49] D J Prockop C A Gregory and J L Spees ldquoOne strategy forcell and gene therapy harnessing the power of adult stemcells torepair tissuesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 1 pp 11917ndash119232003

[50] X He J-P Saint-Jeannet Y Wang J Nathans I Dawid and HVarmus ldquoA member of the Frizzled protein family mediatingaxis induction byWnt-5Ardquo Science vol 275 no 5306 pp 1652ndash1654 1997

Stem Cells International 13

[51] D G Phinney G Kopen R L Isaacson and D J ProckopldquoPlastic adherent stromal cells from the bone marrow ofcommonly used strains of inbred mice variations in yieldgrowth and differentiationrdquo Journal of Cellular Biochemistryvol 72 no 4 pp 570ndash585 1999

[52] N Sasaki T Sasamura H O Ishikawa et al ldquoPolarized exo-cytosis and transcytosis of Notch during its apical localizationin Drosophila epithelial cellsrdquo Genes to Cells vol 12 no 1 pp89ndash103 2007

[53] M Handler X Yang and J Shen ldquoPresenilin-1 regulatesneuronal differentiation during neurogenesisrdquo Developmentvol 127 no 12 pp 2593ndash2606 2000

[54] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006

[55] A W Duncan F M Rattis L N DiMascio et al ldquoIntegrationof Notch andWnt signaling in hematopoietic stem cell mainte-nancerdquo Nature Immunology vol 6 no 3 pp 314ndash322 2005

[56] N Carlesso J C Aster J Sklar and D T Scadden ldquoNotch1-induced delay of human hematopoietic progenitor cell differen-tiation is associated with altered cell cycle kineticsrdquo Blood vol93 no 3 pp 838ndash848 1999

[57] I Maillard U Koch A Dumortier et al ldquoCanonical notch sig-naling is dispensable for the maintenance of adult hematopoi-etic stem cellsrdquo Cell Stem Cell vol 2 no 4 pp 356ndash366 2008

[58] G Pearson F Robinson T B Gibson et al ldquoMitogen-activatedprotein (MAP) kinase pathways regulation and physiologicalfunctionsrdquo Endocrine Reviews vol 22 no 2 pp 153ndash183 2001

[59] R E Dolmetsch R S Lewis C C Goodnow and J I HealyldquoDifferential activation of transcription factors induced by Ca2+response amplitude and durationrdquoNature vol 386 no 6627 pp855ndash858 1997

[60] S Kawano S Shoji S Ichinose K Yamagata M Tagami andM Hiraoka ldquoCharacterization of Ca2+ signaling pathways inhuman mesenchymal stem cellsrdquo Cell Calcium vol 32 no 4pp 165ndash174 2002

[61] B Ye ldquoCa2+ oscillations and its transporters in mesenchymalstem cellsrdquo Physiological Research vol 59 no 3 pp 323ndash3292010

[62] J E Ip Y Wu J Huang L Zhang R E Pratt and V J DzauldquoMesenchymal stem cells use integrin 1205731 not CXC chemokinereceptor 4 for myocardial migration and engraftmentrdquoMolecu-lar Biology of the Cell vol 18 no 8 pp 2873ndash2882 2007

[63] C A Opitz U M Litzenburger C Lutz et al ldquoToll-like recep-tor engagement enhances the immunosuppressive propertiesof human bone marrow-derived mesenchymal stem cells byinducing indoleamine-23-dioxygenase-1 via Interferon-120573 andprotein kinase Rrdquo Stem Cells vol 27 no 4 pp 909ndash919 2009

[64] B Kulterer G Friedl A Jandrositz et al ldquoGene expressionprofiling of human mesenchymal stem cells derived from bonemarrow during expansion and osteoblast differentiationrdquo BMCGenomics vol 8 article 70 2007

[65] W G Li and X X Xu ldquoThe expression of N-cadherinfibronectin during chondrogenic differentiation of MSCinduced by TGF-1205731rdquo Chinese Journal of Traumatology vol 8no 6 pp 349ndash351 2005

[66] F Ng S Boucher S Koh et al ldquoPDGFTGF-120573 and FGF signal-ing is important for differentiation and growth of mesenchymalstem cells (mscs) transcriptional profiling can identify markersand signaling pathways important in differentiation of MSCsinto adipogenic chondrogenic and osteogenic lineagesrdquo Bloodvol 112 no 2 pp 295ndash307 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

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Molecular Biology International

GenomicsInternational Journal of

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BioinformaticsAdvances in

Marine BiologyJournal of

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Signal TransductionJournal of

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Evolutionary BiologyInternational Journal of

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Microbiology

Stem Cells International 7

0000 0010 0020 0030

Negative regulation of signal transduction (GO0009968)

Vascular endothelial growth factor receptor signalingpathway (GO0048010)

Transmembrane receptor protein tyrosine kinase signalingpathway (GO0007169)

Transmembrane receptor protein serinethreonine kinasesignaling pathway (GO0007178)

Regulation of transforming growth factor beta receptorsignaling pathway (GO0017015)

Cell surface receptor linked signal transduction(GO0007166)

BMP signaling pathway (GO0030509)

p value

Signaling pathway

Figure 3 Functional annotations 7 GO classification terms for biological processes in the group signaling pathways in cluster 13 dependingon the 119901 value (119901 lt 004)

Table 2 Gene pathways based on the KEGG classification of an increased expression in CD34minus stem cells from umbilical cord blood

KEGGID 119901 value Odds ratio Exp count Count Size Term4060 0001 2947 14 33 223 Cytokine-cytokine receptor interaction4620 0001 3672 6 17 92 Toll-like receptor signaling pathway5120 0001 3748 4 12 63 Epithelial cell signaling inHelicobacter pylori infection4630 0012 2067 8 15 130 JAK-STAT signaling pathway

processes regulation and signaling pathways In CD34minus cellsfrom umbilical cord blood as compared with CD34minus cellsfrom bone marrow a decreased gene activity was observedof the BMP signaling pathway TGF beta transmembranereceptor of protein serinethreonine kinase transmembranereceptor of tyrosine kinase and VEGF (Figure 3)

Gene pathways whose components are genes belongingto specific clusters underwent an additional analysis whichwas independent of a direct description of cellular processesFor this purpose data from the KEGG database (KyotoEncyclopedia of Genes and Genomes) were used For eachpathway the hypothesis of overrepresentation of specificclusters of genes as compared with the whole scope ofanalyzed genes was verified and precise 119901 values weredetermined For the purpose of this study gene pathwaysfor clusters 9 and 13 were analyzed because they containedgenes discriminating both gene populations On the basisof KEGG database cytokine-cytokine receptor interactionpathway toll-like receptor signaling pathway epithelial cellsignaling in Helicobacter pylori infection and JAK-STATsignaling pathway are overrepresented in CD34minus cells fromumbilical cord blood (Table 2)

In cluster 13 on the basis of KEGG classification theCD34minus cells from umbilical cord blood show a decreasedactivity of focal adhesion signaling pathway cell junctionsand TGF-beta signaling pathway (Table 3)

At the next stage the genes of the remaining clustersnumbered 1ndash8 10ndash12 and 14 of a similar gene expression were

analyzed The analysis was based on the GO classification foreach of the abovementioned clusters The criteria used forchoosing the clusters included the biological processes con-nected with cellular cycle as well as the processes connectedwith signaling pathways activity A similar gene expression inCD34minus cells from umbilical cord blood and CD34minus cells frombone marrow was characteristic for such biological processesas cell division mitosis maintaining telomere endings RNAandDNA processing and a similar activity of Notch andWntsignaling pathways genes

An independent analysis was also carried out for genes ofa similar activity in clusters 1ndash8 10ndash12 and 14 on the basis ofthe KEGG database CD34minus cells from umbilical cord bloodand CD34minus cells from bone marrow showed similar activityof Wnt and Notch signaling pathways genes p53 signalingpathway genes cell cycle PPAR and mTOR (Table 4)

CD34minus cells from umbilical cord blood and CD34minus cellsfrom bone marrow showed similar gene activity of Wntand Notch signaling pathways both on the basis of the GOclassification system and the KEGG database Therefore atthe last stage of the study the activity of single genes of Wntand Notch as well as JAK-STAT signaling pathways chosenfrom 11976 sampled genes was analyzed

In accordance with the KEGG database the JAK-STATsignaling pathway is defined by 155 genes out of whichthere were 128 active genes in the studied material Genesexhibiting increased activity in CD34minus cells from umbilicalcord blood included SPRY2 CISH CSF2 GRB2 IFNGR1

8 Stem Cells International

Table 3 Gene pathways based on the KEGG classification of a decreased expression in CD34minus stem cells from umbilical cord blood

KEGGID 119901 value Odds ratio Exp count Count Size Term4510 0001 7517 7 33 188 Focal adhesion1430 0001 8157 4 22 109 Cell junctions4350 0001 4038 3 10 82 TGF-beta signaling pathway

Table 4 Gene pathways based on the KEGG classification of a similar expression in CD34minus stem cells from umbilical cord blood and CD34minuscells from bone marrow for clusters 1ndash8 10ndash12 and 14-15

KEGGID 119901 value Odds ratio Exp count Count Size Term04310 0018 1880 10 17 131 Wnt signaling pathway04330 0025 2722 3 7 39 Notch signaling pathway04115 0038 2157 5 9 61 p53 signaling pathway04110 0009 2028 10 18 100 Cell cycle03320 0020 2791 3 7 60 PPAR signaling pathway04150 0035 2220 5 9 46 mTOR signaling pathway

IL2RB IL2RG IL7R IL10RA IL15RA PIM1 PTPN6 STAT1STAT5A and CCND3 Genes exhibiting decreased activityincluded PIAS3 IL11RA and CBLB

On the basis of the KEGG database the Wnt signalingpathway is defined by 151 genes out of which there were132 active genes in the studied material Genes exhibit-ing increased activity in CD34minus cells from umbilical cordblood include MMP7 PRKCB PSEN1 RAC2 and CCND3Genes exhibiting decreased activity include DKK1 WNT5AWNT5B FZD7 and TCF7L1

On the basis of the KEGG database the Notch signalingpathway is defined by 47 genes out of which there were38 active genes in the studied material Genes exhibitingincreased activity in CD34minus cells from umbilical cord bloodincluded DTX4 and PSEN1

4 Discussion

Nonhematopoietic stem cells were first identified by Frieden-stein et al who described the progenitor cells of rat bonemarrow [21] Currently the nonhematopoietic stem cellswhich are frequently referred to although not quite correctlyas the mesenchymal stem cells are isolated from varioustissues including the umbilical cord blood placenta andumbilical cord A constantly increasing number of studieson MSCs have proved that in appropriate environmentalconditions they are capable of differentiating into special-ized mesodermal endodermal and even ectodermal celllineages [2 22ndash24] In natural conditions they play an impor-tant role in cell regeneration processes and in repairingthe tissues damaged by degenerating processes Anotherintriguing characteristic of MSCs is their ability to avoidimmunological identification and response therefore theyare referred to as the hypoimmunogenic cells Despite greatattention paid to mesenchymal stem cells their biology isnot yet fully understood Introducing multipotential stemcells into clinical application requires a full understandingof the mechanisms which control key properties of thecell such as cell self-replication versus differentiation and

mobilization versus tissue homing The understanding ofthese mechanisms must be based on strict definitions ofhuman multipotential stem cells at the molecular level Inaddition understanding of molecular mechanisms of self-replicating pluripotentiality plasticity and differentiation ofstem cells not only is a necessary condition to overcomelimitations of stem cells application as a therapeutic factorbut also is key to understand a fundamental mystery of thedevelopment of human organism

This paper presents a comparison of gene activity of thenonhematopoietic cells of umbilical cord blood and bonemarrow using oligonucleotidemicroarrays Gene activity wastested in stem cells CD34 and included 11 979 genes obtainedafter completing procedures to increase the purity of cellsamples Cell populations included in the study were alsolimited based on the noise threshold revealed in the finalcomparative analysis The gene expression profiles of thecompared populations of CD34minus stem cells from umbilicalcord blood and CD34minus stem cells from bone marrow werevery similar (Figure 1) For a better illustration of the biolog-ical molecular activity and the activity of signaling pathwaysin the studied cell populations a transcription profile analysiswith the GO and KEGG databases was completed In termsof fundamental signaling pathways for biological activityof stem cells like JAK-STAT Wnt and Notch we recordeddiscrete differences among expressed genes from umbilicalcord blood cells as compared to bone marrow cells

The analysis regarding signaling pathways showedincreased activity of the JAK-STAT pathway in CD34minusstem cells from umbilical cord blood both on the basis ofthe gene ontology classification and the KEGG database(Figure 2 and Table 2) The increased activity regarded 15genes which take part in the JAK-STAT cascade that isSPRY2 CISH CSF2 GRB2 IFNGR1 IL2RB IL2RG IL7RIL10RA IL15RA PIM1 PTPN6 and CCND3 including thegenes which code transcription factors STAT1 and STAT5AIn previous publications the significance of the JAK-STATsignaling pathway is best described in the embryonic andhematopoietic stem cells Ramalho-Santos et al searchedfor a transcription profile describing both the embryonic

Stem Cells International 9

stem cells and the stem cells of an adult organism on thebasis of the study of embryonic cells neural stem cellsand hematopoietic stem cells activity [25] These studiesattempted to create a molecular signature of the stem cells inrelation to the self-regeneration and differentiation abilities ofthe cells The active JAK-STAT pathway belongs to the basicattributes defining the so-called ldquostemnessrdquo phenomenon Inthe embryonic stem cells the JAK-STAT signaling pathwaypromotes proliferation in the form of self-replicationThe activating factor LIF (Leukemia Inhibitory Factor)provokes phosphorylation and dimerisation of STAT3which after translocation to the cell nucleus stimulates theexpression of gene group responsible for self-replication ofthe embryonic stem cell However the JAK-STAT signalingpathway is not the only factor responsible for proliferationregulation of the embryonic stem cells Internal processesregulating the multiplication processes independent of theSTAT3 activity such as the Nanog and Oct-4 activity arealso important [15 17 26 27] In our study material theNanog and Oct-4 genes were not active in both types ofnonhematopoietic stem cells which is in agreement withother publications devoted to this subject [28 29] TheSTAT1 transcription factor overactive in our study in CD34minuscells of umbilical cord blood is responsible among othersfor the expression of interferon-activated genes involved incell defense against pathogenic factors [30] Moreover itis believed that STAT1 is a medium in expression of genegroup which is important for cell viability in response tostimulating factors In 2006 Kimrsquos team described STAT1as a highly overactive gene in human mesenchymal stemcells [31] In comparison the transcription factor STAT5which functions in the form of two isoforms STAT5a andSTAT5b is involved in the cellular proproliferation responsesand antiapoptosis processes [32 33] STAT5 is activatedby various cytokine receptors In our study the isoformSTAT5A showed increased activity It is believed that theSTAT5 factor activates specifically the cellular cycle genesthat is cyclins [34 35] In human stem cells the STAT factorsactivate the expression of cyclin Dwhich codes the regulatingsubunit of the CYCDCDK4 complex driving the G1S phasetransition which in turn activates the cell cycle progression[32 36] STAT5a and STAT5b also play an important rolein expression of genes promoting hematopoietic stem cellssurvival [37] Gene expression profiling over a range ofSTAT5 activities in human CB cells revealed subsets of genesthat are associated with the self-renewal and long-termexpansion phenotype Taking into account the fact thatin STAT5minusminus mice myelopoiesis appears to be relativelyunaffected it can be assumed that STAT5 can play a keyrole in assessing the quality of cells [38] Additionally notonly is the JAK-STAT signaling pathway a significant signaltransduction component which controls cell proliferationand purpose but it also regulates cell migration which isimportant both in the primary processes of gastrulationand in the tissue regeneration For instance the JAK-STATsignaling pathway is connected with cardioprotection [39]as well as with processes of regeneration and remodeling ofskeleton [40 41] The active JAK-STAT signaling pathwayhelps to regulate cell organization within cell grouping

[42] The above results may suggest a greater self-renewalpotential of CD34minus stem cells of umbilical cord bloodlong-term expansion phenotype and their higher ability interms of tissue regeneration

The Wnt signaling pathway is perceived as a signalingcascade involved in the control of fundamental processesof nondifferentiated mesenchymal stem cells such as cellreproduction and directed differentiation Both methods ofgene activity classification that is GO and KEGG showeda similar activity of the majority of Wnt signaling pathwaygenes (Table 4) In addition the analysis of separate genestaking part in the Wnt signaling pathway and coding 19glycosylated molecules of extracellular signaling of the Wntfamily cellular surface receptors including Frizzled receptorsintracellular signaling molecules and genes involved in cellcycle and taking part in regulation of proliferation growthand modification of proteins showed that the expressionprofilewas very similar except for single genes of an increasedand decreased expression in CD34minus stem cells of umbilicalcord blood in our study that is MMP7 PRKCB PSEN1RAC2 and CCND3 respectively as well as DKK1 WNT5AWNT5B FZD7 and TCF7L1 The Wnt5a ligand is involvedin the noncanonical Wnt signaling pathway transmissionand it is assumed that it may inhibit the canonical Wntpathway in mouse embryonic cells similarly to Dkk1 Therole of the noncanonical Wnt signaling pathway which inour results seemed to prevail in CD34minus stem cells of bonemarrow is not clear It is believed that the noncanonical Wntsignaling pathway controls the reorganization of cytoskeletonmuscle proteins tissue polarity and cell movement [43] Incontrast the canonical signaling pathway which activatestarget gene transcription through the increase of beta-cateninconcentration seems to be more active in CD34minus stemcells from umbilical cord cells Its role is to control thetissue-specific cell purpose during embryogenesis as well asregulation of proliferation in adult tissues [44] Additionallyhigh activity of WNT5A gene is noticeable in the humanMSCs differentiation into osteoblasts as well as in early stagesof adipogenesis [45 46] According to the publication ofYang et al the Wnt5a and Wnt5b glycoproteins are alsoresponsible for coordination of chondrocyte proliferation anddifferentiation [47] Wnt5a is believed to be an importantfactor taking part in the positive autocrinic regulation ofHSC repopulation ability [48] In their publication Prockopet al suggest that a high WNT5A expression in MSCs ischaracteristic for the end of the exponential and stationarygrowth phases in cell cultures in in vitro conditions [49]Slight changes in theWnt gene activity especially inWNT5Aactivity may result from the role played by MSCs in theirenvironmental niche Bone marrow MSCs seem to be moreinvolved in the renewal processes of the progenitor cells poolwhich reside in bone marrow The researches show that theexpression of Wnt ligands and Fz receptors in MSCs showssome correlation of WntFz pairs such as Wnt5aFz5 [50]The above discussed study results showed that there wasa coexpression of WNT5AFZD7 Their higher expressionwas characteristic for the bone marrow cells while thepreviously published data suggested differences in WntFzactivity depending on the origin-mouse versus humanMSCs

10 Stem Cells International

[51] Currently available data suggest differences which maybe characteristic not only for proliferation and differentiationprograms but also for the phenotype of MSCs from umbilicalcord blood and bone marrow which is in agreement withthe results published by Baksh et al [7] The remaining genestake part in theWnt signaling cascade of increased activity inCD34minus from umbilical cord blood such as MMP7 belongingto metalloproteinases which play a role in the extracellulararray remodeling and wound healing [5]

The Notch signaling pathway belongs to the processes ofintercellular communication which regulate gene expressionpatterns defining a dualistic character of stem cells reflectedby the asymmetric cell division [52] Additionally the Notchsignaling cascade controls various cells biological processesnot only proliferation and differentiation but also apoptosisand regeneration The signaling activity of Notch pathwaygenes determined in the study of 38 genes showed theirsimilar expression as defined by the KEGG classification(Table 4) A similar activity level was noted for all four Notch1ndash4 receptors as well as for JAG1 and JAG2 ligands The dif-ferences were related to two genes DTX4 and PSEN1 whichwere overactive in CD34minus cells from umbilical cord bloodThe function of the PSEN1 gene is mainly developmentalprocesses regarding cell differentiation during neurogenesisAnd the lack of this gene leads to a premature directeddifferentiation of neural progenitor cells [53] The activity ofthe Notch gene cascade plays an important role at all levelsof stem cell development starting from the embryonic stemcell through fetal cells to adult organism stem cells Notchis significantly involved in the asymmetric segregation offactors determining cell purpose during replication divisionsof the stem cell poolThe significance of receptors and ligandsis best described in the hematopoietic stem cells Expressionof the constitutively active Notch1 gene in hematopoieticstem cells induces the immortalized cell lineages which canreproduce the myeloid and lymphoid cell lineage in a long-term repopulation in animal studies [54 55] Apart from thatit delays differentiation of human hematopoietic progenitorstem cells in vitro [56] The studies suggest that Notch1 is animportant factor inHSC self-renewal and can also participatein the inhibition of stem cell differentiation The scientificfindings reveal that a suppression of the Notch signalingpathway via deletion of key genes in HSC did not cause cellsto lose their ability of long-term hematopoiesis renewal inanimal models [57] Therefore the role of ligands and Notchreceptors in HSC has not been precisely defined yet andcurrent publications do not provide precise data regardingthe role played by particular Notch signaling pathway genesin mesenchymal stem cells The only phenomenon whichappears to be superior and characteristic for all stem cellsincluding MSCs is the significance of the Notch cascadein asymmetrical cell divisions This is a significant com-ponent of stem cells population self-renewal as well asthe functionality of the cascade in the stem niche Thisphenomenon constitutes an integral part of the hypothesisabout stem cell population aging However further inves-tigation will be required to determine if the differentiallyexpressed genes described in the studies above could serve asstemness genes

Remaining signaling pathways which revealed increasedactivity in the studied CD34minus cells of umbilical cord bloodincludedMAPK (Mitogen-Activated Protein Kinase Figure 2)which regulates a wide range of cell biological activities suchas gene expression mitosis proliferation differentiationmigration and apoptosis processes [58]TheMAPKpathwaystake part in regulation of early embryonic development andin defining the role of embryonic stem cells from the earlydevelopmental phases until the moment of formation ofdifferentiated adult cells However this precise role played bythe MAPK pathways in various stem cell types has not yetbeen defined

Transmission via calcium ions was also singled out inthe group of the overactive signaling pathways of the studiedCD34minus stem cells of umbilical cord blood versus CD34minus stemcells of bone marrow (Figure 2) The ions are a vital andcommonly present mediator in the process of extracellularsignals transmission and conversion into a cellular responseVariations of calcium ions concentrations regulate geneexpression through an impact on the activity of numeroussignaling pathways Intracellular increase of calcium ionsconcentration also regulates cell activities including adhe-sion motility gene expression and proliferation [59] Thesignificance of transmission via calcium ions and the ways oftheir concentration changes in mesenchymal stem cells stillhave not been studied Kawano et al were the first to reportthat in the nondifferentiated MSCs there are spontaneouschanges of calcium ions concentrations despite the lack ofany stimulating factors [60] As reported the calcium iontransmission through the activation of different gene groupsmay direct the cells onto specific developmental pathways[61]

The integrin-mediated signaling pathway takes part inextracellular array remodeling which means that it con-trols cell behavior and tissue organization Its increasedactivity was observed in our study in CD34minus stem cells ofumbilical cord blood (Figure 2) In recent years the role ofintegrins and selectins has been stressed in the adhesion andtransendothelial migration which has a significant meaningin the processes of repopulation of tissues damaged bydiseases and their regeneration via the circulating MSCs Forinstance Ip et al showed that blocking of beta 1-integrinwhich constitutes a component of the adhesive moleculeVLA-4 in MSC weakened the ability of mesenchymal stemcells to engraft into the infarction-changedmyocardium [62]The above results may suggest a better migration abilityof CD34minus stem cells of umbilical cord blood into the areaof pathologic tissue MSCs own cytokinetic activity thathas similar significance in terms of tissue regenerationThe study of cytokinetic activity of CD34minus stem cells ofumbilical cord blood showed increased activity of signalingpathways activated via cytokines (Figure 2 Table 2) On theother hand the TLR signaling pathway (toll-like receptorsignaling) which in our own results revealed a significantincreased activity in CD34minus stem cells of umbilical cordblood (Figure 2) increases the immunosuppressive charac-teristics of MSCs Weakening of the immunological responsegenerated by the TLR pathway depends on the produc-tion of immunosuppressive kynurenines by the indoleamine

Stem Cells International 11

23-dioxygenase enzyme which degrades tryptophan Induc-tion of indoleamine 23-dioxygenase by TLR entails autocrinesignaling loop of interferon-beta which plays a role in MSCsimmunomodulative effects [63]

According to the GO and KEGG classifications carriedout for cluster 13 with the gene pool of a decreased expressiona statistically significant decrease of gene activity was shownfor the TGF-beta and BMP pathways (Figure 3 Table 3) Itis believed that the family of TGF-beta members whichalso includes BMP plays an important role in initiating theprocess of a directed MSCs differentiation TGF-beta sig-naling transmission runs through specific serine-threoninekinase receptors and their nuclear receptors called the Smadproteins TGF-beta signaling transmission promotes theprocess of early differentiation stages into the chondrogenicand osteogenic lineages [64 65] However cell differentiatinginto the adipogenic lineage results in inhibition of the TGF-beta signaling pathway This was confirmed in experimentalstudies developed by Ng et al where the inhibition of TGF-beta transmission resulted in an increase of adipogenesisand a decrease of differentiating ability towards chondrocytes[66] The BMP signaling pathway strongly promotes differ-entiation towards osteoblasts but it also leads to inhibitionof myogenesis and adipogenesis Such a gene expressionprofile may suggest that the MSCs biological characteristicsare strongly influenced by the significance of stem cell nicheTherefore the mesenchymal stem cells from bone marrowmay be more involved in the promotion of the phenomenawhich maintain the stability of bone marrow environment

5 Conclusions

Multipotentiality of the mesenchymal stem cells their highproliferation ability and not too-demanding culturing con-ditions reveal their potential for wide application in variousmedical areas These are the reasons for the common pursuitto understand their biology based onmore andmore sublimebiomolecular procedures Scientists have been searchingfor an unequivocal marker which would characterize themesenchymal stem cells and simultaneously would enable aseparation of a homogenous MSC population Understand-ing of molecular mechanisms controlling self-replication anddifferentiation is becoming a fundamental issue for clinicalapplication of MSC In this respect the activity of signalingpathways is important yet the interpretation of the pathwaysespecially their interdependence constitutes a great challengefor the scientific world Moreover the answer to the questionregarding MSC innidation and its survival ability in hosttissues after transplantation is yet to be found Explanationof the abovementioned issues will require a more globalunderstanding of biological activity of these cells especiallyon the basis of the significance of the mesenchymal nicheenvironment of stem cells and its impact on the gene andepigenetic activity It should be assumed that continuedprogress in the understanding of MSCs will finally lead totheir common therapeutic and preventive application andconsequently to the improvement of the quality and lengthof human life

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

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[2] M F Pittenger A M Mackay S C Beck et al ldquoMultilineagepotential of adult human mesenchymal stem cellsrdquo Science vol284 no 5411 pp 143ndash147 1999

[3] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006

[4] Y-J Chang D T-B Shih C-P Tseng T-B Hsieh D-C Leeand S-M Hwang ldquoDisparate mesenchyme-lineage tendenciesin mesenchymal stem cells from human bone marrow andumbilical cord bloodrdquo Stem Cells vol 24 no 3 pp 679ndash6852006

[5] R A Panepucci J L C Siufi W A Silva Jr et al ldquoComparisonof gene expression of umbilical cord vein and bone marrowndashderived mesenchymal stem cellsrdquo STEM CELLS vol 22 no 7pp 1263ndash1278 2004

[6] C K Rebelatto A M Aguiar M P Moretao et al ldquoDissimilardifferentiation of mesenchymal stem cells from bone marrowumbilical cord blood and adipose tissuerdquo Experimental Biologyand Medicine vol 233 no 7 pp 901ndash913 2008

[7] D Baksh R Yao and R S Tuan ldquoComparison of proliferativeand multilineage differentiation potential of human mesenchy-mal stem cells derived from umbilical cord and bone marrowrdquoStem Cells vol 25 no 6 pp 1384ndash1392 2007

[8] D L Clarke C B Johansson J Wilbertz et al ldquoGeneralizedpotential of adult neural stem cellsrdquo Science vol 288 no 5471pp 1660ndash1663 2000

[9] D J Anderson F H Gage and I L Weissman ldquoCan stem cellscross lineage boundariesrdquo Nature Medicine vol 7 no 4 pp393ndash395 2001

[10] D L Stemple and D J Anderson ldquoIsolation of a stem cell forneurons and glia from the mammalian neural crestrdquo Cell vol71 no 6 pp 973ndash985 1992

[11] R L Rietze H Valcanis G F Brooker T Thomas A K Vossand P F Bartlett ldquoPurification of a pluripotent neural stem cellfrom the adult mouse brainrdquoNature vol 412 no 6848 pp 736ndash739 2001

[12] R M Seaberg S R Smukler T J Kieffer et al ldquoClonalidentification of multipotent precursors from adult mousepancreas that generate neural and pancreatic lineagesrdquo NatureBiotechnology vol 22 no 9 pp 1115ndash1124 2004

[13] A P Beltrami L Barlucchi D Torella et al ldquoAdult cardiac stemcells are multipotent and support myocardial regenerationrdquoCell vol 114 no 6 pp 763ndash776 2003

[14] L Yang S Li H Hatch et al ldquoIn vitro trans-differentiation ofadult hepatic stem cells into pancreatic endocrine hormone-producing cellsrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 99 no 12 pp 8078ndash80832002

[15] I Chambers D Colby M Robertson et al ldquoFunctional expres-sion cloning of Nanog a pluripotency sustaining factor inembryonic stem cellsrdquo Cell vol 113 no 5 pp 643ndash655 2003

12 Stem Cells International

[16] M Dai P Wang A D Boyd et al ldquoEvolving genetranscriptdefinitions significantly alter the interpretation of GeneChipdatardquo Nucleic Acids Research vol 33 pp 175ndash179 2005

[17] RA Irizarry BHobbs F Collin et al ldquoExploration normaliza-tion and summaries of high density oligonucleotide array probelevel datardquo Biostatistics vol 4 no 2 pp 249ndash264 2003

[18] R B Dean and W J Dixon ldquoSimplified statistics for smallnumbers of observationsrdquo Analytical Chemistry vol 23 no 4pp 636ndash638 1951

[19] G J McLachlan and D Peel ldquoOn computational aspectsof clustering via mixtures of normal and t-componentsrdquo inProceedings of the American Statistical Association (BayesianStatistical Science Section) American Statistical AssociationIndianapolis Ind USA 2000

[20] J Polanska P Widlak J Rzeszowska-Wolny M Kimmel andA Polanski ldquoGaussian mixture decomposition of time-courseDNA microarray datardquo in Mathematical Modeling of BiologicalSystems Volume I pp 351ndash359 Birkhauser Boston Mass USA2007

[21] A J Friedenstein U F Gorskaja and N N Kulagina ldquoFibrob-last precursors in normal and irradiated mouse hematopoieticorgansrdquo Experimental Hematology vol 4 no 5 pp 267ndash2741976

[22] S Wakitani T Saito and A I Caplan ldquoMyogenic cells derivedfrom rat bone marrow mesenchymal stem cells exposed to 5-azacytidinerdquoMuscle ampNerve vol 18 no 12 pp 1417ndash1426 1995

[23] D Woodbury E J Schwarz D J Prockop and I B BlackldquoAdult rat and human bone marrow stromal cells differentiateinto neuronsrdquo Journal of Neuroscience Research vol 61 no 4pp 364ndash370 2000

[24] M Reyes T Lund T Lenvik D Aguiar L Koodie and CM Verfaillie ldquoPurification and ex vivo expansion of postnatalhumanmarrowmesodermal progenitor cellsrdquoBlood vol 98 no9 pp 2615ndash2625 2001

[25] M Ramalho-Santos S Yoon Y Matsuzaki R C Mulliganand D A Melton ldquolsquoStemnessrsquo transcriptional profiling ofembryonic and adult stem cellsrdquo Science vol 298 no 5593 pp597ndash600 2002

[26] KMitsui Y Tokuzawa H Itoh et al ldquoThe homeoprotein nanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003

[27] S Okumura-Nakanishi M Saito H Niwa and F IshikawaldquoOct-34 and Sox2 regulate Oct-34 gene in embryonic stemcellsrdquo The Journal of Biological Chemistry vol 280 no 7 pp5307ndash5317 2005

[28] F Ulloa-Montoya B L Kidder K A Pauwelyn et al ldquoCompar-ative transcriptome analysis of embryonic and adult stem cellswith extended and limited differentiation capacityrdquo GenomeBiology vol 8 no 8 article R163 2007

[29] L Song N EWebb Y Song and R S Tuan ldquoIdentification andfunctional analysis of candidate genes regulating mesenchymalstem cell self-renewal and multipotencyrdquo STEMCELLS vol 24no 7 pp 1707ndash1718 2006

[30] A H H Van Boxel-Dezaire M R S Rani and G R StarkldquoComplexmodulation of cell type-specific signaling in responseto type I interferonsrdquo Immunity vol 25 no 3 pp 361ndash372 2006

[31] C G Kim J J Lee D Y Jung et al ldquoProfiling of differentiallyexpressed genes in human stem cells by cDNA microarrayrdquoMolecules and Cells vol 21 no 3 pp 343ndash355 2006

[32] T Bowman R Garcia J Turkson and R Jove ldquoSTATs inoncogenesisrdquo Oncogene vol 19 no 21 pp 2474ndash2488 2000

[33] A Stephanou andD S Latchman ldquoOpposing actions of STAT-1and STAT-3rdquo Growth Factors vol 23 no 3 pp 177ndash182 2005

[34] A Martino J H Holmes J D Lord J J Moon and B HNelson ldquoStat5 and Sp1 regulate transcription of the cyclin D2gene in response to IL-2rdquo The Journal of Immunology vol 166no 3 pp 1723ndash1729 2001

[35] I Matsumura T Kitamura H Wakao et al ldquoTranscriptionalregulation of the cyclin D1 promoter by STAT5 its involvementin cytokine-dependent growth of hematopoietic cellsrdquo TheEMBO Journal vol 18 no 5 pp 1367ndash1377 1999

[36] V Calo M Migliavacca V Bazan et al ldquoSTAT proteins fromnormal control of cellular events to tumorigenesisrdquo Journal ofCellular Physiology vol 197 no 2 pp 157ndash168 2003

[37] M Socolovsky A E J Fallon S Wang C Brugnara and HF Lodish ldquoFetal anemia and apoptosis of red cell progenitorsin Stat5aminusminus5bminusminus mice a direct role for stat5 in Bcl-X(L)inductionrdquo Cell vol 98 no 2 pp 181ndash191 1999

[38] G Li Z Wang Y Zhang et al ldquoSTAT5 requires the N-domainto maintain hematopoietic stem cell repopulating function andappropriate lymphoid-myeloid lineage outputrdquo ExperimentalHematology vol 35 no 11 pp 1684ndash1694 2007

[39] S Negoro K Kunisada E Tone et al ldquoActivation of JAKSTATpathway transduces cytoprotective signal in rat acute myocar-dial infarctionrdquo Cardiovascular Research vol 47 no 4 pp 797ndash805 2000

[40] H C Blair L J Robinson and M Zaidi ldquoOsteoclast signallingpathwaysrdquo Biochemical and Biophysical Research Communica-tions vol 328 no 3 pp 728ndash738 2005

[41] C J Malemud ldquoProtein kinases in chondrocyte signaling andosteoarthritisrdquo Clinical Orthopaedics and Related Research no427 supplement pp S145ndashS151 2004

[42] E M Pinheiro and D J Montell ldquoRequirement for Par-6 andBazooka inDrosophila border cell migrationrdquoDevelopment vol131 no 21 pp 5243ndash5251 2004

[43] D Strutt ldquoFrizzled signalling and cell polarisation inDrosophilaand vertebratesrdquo Development vol 130 no 19 pp 4501ndash45132003

[44] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[45] J Guo J Jin and L F Cooper ldquoDissection of sets of genes thatcontrol the character of wnt5a-deficient mouse calvarial cellsrdquoBone vol 43 no 5 pp 961ndash971 2008

[46] M Nishizuka A Koyanagi S Osada and M Imagawa ldquoWnt4and Wnt5a promote adipocyte differentiationrdquo FEBS Lettersvol 582 no 21-22 pp 3201ndash3205 2008

[47] Y Yang L Topol H Lee and J Wu ldquoWnt5a andWnt5b exhibitdistinct activities in coordinating chondrocyte proliferation anddifferentiationrdquo Development vol 130 no 5 pp 1003ndash10152003

[48] D J Van Den Berg A K Sharma E Bruno and R Hoff-man ldquoRole of members of the Wnt gene family in humanhematopoiesisrdquo Blood vol 92 no 9 pp 3189ndash3202 1998

[49] D J Prockop C A Gregory and J L Spees ldquoOne strategy forcell and gene therapy harnessing the power of adult stemcells torepair tissuesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 1 pp 11917ndash119232003

[50] X He J-P Saint-Jeannet Y Wang J Nathans I Dawid and HVarmus ldquoA member of the Frizzled protein family mediatingaxis induction byWnt-5Ardquo Science vol 275 no 5306 pp 1652ndash1654 1997

Stem Cells International 13

[51] D G Phinney G Kopen R L Isaacson and D J ProckopldquoPlastic adherent stromal cells from the bone marrow ofcommonly used strains of inbred mice variations in yieldgrowth and differentiationrdquo Journal of Cellular Biochemistryvol 72 no 4 pp 570ndash585 1999

[52] N Sasaki T Sasamura H O Ishikawa et al ldquoPolarized exo-cytosis and transcytosis of Notch during its apical localizationin Drosophila epithelial cellsrdquo Genes to Cells vol 12 no 1 pp89ndash103 2007

[53] M Handler X Yang and J Shen ldquoPresenilin-1 regulatesneuronal differentiation during neurogenesisrdquo Developmentvol 127 no 12 pp 2593ndash2606 2000

[54] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006

[55] A W Duncan F M Rattis L N DiMascio et al ldquoIntegrationof Notch andWnt signaling in hematopoietic stem cell mainte-nancerdquo Nature Immunology vol 6 no 3 pp 314ndash322 2005

[56] N Carlesso J C Aster J Sklar and D T Scadden ldquoNotch1-induced delay of human hematopoietic progenitor cell differen-tiation is associated with altered cell cycle kineticsrdquo Blood vol93 no 3 pp 838ndash848 1999

[57] I Maillard U Koch A Dumortier et al ldquoCanonical notch sig-naling is dispensable for the maintenance of adult hematopoi-etic stem cellsrdquo Cell Stem Cell vol 2 no 4 pp 356ndash366 2008

[58] G Pearson F Robinson T B Gibson et al ldquoMitogen-activatedprotein (MAP) kinase pathways regulation and physiologicalfunctionsrdquo Endocrine Reviews vol 22 no 2 pp 153ndash183 2001

[59] R E Dolmetsch R S Lewis C C Goodnow and J I HealyldquoDifferential activation of transcription factors induced by Ca2+response amplitude and durationrdquoNature vol 386 no 6627 pp855ndash858 1997

[60] S Kawano S Shoji S Ichinose K Yamagata M Tagami andM Hiraoka ldquoCharacterization of Ca2+ signaling pathways inhuman mesenchymal stem cellsrdquo Cell Calcium vol 32 no 4pp 165ndash174 2002

[61] B Ye ldquoCa2+ oscillations and its transporters in mesenchymalstem cellsrdquo Physiological Research vol 59 no 3 pp 323ndash3292010

[62] J E Ip Y Wu J Huang L Zhang R E Pratt and V J DzauldquoMesenchymal stem cells use integrin 1205731 not CXC chemokinereceptor 4 for myocardial migration and engraftmentrdquoMolecu-lar Biology of the Cell vol 18 no 8 pp 2873ndash2882 2007

[63] C A Opitz U M Litzenburger C Lutz et al ldquoToll-like recep-tor engagement enhances the immunosuppressive propertiesof human bone marrow-derived mesenchymal stem cells byinducing indoleamine-23-dioxygenase-1 via Interferon-120573 andprotein kinase Rrdquo Stem Cells vol 27 no 4 pp 909ndash919 2009

[64] B Kulterer G Friedl A Jandrositz et al ldquoGene expressionprofiling of human mesenchymal stem cells derived from bonemarrow during expansion and osteoblast differentiationrdquo BMCGenomics vol 8 article 70 2007

[65] W G Li and X X Xu ldquoThe expression of N-cadherinfibronectin during chondrogenic differentiation of MSCinduced by TGF-1205731rdquo Chinese Journal of Traumatology vol 8no 6 pp 349ndash351 2005

[66] F Ng S Boucher S Koh et al ldquoPDGFTGF-120573 and FGF signal-ing is important for differentiation and growth of mesenchymalstem cells (mscs) transcriptional profiling can identify markersand signaling pathways important in differentiation of MSCsinto adipogenic chondrogenic and osteogenic lineagesrdquo Bloodvol 112 no 2 pp 295ndash307 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Microbiology

8 Stem Cells International

Table 3 Gene pathways based on the KEGG classification of a decreased expression in CD34minus stem cells from umbilical cord blood

KEGGID 119901 value Odds ratio Exp count Count Size Term4510 0001 7517 7 33 188 Focal adhesion1430 0001 8157 4 22 109 Cell junctions4350 0001 4038 3 10 82 TGF-beta signaling pathway

Table 4 Gene pathways based on the KEGG classification of a similar expression in CD34minus stem cells from umbilical cord blood and CD34minuscells from bone marrow for clusters 1ndash8 10ndash12 and 14-15

KEGGID 119901 value Odds ratio Exp count Count Size Term04310 0018 1880 10 17 131 Wnt signaling pathway04330 0025 2722 3 7 39 Notch signaling pathway04115 0038 2157 5 9 61 p53 signaling pathway04110 0009 2028 10 18 100 Cell cycle03320 0020 2791 3 7 60 PPAR signaling pathway04150 0035 2220 5 9 46 mTOR signaling pathway

IL2RB IL2RG IL7R IL10RA IL15RA PIM1 PTPN6 STAT1STAT5A and CCND3 Genes exhibiting decreased activityincluded PIAS3 IL11RA and CBLB

On the basis of the KEGG database the Wnt signalingpathway is defined by 151 genes out of which there were132 active genes in the studied material Genes exhibit-ing increased activity in CD34minus cells from umbilical cordblood include MMP7 PRKCB PSEN1 RAC2 and CCND3Genes exhibiting decreased activity include DKK1 WNT5AWNT5B FZD7 and TCF7L1

On the basis of the KEGG database the Notch signalingpathway is defined by 47 genes out of which there were38 active genes in the studied material Genes exhibitingincreased activity in CD34minus cells from umbilical cord bloodincluded DTX4 and PSEN1

4 Discussion

Nonhematopoietic stem cells were first identified by Frieden-stein et al who described the progenitor cells of rat bonemarrow [21] Currently the nonhematopoietic stem cellswhich are frequently referred to although not quite correctlyas the mesenchymal stem cells are isolated from varioustissues including the umbilical cord blood placenta andumbilical cord A constantly increasing number of studieson MSCs have proved that in appropriate environmentalconditions they are capable of differentiating into special-ized mesodermal endodermal and even ectodermal celllineages [2 22ndash24] In natural conditions they play an impor-tant role in cell regeneration processes and in repairingthe tissues damaged by degenerating processes Anotherintriguing characteristic of MSCs is their ability to avoidimmunological identification and response therefore theyare referred to as the hypoimmunogenic cells Despite greatattention paid to mesenchymal stem cells their biology isnot yet fully understood Introducing multipotential stemcells into clinical application requires a full understandingof the mechanisms which control key properties of thecell such as cell self-replication versus differentiation and

mobilization versus tissue homing The understanding ofthese mechanisms must be based on strict definitions ofhuman multipotential stem cells at the molecular level Inaddition understanding of molecular mechanisms of self-replicating pluripotentiality plasticity and differentiation ofstem cells not only is a necessary condition to overcomelimitations of stem cells application as a therapeutic factorbut also is key to understand a fundamental mystery of thedevelopment of human organism

This paper presents a comparison of gene activity of thenonhematopoietic cells of umbilical cord blood and bonemarrow using oligonucleotidemicroarrays Gene activity wastested in stem cells CD34 and included 11 979 genes obtainedafter completing procedures to increase the purity of cellsamples Cell populations included in the study were alsolimited based on the noise threshold revealed in the finalcomparative analysis The gene expression profiles of thecompared populations of CD34minus stem cells from umbilicalcord blood and CD34minus stem cells from bone marrow werevery similar (Figure 1) For a better illustration of the biolog-ical molecular activity and the activity of signaling pathwaysin the studied cell populations a transcription profile analysiswith the GO and KEGG databases was completed In termsof fundamental signaling pathways for biological activityof stem cells like JAK-STAT Wnt and Notch we recordeddiscrete differences among expressed genes from umbilicalcord blood cells as compared to bone marrow cells

The analysis regarding signaling pathways showedincreased activity of the JAK-STAT pathway in CD34minusstem cells from umbilical cord blood both on the basis ofthe gene ontology classification and the KEGG database(Figure 2 and Table 2) The increased activity regarded 15genes which take part in the JAK-STAT cascade that isSPRY2 CISH CSF2 GRB2 IFNGR1 IL2RB IL2RG IL7RIL10RA IL15RA PIM1 PTPN6 and CCND3 including thegenes which code transcription factors STAT1 and STAT5AIn previous publications the significance of the JAK-STATsignaling pathway is best described in the embryonic andhematopoietic stem cells Ramalho-Santos et al searchedfor a transcription profile describing both the embryonic

Stem Cells International 9

stem cells and the stem cells of an adult organism on thebasis of the study of embryonic cells neural stem cellsand hematopoietic stem cells activity [25] These studiesattempted to create a molecular signature of the stem cells inrelation to the self-regeneration and differentiation abilities ofthe cells The active JAK-STAT pathway belongs to the basicattributes defining the so-called ldquostemnessrdquo phenomenon Inthe embryonic stem cells the JAK-STAT signaling pathwaypromotes proliferation in the form of self-replicationThe activating factor LIF (Leukemia Inhibitory Factor)provokes phosphorylation and dimerisation of STAT3which after translocation to the cell nucleus stimulates theexpression of gene group responsible for self-replication ofthe embryonic stem cell However the JAK-STAT signalingpathway is not the only factor responsible for proliferationregulation of the embryonic stem cells Internal processesregulating the multiplication processes independent of theSTAT3 activity such as the Nanog and Oct-4 activity arealso important [15 17 26 27] In our study material theNanog and Oct-4 genes were not active in both types ofnonhematopoietic stem cells which is in agreement withother publications devoted to this subject [28 29] TheSTAT1 transcription factor overactive in our study in CD34minuscells of umbilical cord blood is responsible among othersfor the expression of interferon-activated genes involved incell defense against pathogenic factors [30] Moreover itis believed that STAT1 is a medium in expression of genegroup which is important for cell viability in response tostimulating factors In 2006 Kimrsquos team described STAT1as a highly overactive gene in human mesenchymal stemcells [31] In comparison the transcription factor STAT5which functions in the form of two isoforms STAT5a andSTAT5b is involved in the cellular proproliferation responsesand antiapoptosis processes [32 33] STAT5 is activatedby various cytokine receptors In our study the isoformSTAT5A showed increased activity It is believed that theSTAT5 factor activates specifically the cellular cycle genesthat is cyclins [34 35] In human stem cells the STAT factorsactivate the expression of cyclin Dwhich codes the regulatingsubunit of the CYCDCDK4 complex driving the G1S phasetransition which in turn activates the cell cycle progression[32 36] STAT5a and STAT5b also play an important rolein expression of genes promoting hematopoietic stem cellssurvival [37] Gene expression profiling over a range ofSTAT5 activities in human CB cells revealed subsets of genesthat are associated with the self-renewal and long-termexpansion phenotype Taking into account the fact thatin STAT5minusminus mice myelopoiesis appears to be relativelyunaffected it can be assumed that STAT5 can play a keyrole in assessing the quality of cells [38] Additionally notonly is the JAK-STAT signaling pathway a significant signaltransduction component which controls cell proliferationand purpose but it also regulates cell migration which isimportant both in the primary processes of gastrulationand in the tissue regeneration For instance the JAK-STATsignaling pathway is connected with cardioprotection [39]as well as with processes of regeneration and remodeling ofskeleton [40 41] The active JAK-STAT signaling pathwayhelps to regulate cell organization within cell grouping

[42] The above results may suggest a greater self-renewalpotential of CD34minus stem cells of umbilical cord bloodlong-term expansion phenotype and their higher ability interms of tissue regeneration

The Wnt signaling pathway is perceived as a signalingcascade involved in the control of fundamental processesof nondifferentiated mesenchymal stem cells such as cellreproduction and directed differentiation Both methods ofgene activity classification that is GO and KEGG showeda similar activity of the majority of Wnt signaling pathwaygenes (Table 4) In addition the analysis of separate genestaking part in the Wnt signaling pathway and coding 19glycosylated molecules of extracellular signaling of the Wntfamily cellular surface receptors including Frizzled receptorsintracellular signaling molecules and genes involved in cellcycle and taking part in regulation of proliferation growthand modification of proteins showed that the expressionprofilewas very similar except for single genes of an increasedand decreased expression in CD34minus stem cells of umbilicalcord blood in our study that is MMP7 PRKCB PSEN1RAC2 and CCND3 respectively as well as DKK1 WNT5AWNT5B FZD7 and TCF7L1 The Wnt5a ligand is involvedin the noncanonical Wnt signaling pathway transmissionand it is assumed that it may inhibit the canonical Wntpathway in mouse embryonic cells similarly to Dkk1 Therole of the noncanonical Wnt signaling pathway which inour results seemed to prevail in CD34minus stem cells of bonemarrow is not clear It is believed that the noncanonical Wntsignaling pathway controls the reorganization of cytoskeletonmuscle proteins tissue polarity and cell movement [43] Incontrast the canonical signaling pathway which activatestarget gene transcription through the increase of beta-cateninconcentration seems to be more active in CD34minus stemcells from umbilical cord cells Its role is to control thetissue-specific cell purpose during embryogenesis as well asregulation of proliferation in adult tissues [44] Additionallyhigh activity of WNT5A gene is noticeable in the humanMSCs differentiation into osteoblasts as well as in early stagesof adipogenesis [45 46] According to the publication ofYang et al the Wnt5a and Wnt5b glycoproteins are alsoresponsible for coordination of chondrocyte proliferation anddifferentiation [47] Wnt5a is believed to be an importantfactor taking part in the positive autocrinic regulation ofHSC repopulation ability [48] In their publication Prockopet al suggest that a high WNT5A expression in MSCs ischaracteristic for the end of the exponential and stationarygrowth phases in cell cultures in in vitro conditions [49]Slight changes in theWnt gene activity especially inWNT5Aactivity may result from the role played by MSCs in theirenvironmental niche Bone marrow MSCs seem to be moreinvolved in the renewal processes of the progenitor cells poolwhich reside in bone marrow The researches show that theexpression of Wnt ligands and Fz receptors in MSCs showssome correlation of WntFz pairs such as Wnt5aFz5 [50]The above discussed study results showed that there wasa coexpression of WNT5AFZD7 Their higher expressionwas characteristic for the bone marrow cells while thepreviously published data suggested differences in WntFzactivity depending on the origin-mouse versus humanMSCs

10 Stem Cells International

[51] Currently available data suggest differences which maybe characteristic not only for proliferation and differentiationprograms but also for the phenotype of MSCs from umbilicalcord blood and bone marrow which is in agreement withthe results published by Baksh et al [7] The remaining genestake part in theWnt signaling cascade of increased activity inCD34minus from umbilical cord blood such as MMP7 belongingto metalloproteinases which play a role in the extracellulararray remodeling and wound healing [5]

The Notch signaling pathway belongs to the processes ofintercellular communication which regulate gene expressionpatterns defining a dualistic character of stem cells reflectedby the asymmetric cell division [52] Additionally the Notchsignaling cascade controls various cells biological processesnot only proliferation and differentiation but also apoptosisand regeneration The signaling activity of Notch pathwaygenes determined in the study of 38 genes showed theirsimilar expression as defined by the KEGG classification(Table 4) A similar activity level was noted for all four Notch1ndash4 receptors as well as for JAG1 and JAG2 ligands The dif-ferences were related to two genes DTX4 and PSEN1 whichwere overactive in CD34minus cells from umbilical cord bloodThe function of the PSEN1 gene is mainly developmentalprocesses regarding cell differentiation during neurogenesisAnd the lack of this gene leads to a premature directeddifferentiation of neural progenitor cells [53] The activity ofthe Notch gene cascade plays an important role at all levelsof stem cell development starting from the embryonic stemcell through fetal cells to adult organism stem cells Notchis significantly involved in the asymmetric segregation offactors determining cell purpose during replication divisionsof the stem cell poolThe significance of receptors and ligandsis best described in the hematopoietic stem cells Expressionof the constitutively active Notch1 gene in hematopoieticstem cells induces the immortalized cell lineages which canreproduce the myeloid and lymphoid cell lineage in a long-term repopulation in animal studies [54 55] Apart from thatit delays differentiation of human hematopoietic progenitorstem cells in vitro [56] The studies suggest that Notch1 is animportant factor inHSC self-renewal and can also participatein the inhibition of stem cell differentiation The scientificfindings reveal that a suppression of the Notch signalingpathway via deletion of key genes in HSC did not cause cellsto lose their ability of long-term hematopoiesis renewal inanimal models [57] Therefore the role of ligands and Notchreceptors in HSC has not been precisely defined yet andcurrent publications do not provide precise data regardingthe role played by particular Notch signaling pathway genesin mesenchymal stem cells The only phenomenon whichappears to be superior and characteristic for all stem cellsincluding MSCs is the significance of the Notch cascadein asymmetrical cell divisions This is a significant com-ponent of stem cells population self-renewal as well asthe functionality of the cascade in the stem niche Thisphenomenon constitutes an integral part of the hypothesisabout stem cell population aging However further inves-tigation will be required to determine if the differentiallyexpressed genes described in the studies above could serve asstemness genes

Remaining signaling pathways which revealed increasedactivity in the studied CD34minus cells of umbilical cord bloodincludedMAPK (Mitogen-Activated Protein Kinase Figure 2)which regulates a wide range of cell biological activities suchas gene expression mitosis proliferation differentiationmigration and apoptosis processes [58]TheMAPKpathwaystake part in regulation of early embryonic development andin defining the role of embryonic stem cells from the earlydevelopmental phases until the moment of formation ofdifferentiated adult cells However this precise role played bythe MAPK pathways in various stem cell types has not yetbeen defined

Transmission via calcium ions was also singled out inthe group of the overactive signaling pathways of the studiedCD34minus stem cells of umbilical cord blood versus CD34minus stemcells of bone marrow (Figure 2) The ions are a vital andcommonly present mediator in the process of extracellularsignals transmission and conversion into a cellular responseVariations of calcium ions concentrations regulate geneexpression through an impact on the activity of numeroussignaling pathways Intracellular increase of calcium ionsconcentration also regulates cell activities including adhe-sion motility gene expression and proliferation [59] Thesignificance of transmission via calcium ions and the ways oftheir concentration changes in mesenchymal stem cells stillhave not been studied Kawano et al were the first to reportthat in the nondifferentiated MSCs there are spontaneouschanges of calcium ions concentrations despite the lack ofany stimulating factors [60] As reported the calcium iontransmission through the activation of different gene groupsmay direct the cells onto specific developmental pathways[61]

The integrin-mediated signaling pathway takes part inextracellular array remodeling which means that it con-trols cell behavior and tissue organization Its increasedactivity was observed in our study in CD34minus stem cells ofumbilical cord blood (Figure 2) In recent years the role ofintegrins and selectins has been stressed in the adhesion andtransendothelial migration which has a significant meaningin the processes of repopulation of tissues damaged bydiseases and their regeneration via the circulating MSCs Forinstance Ip et al showed that blocking of beta 1-integrinwhich constitutes a component of the adhesive moleculeVLA-4 in MSC weakened the ability of mesenchymal stemcells to engraft into the infarction-changedmyocardium [62]The above results may suggest a better migration abilityof CD34minus stem cells of umbilical cord blood into the areaof pathologic tissue MSCs own cytokinetic activity thathas similar significance in terms of tissue regenerationThe study of cytokinetic activity of CD34minus stem cells ofumbilical cord blood showed increased activity of signalingpathways activated via cytokines (Figure 2 Table 2) On theother hand the TLR signaling pathway (toll-like receptorsignaling) which in our own results revealed a significantincreased activity in CD34minus stem cells of umbilical cordblood (Figure 2) increases the immunosuppressive charac-teristics of MSCs Weakening of the immunological responsegenerated by the TLR pathway depends on the produc-tion of immunosuppressive kynurenines by the indoleamine

Stem Cells International 11

23-dioxygenase enzyme which degrades tryptophan Induc-tion of indoleamine 23-dioxygenase by TLR entails autocrinesignaling loop of interferon-beta which plays a role in MSCsimmunomodulative effects [63]

According to the GO and KEGG classifications carriedout for cluster 13 with the gene pool of a decreased expressiona statistically significant decrease of gene activity was shownfor the TGF-beta and BMP pathways (Figure 3 Table 3) Itis believed that the family of TGF-beta members whichalso includes BMP plays an important role in initiating theprocess of a directed MSCs differentiation TGF-beta sig-naling transmission runs through specific serine-threoninekinase receptors and their nuclear receptors called the Smadproteins TGF-beta signaling transmission promotes theprocess of early differentiation stages into the chondrogenicand osteogenic lineages [64 65] However cell differentiatinginto the adipogenic lineage results in inhibition of the TGF-beta signaling pathway This was confirmed in experimentalstudies developed by Ng et al where the inhibition of TGF-beta transmission resulted in an increase of adipogenesisand a decrease of differentiating ability towards chondrocytes[66] The BMP signaling pathway strongly promotes differ-entiation towards osteoblasts but it also leads to inhibitionof myogenesis and adipogenesis Such a gene expressionprofile may suggest that the MSCs biological characteristicsare strongly influenced by the significance of stem cell nicheTherefore the mesenchymal stem cells from bone marrowmay be more involved in the promotion of the phenomenawhich maintain the stability of bone marrow environment

5 Conclusions

Multipotentiality of the mesenchymal stem cells their highproliferation ability and not too-demanding culturing con-ditions reveal their potential for wide application in variousmedical areas These are the reasons for the common pursuitto understand their biology based onmore andmore sublimebiomolecular procedures Scientists have been searchingfor an unequivocal marker which would characterize themesenchymal stem cells and simultaneously would enable aseparation of a homogenous MSC population Understand-ing of molecular mechanisms controlling self-replication anddifferentiation is becoming a fundamental issue for clinicalapplication of MSC In this respect the activity of signalingpathways is important yet the interpretation of the pathwaysespecially their interdependence constitutes a great challengefor the scientific world Moreover the answer to the questionregarding MSC innidation and its survival ability in hosttissues after transplantation is yet to be found Explanationof the abovementioned issues will require a more globalunderstanding of biological activity of these cells especiallyon the basis of the significance of the mesenchymal nicheenvironment of stem cells and its impact on the gene andepigenetic activity It should be assumed that continuedprogress in the understanding of MSCs will finally lead totheir common therapeutic and preventive application andconsequently to the improvement of the quality and lengthof human life

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] R J Deans andA BMoseley ldquoMesenchymal stem cells biologyand potential clinical usesrdquo Experimental Hematology vol 28no 8 pp 875ndash884 2000

[2] M F Pittenger A M Mackay S C Beck et al ldquoMultilineagepotential of adult human mesenchymal stem cellsrdquo Science vol284 no 5411 pp 143ndash147 1999

[3] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006

[4] Y-J Chang D T-B Shih C-P Tseng T-B Hsieh D-C Leeand S-M Hwang ldquoDisparate mesenchyme-lineage tendenciesin mesenchymal stem cells from human bone marrow andumbilical cord bloodrdquo Stem Cells vol 24 no 3 pp 679ndash6852006

[5] R A Panepucci J L C Siufi W A Silva Jr et al ldquoComparisonof gene expression of umbilical cord vein and bone marrowndashderived mesenchymal stem cellsrdquo STEM CELLS vol 22 no 7pp 1263ndash1278 2004

[6] C K Rebelatto A M Aguiar M P Moretao et al ldquoDissimilardifferentiation of mesenchymal stem cells from bone marrowumbilical cord blood and adipose tissuerdquo Experimental Biologyand Medicine vol 233 no 7 pp 901ndash913 2008

[7] D Baksh R Yao and R S Tuan ldquoComparison of proliferativeand multilineage differentiation potential of human mesenchy-mal stem cells derived from umbilical cord and bone marrowrdquoStem Cells vol 25 no 6 pp 1384ndash1392 2007

[8] D L Clarke C B Johansson J Wilbertz et al ldquoGeneralizedpotential of adult neural stem cellsrdquo Science vol 288 no 5471pp 1660ndash1663 2000

[9] D J Anderson F H Gage and I L Weissman ldquoCan stem cellscross lineage boundariesrdquo Nature Medicine vol 7 no 4 pp393ndash395 2001

[10] D L Stemple and D J Anderson ldquoIsolation of a stem cell forneurons and glia from the mammalian neural crestrdquo Cell vol71 no 6 pp 973ndash985 1992

[11] R L Rietze H Valcanis G F Brooker T Thomas A K Vossand P F Bartlett ldquoPurification of a pluripotent neural stem cellfrom the adult mouse brainrdquoNature vol 412 no 6848 pp 736ndash739 2001

[12] R M Seaberg S R Smukler T J Kieffer et al ldquoClonalidentification of multipotent precursors from adult mousepancreas that generate neural and pancreatic lineagesrdquo NatureBiotechnology vol 22 no 9 pp 1115ndash1124 2004

[13] A P Beltrami L Barlucchi D Torella et al ldquoAdult cardiac stemcells are multipotent and support myocardial regenerationrdquoCell vol 114 no 6 pp 763ndash776 2003

[14] L Yang S Li H Hatch et al ldquoIn vitro trans-differentiation ofadult hepatic stem cells into pancreatic endocrine hormone-producing cellsrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 99 no 12 pp 8078ndash80832002

[15] I Chambers D Colby M Robertson et al ldquoFunctional expres-sion cloning of Nanog a pluripotency sustaining factor inembryonic stem cellsrdquo Cell vol 113 no 5 pp 643ndash655 2003

12 Stem Cells International

[16] M Dai P Wang A D Boyd et al ldquoEvolving genetranscriptdefinitions significantly alter the interpretation of GeneChipdatardquo Nucleic Acids Research vol 33 pp 175ndash179 2005

[17] RA Irizarry BHobbs F Collin et al ldquoExploration normaliza-tion and summaries of high density oligonucleotide array probelevel datardquo Biostatistics vol 4 no 2 pp 249ndash264 2003

[18] R B Dean and W J Dixon ldquoSimplified statistics for smallnumbers of observationsrdquo Analytical Chemistry vol 23 no 4pp 636ndash638 1951

[19] G J McLachlan and D Peel ldquoOn computational aspectsof clustering via mixtures of normal and t-componentsrdquo inProceedings of the American Statistical Association (BayesianStatistical Science Section) American Statistical AssociationIndianapolis Ind USA 2000

[20] J Polanska P Widlak J Rzeszowska-Wolny M Kimmel andA Polanski ldquoGaussian mixture decomposition of time-courseDNA microarray datardquo in Mathematical Modeling of BiologicalSystems Volume I pp 351ndash359 Birkhauser Boston Mass USA2007

[21] A J Friedenstein U F Gorskaja and N N Kulagina ldquoFibrob-last precursors in normal and irradiated mouse hematopoieticorgansrdquo Experimental Hematology vol 4 no 5 pp 267ndash2741976

[22] S Wakitani T Saito and A I Caplan ldquoMyogenic cells derivedfrom rat bone marrow mesenchymal stem cells exposed to 5-azacytidinerdquoMuscle ampNerve vol 18 no 12 pp 1417ndash1426 1995

[23] D Woodbury E J Schwarz D J Prockop and I B BlackldquoAdult rat and human bone marrow stromal cells differentiateinto neuronsrdquo Journal of Neuroscience Research vol 61 no 4pp 364ndash370 2000

[24] M Reyes T Lund T Lenvik D Aguiar L Koodie and CM Verfaillie ldquoPurification and ex vivo expansion of postnatalhumanmarrowmesodermal progenitor cellsrdquoBlood vol 98 no9 pp 2615ndash2625 2001

[25] M Ramalho-Santos S Yoon Y Matsuzaki R C Mulliganand D A Melton ldquolsquoStemnessrsquo transcriptional profiling ofembryonic and adult stem cellsrdquo Science vol 298 no 5593 pp597ndash600 2002

[26] KMitsui Y Tokuzawa H Itoh et al ldquoThe homeoprotein nanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003

[27] S Okumura-Nakanishi M Saito H Niwa and F IshikawaldquoOct-34 and Sox2 regulate Oct-34 gene in embryonic stemcellsrdquo The Journal of Biological Chemistry vol 280 no 7 pp5307ndash5317 2005

[28] F Ulloa-Montoya B L Kidder K A Pauwelyn et al ldquoCompar-ative transcriptome analysis of embryonic and adult stem cellswith extended and limited differentiation capacityrdquo GenomeBiology vol 8 no 8 article R163 2007

[29] L Song N EWebb Y Song and R S Tuan ldquoIdentification andfunctional analysis of candidate genes regulating mesenchymalstem cell self-renewal and multipotencyrdquo STEMCELLS vol 24no 7 pp 1707ndash1718 2006

[30] A H H Van Boxel-Dezaire M R S Rani and G R StarkldquoComplexmodulation of cell type-specific signaling in responseto type I interferonsrdquo Immunity vol 25 no 3 pp 361ndash372 2006

[31] C G Kim J J Lee D Y Jung et al ldquoProfiling of differentiallyexpressed genes in human stem cells by cDNA microarrayrdquoMolecules and Cells vol 21 no 3 pp 343ndash355 2006

[32] T Bowman R Garcia J Turkson and R Jove ldquoSTATs inoncogenesisrdquo Oncogene vol 19 no 21 pp 2474ndash2488 2000

[33] A Stephanou andD S Latchman ldquoOpposing actions of STAT-1and STAT-3rdquo Growth Factors vol 23 no 3 pp 177ndash182 2005

[34] A Martino J H Holmes J D Lord J J Moon and B HNelson ldquoStat5 and Sp1 regulate transcription of the cyclin D2gene in response to IL-2rdquo The Journal of Immunology vol 166no 3 pp 1723ndash1729 2001

[35] I Matsumura T Kitamura H Wakao et al ldquoTranscriptionalregulation of the cyclin D1 promoter by STAT5 its involvementin cytokine-dependent growth of hematopoietic cellsrdquo TheEMBO Journal vol 18 no 5 pp 1367ndash1377 1999

[36] V Calo M Migliavacca V Bazan et al ldquoSTAT proteins fromnormal control of cellular events to tumorigenesisrdquo Journal ofCellular Physiology vol 197 no 2 pp 157ndash168 2003

[37] M Socolovsky A E J Fallon S Wang C Brugnara and HF Lodish ldquoFetal anemia and apoptosis of red cell progenitorsin Stat5aminusminus5bminusminus mice a direct role for stat5 in Bcl-X(L)inductionrdquo Cell vol 98 no 2 pp 181ndash191 1999

[38] G Li Z Wang Y Zhang et al ldquoSTAT5 requires the N-domainto maintain hematopoietic stem cell repopulating function andappropriate lymphoid-myeloid lineage outputrdquo ExperimentalHematology vol 35 no 11 pp 1684ndash1694 2007

[39] S Negoro K Kunisada E Tone et al ldquoActivation of JAKSTATpathway transduces cytoprotective signal in rat acute myocar-dial infarctionrdquo Cardiovascular Research vol 47 no 4 pp 797ndash805 2000

[40] H C Blair L J Robinson and M Zaidi ldquoOsteoclast signallingpathwaysrdquo Biochemical and Biophysical Research Communica-tions vol 328 no 3 pp 728ndash738 2005

[41] C J Malemud ldquoProtein kinases in chondrocyte signaling andosteoarthritisrdquo Clinical Orthopaedics and Related Research no427 supplement pp S145ndashS151 2004

[42] E M Pinheiro and D J Montell ldquoRequirement for Par-6 andBazooka inDrosophila border cell migrationrdquoDevelopment vol131 no 21 pp 5243ndash5251 2004

[43] D Strutt ldquoFrizzled signalling and cell polarisation inDrosophilaand vertebratesrdquo Development vol 130 no 19 pp 4501ndash45132003

[44] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[45] J Guo J Jin and L F Cooper ldquoDissection of sets of genes thatcontrol the character of wnt5a-deficient mouse calvarial cellsrdquoBone vol 43 no 5 pp 961ndash971 2008

[46] M Nishizuka A Koyanagi S Osada and M Imagawa ldquoWnt4and Wnt5a promote adipocyte differentiationrdquo FEBS Lettersvol 582 no 21-22 pp 3201ndash3205 2008

[47] Y Yang L Topol H Lee and J Wu ldquoWnt5a andWnt5b exhibitdistinct activities in coordinating chondrocyte proliferation anddifferentiationrdquo Development vol 130 no 5 pp 1003ndash10152003

[48] D J Van Den Berg A K Sharma E Bruno and R Hoff-man ldquoRole of members of the Wnt gene family in humanhematopoiesisrdquo Blood vol 92 no 9 pp 3189ndash3202 1998

[49] D J Prockop C A Gregory and J L Spees ldquoOne strategy forcell and gene therapy harnessing the power of adult stemcells torepair tissuesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 1 pp 11917ndash119232003

[50] X He J-P Saint-Jeannet Y Wang J Nathans I Dawid and HVarmus ldquoA member of the Frizzled protein family mediatingaxis induction byWnt-5Ardquo Science vol 275 no 5306 pp 1652ndash1654 1997

Stem Cells International 13

[51] D G Phinney G Kopen R L Isaacson and D J ProckopldquoPlastic adherent stromal cells from the bone marrow ofcommonly used strains of inbred mice variations in yieldgrowth and differentiationrdquo Journal of Cellular Biochemistryvol 72 no 4 pp 570ndash585 1999

[52] N Sasaki T Sasamura H O Ishikawa et al ldquoPolarized exo-cytosis and transcytosis of Notch during its apical localizationin Drosophila epithelial cellsrdquo Genes to Cells vol 12 no 1 pp89ndash103 2007

[53] M Handler X Yang and J Shen ldquoPresenilin-1 regulatesneuronal differentiation during neurogenesisrdquo Developmentvol 127 no 12 pp 2593ndash2606 2000

[54] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006

[55] A W Duncan F M Rattis L N DiMascio et al ldquoIntegrationof Notch andWnt signaling in hematopoietic stem cell mainte-nancerdquo Nature Immunology vol 6 no 3 pp 314ndash322 2005

[56] N Carlesso J C Aster J Sklar and D T Scadden ldquoNotch1-induced delay of human hematopoietic progenitor cell differen-tiation is associated with altered cell cycle kineticsrdquo Blood vol93 no 3 pp 838ndash848 1999

[57] I Maillard U Koch A Dumortier et al ldquoCanonical notch sig-naling is dispensable for the maintenance of adult hematopoi-etic stem cellsrdquo Cell Stem Cell vol 2 no 4 pp 356ndash366 2008

[58] G Pearson F Robinson T B Gibson et al ldquoMitogen-activatedprotein (MAP) kinase pathways regulation and physiologicalfunctionsrdquo Endocrine Reviews vol 22 no 2 pp 153ndash183 2001

[59] R E Dolmetsch R S Lewis C C Goodnow and J I HealyldquoDifferential activation of transcription factors induced by Ca2+response amplitude and durationrdquoNature vol 386 no 6627 pp855ndash858 1997

[60] S Kawano S Shoji S Ichinose K Yamagata M Tagami andM Hiraoka ldquoCharacterization of Ca2+ signaling pathways inhuman mesenchymal stem cellsrdquo Cell Calcium vol 32 no 4pp 165ndash174 2002

[61] B Ye ldquoCa2+ oscillations and its transporters in mesenchymalstem cellsrdquo Physiological Research vol 59 no 3 pp 323ndash3292010

[62] J E Ip Y Wu J Huang L Zhang R E Pratt and V J DzauldquoMesenchymal stem cells use integrin 1205731 not CXC chemokinereceptor 4 for myocardial migration and engraftmentrdquoMolecu-lar Biology of the Cell vol 18 no 8 pp 2873ndash2882 2007

[63] C A Opitz U M Litzenburger C Lutz et al ldquoToll-like recep-tor engagement enhances the immunosuppressive propertiesof human bone marrow-derived mesenchymal stem cells byinducing indoleamine-23-dioxygenase-1 via Interferon-120573 andprotein kinase Rrdquo Stem Cells vol 27 no 4 pp 909ndash919 2009

[64] B Kulterer G Friedl A Jandrositz et al ldquoGene expressionprofiling of human mesenchymal stem cells derived from bonemarrow during expansion and osteoblast differentiationrdquo BMCGenomics vol 8 article 70 2007

[65] W G Li and X X Xu ldquoThe expression of N-cadherinfibronectin during chondrogenic differentiation of MSCinduced by TGF-1205731rdquo Chinese Journal of Traumatology vol 8no 6 pp 349ndash351 2005

[66] F Ng S Boucher S Koh et al ldquoPDGFTGF-120573 and FGF signal-ing is important for differentiation and growth of mesenchymalstem cells (mscs) transcriptional profiling can identify markersand signaling pathways important in differentiation of MSCsinto adipogenic chondrogenic and osteogenic lineagesrdquo Bloodvol 112 no 2 pp 295ndash307 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Signal TransductionJournal of

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Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

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Advances in

Virolog y

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Nucleic AcidsJournal of

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Enzyme Research

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International Journal of

Microbiology

Stem Cells International 9

stem cells and the stem cells of an adult organism on thebasis of the study of embryonic cells neural stem cellsand hematopoietic stem cells activity [25] These studiesattempted to create a molecular signature of the stem cells inrelation to the self-regeneration and differentiation abilities ofthe cells The active JAK-STAT pathway belongs to the basicattributes defining the so-called ldquostemnessrdquo phenomenon Inthe embryonic stem cells the JAK-STAT signaling pathwaypromotes proliferation in the form of self-replicationThe activating factor LIF (Leukemia Inhibitory Factor)provokes phosphorylation and dimerisation of STAT3which after translocation to the cell nucleus stimulates theexpression of gene group responsible for self-replication ofthe embryonic stem cell However the JAK-STAT signalingpathway is not the only factor responsible for proliferationregulation of the embryonic stem cells Internal processesregulating the multiplication processes independent of theSTAT3 activity such as the Nanog and Oct-4 activity arealso important [15 17 26 27] In our study material theNanog and Oct-4 genes were not active in both types ofnonhematopoietic stem cells which is in agreement withother publications devoted to this subject [28 29] TheSTAT1 transcription factor overactive in our study in CD34minuscells of umbilical cord blood is responsible among othersfor the expression of interferon-activated genes involved incell defense against pathogenic factors [30] Moreover itis believed that STAT1 is a medium in expression of genegroup which is important for cell viability in response tostimulating factors In 2006 Kimrsquos team described STAT1as a highly overactive gene in human mesenchymal stemcells [31] In comparison the transcription factor STAT5which functions in the form of two isoforms STAT5a andSTAT5b is involved in the cellular proproliferation responsesand antiapoptosis processes [32 33] STAT5 is activatedby various cytokine receptors In our study the isoformSTAT5A showed increased activity It is believed that theSTAT5 factor activates specifically the cellular cycle genesthat is cyclins [34 35] In human stem cells the STAT factorsactivate the expression of cyclin Dwhich codes the regulatingsubunit of the CYCDCDK4 complex driving the G1S phasetransition which in turn activates the cell cycle progression[32 36] STAT5a and STAT5b also play an important rolein expression of genes promoting hematopoietic stem cellssurvival [37] Gene expression profiling over a range ofSTAT5 activities in human CB cells revealed subsets of genesthat are associated with the self-renewal and long-termexpansion phenotype Taking into account the fact thatin STAT5minusminus mice myelopoiesis appears to be relativelyunaffected it can be assumed that STAT5 can play a keyrole in assessing the quality of cells [38] Additionally notonly is the JAK-STAT signaling pathway a significant signaltransduction component which controls cell proliferationand purpose but it also regulates cell migration which isimportant both in the primary processes of gastrulationand in the tissue regeneration For instance the JAK-STATsignaling pathway is connected with cardioprotection [39]as well as with processes of regeneration and remodeling ofskeleton [40 41] The active JAK-STAT signaling pathwayhelps to regulate cell organization within cell grouping

[42] The above results may suggest a greater self-renewalpotential of CD34minus stem cells of umbilical cord bloodlong-term expansion phenotype and their higher ability interms of tissue regeneration

The Wnt signaling pathway is perceived as a signalingcascade involved in the control of fundamental processesof nondifferentiated mesenchymal stem cells such as cellreproduction and directed differentiation Both methods ofgene activity classification that is GO and KEGG showeda similar activity of the majority of Wnt signaling pathwaygenes (Table 4) In addition the analysis of separate genestaking part in the Wnt signaling pathway and coding 19glycosylated molecules of extracellular signaling of the Wntfamily cellular surface receptors including Frizzled receptorsintracellular signaling molecules and genes involved in cellcycle and taking part in regulation of proliferation growthand modification of proteins showed that the expressionprofilewas very similar except for single genes of an increasedand decreased expression in CD34minus stem cells of umbilicalcord blood in our study that is MMP7 PRKCB PSEN1RAC2 and CCND3 respectively as well as DKK1 WNT5AWNT5B FZD7 and TCF7L1 The Wnt5a ligand is involvedin the noncanonical Wnt signaling pathway transmissionand it is assumed that it may inhibit the canonical Wntpathway in mouse embryonic cells similarly to Dkk1 Therole of the noncanonical Wnt signaling pathway which inour results seemed to prevail in CD34minus stem cells of bonemarrow is not clear It is believed that the noncanonical Wntsignaling pathway controls the reorganization of cytoskeletonmuscle proteins tissue polarity and cell movement [43] Incontrast the canonical signaling pathway which activatestarget gene transcription through the increase of beta-cateninconcentration seems to be more active in CD34minus stemcells from umbilical cord cells Its role is to control thetissue-specific cell purpose during embryogenesis as well asregulation of proliferation in adult tissues [44] Additionallyhigh activity of WNT5A gene is noticeable in the humanMSCs differentiation into osteoblasts as well as in early stagesof adipogenesis [45 46] According to the publication ofYang et al the Wnt5a and Wnt5b glycoproteins are alsoresponsible for coordination of chondrocyte proliferation anddifferentiation [47] Wnt5a is believed to be an importantfactor taking part in the positive autocrinic regulation ofHSC repopulation ability [48] In their publication Prockopet al suggest that a high WNT5A expression in MSCs ischaracteristic for the end of the exponential and stationarygrowth phases in cell cultures in in vitro conditions [49]Slight changes in theWnt gene activity especially inWNT5Aactivity may result from the role played by MSCs in theirenvironmental niche Bone marrow MSCs seem to be moreinvolved in the renewal processes of the progenitor cells poolwhich reside in bone marrow The researches show that theexpression of Wnt ligands and Fz receptors in MSCs showssome correlation of WntFz pairs such as Wnt5aFz5 [50]The above discussed study results showed that there wasa coexpression of WNT5AFZD7 Their higher expressionwas characteristic for the bone marrow cells while thepreviously published data suggested differences in WntFzactivity depending on the origin-mouse versus humanMSCs

10 Stem Cells International

[51] Currently available data suggest differences which maybe characteristic not only for proliferation and differentiationprograms but also for the phenotype of MSCs from umbilicalcord blood and bone marrow which is in agreement withthe results published by Baksh et al [7] The remaining genestake part in theWnt signaling cascade of increased activity inCD34minus from umbilical cord blood such as MMP7 belongingto metalloproteinases which play a role in the extracellulararray remodeling and wound healing [5]

The Notch signaling pathway belongs to the processes ofintercellular communication which regulate gene expressionpatterns defining a dualistic character of stem cells reflectedby the asymmetric cell division [52] Additionally the Notchsignaling cascade controls various cells biological processesnot only proliferation and differentiation but also apoptosisand regeneration The signaling activity of Notch pathwaygenes determined in the study of 38 genes showed theirsimilar expression as defined by the KEGG classification(Table 4) A similar activity level was noted for all four Notch1ndash4 receptors as well as for JAG1 and JAG2 ligands The dif-ferences were related to two genes DTX4 and PSEN1 whichwere overactive in CD34minus cells from umbilical cord bloodThe function of the PSEN1 gene is mainly developmentalprocesses regarding cell differentiation during neurogenesisAnd the lack of this gene leads to a premature directeddifferentiation of neural progenitor cells [53] The activity ofthe Notch gene cascade plays an important role at all levelsof stem cell development starting from the embryonic stemcell through fetal cells to adult organism stem cells Notchis significantly involved in the asymmetric segregation offactors determining cell purpose during replication divisionsof the stem cell poolThe significance of receptors and ligandsis best described in the hematopoietic stem cells Expressionof the constitutively active Notch1 gene in hematopoieticstem cells induces the immortalized cell lineages which canreproduce the myeloid and lymphoid cell lineage in a long-term repopulation in animal studies [54 55] Apart from thatit delays differentiation of human hematopoietic progenitorstem cells in vitro [56] The studies suggest that Notch1 is animportant factor inHSC self-renewal and can also participatein the inhibition of stem cell differentiation The scientificfindings reveal that a suppression of the Notch signalingpathway via deletion of key genes in HSC did not cause cellsto lose their ability of long-term hematopoiesis renewal inanimal models [57] Therefore the role of ligands and Notchreceptors in HSC has not been precisely defined yet andcurrent publications do not provide precise data regardingthe role played by particular Notch signaling pathway genesin mesenchymal stem cells The only phenomenon whichappears to be superior and characteristic for all stem cellsincluding MSCs is the significance of the Notch cascadein asymmetrical cell divisions This is a significant com-ponent of stem cells population self-renewal as well asthe functionality of the cascade in the stem niche Thisphenomenon constitutes an integral part of the hypothesisabout stem cell population aging However further inves-tigation will be required to determine if the differentiallyexpressed genes described in the studies above could serve asstemness genes

Remaining signaling pathways which revealed increasedactivity in the studied CD34minus cells of umbilical cord bloodincludedMAPK (Mitogen-Activated Protein Kinase Figure 2)which regulates a wide range of cell biological activities suchas gene expression mitosis proliferation differentiationmigration and apoptosis processes [58]TheMAPKpathwaystake part in regulation of early embryonic development andin defining the role of embryonic stem cells from the earlydevelopmental phases until the moment of formation ofdifferentiated adult cells However this precise role played bythe MAPK pathways in various stem cell types has not yetbeen defined

Transmission via calcium ions was also singled out inthe group of the overactive signaling pathways of the studiedCD34minus stem cells of umbilical cord blood versus CD34minus stemcells of bone marrow (Figure 2) The ions are a vital andcommonly present mediator in the process of extracellularsignals transmission and conversion into a cellular responseVariations of calcium ions concentrations regulate geneexpression through an impact on the activity of numeroussignaling pathways Intracellular increase of calcium ionsconcentration also regulates cell activities including adhe-sion motility gene expression and proliferation [59] Thesignificance of transmission via calcium ions and the ways oftheir concentration changes in mesenchymal stem cells stillhave not been studied Kawano et al were the first to reportthat in the nondifferentiated MSCs there are spontaneouschanges of calcium ions concentrations despite the lack ofany stimulating factors [60] As reported the calcium iontransmission through the activation of different gene groupsmay direct the cells onto specific developmental pathways[61]

The integrin-mediated signaling pathway takes part inextracellular array remodeling which means that it con-trols cell behavior and tissue organization Its increasedactivity was observed in our study in CD34minus stem cells ofumbilical cord blood (Figure 2) In recent years the role ofintegrins and selectins has been stressed in the adhesion andtransendothelial migration which has a significant meaningin the processes of repopulation of tissues damaged bydiseases and their regeneration via the circulating MSCs Forinstance Ip et al showed that blocking of beta 1-integrinwhich constitutes a component of the adhesive moleculeVLA-4 in MSC weakened the ability of mesenchymal stemcells to engraft into the infarction-changedmyocardium [62]The above results may suggest a better migration abilityof CD34minus stem cells of umbilical cord blood into the areaof pathologic tissue MSCs own cytokinetic activity thathas similar significance in terms of tissue regenerationThe study of cytokinetic activity of CD34minus stem cells ofumbilical cord blood showed increased activity of signalingpathways activated via cytokines (Figure 2 Table 2) On theother hand the TLR signaling pathway (toll-like receptorsignaling) which in our own results revealed a significantincreased activity in CD34minus stem cells of umbilical cordblood (Figure 2) increases the immunosuppressive charac-teristics of MSCs Weakening of the immunological responsegenerated by the TLR pathway depends on the produc-tion of immunosuppressive kynurenines by the indoleamine

Stem Cells International 11

23-dioxygenase enzyme which degrades tryptophan Induc-tion of indoleamine 23-dioxygenase by TLR entails autocrinesignaling loop of interferon-beta which plays a role in MSCsimmunomodulative effects [63]

According to the GO and KEGG classifications carriedout for cluster 13 with the gene pool of a decreased expressiona statistically significant decrease of gene activity was shownfor the TGF-beta and BMP pathways (Figure 3 Table 3) Itis believed that the family of TGF-beta members whichalso includes BMP plays an important role in initiating theprocess of a directed MSCs differentiation TGF-beta sig-naling transmission runs through specific serine-threoninekinase receptors and their nuclear receptors called the Smadproteins TGF-beta signaling transmission promotes theprocess of early differentiation stages into the chondrogenicand osteogenic lineages [64 65] However cell differentiatinginto the adipogenic lineage results in inhibition of the TGF-beta signaling pathway This was confirmed in experimentalstudies developed by Ng et al where the inhibition of TGF-beta transmission resulted in an increase of adipogenesisand a decrease of differentiating ability towards chondrocytes[66] The BMP signaling pathway strongly promotes differ-entiation towards osteoblasts but it also leads to inhibitionof myogenesis and adipogenesis Such a gene expressionprofile may suggest that the MSCs biological characteristicsare strongly influenced by the significance of stem cell nicheTherefore the mesenchymal stem cells from bone marrowmay be more involved in the promotion of the phenomenawhich maintain the stability of bone marrow environment

5 Conclusions

Multipotentiality of the mesenchymal stem cells their highproliferation ability and not too-demanding culturing con-ditions reveal their potential for wide application in variousmedical areas These are the reasons for the common pursuitto understand their biology based onmore andmore sublimebiomolecular procedures Scientists have been searchingfor an unequivocal marker which would characterize themesenchymal stem cells and simultaneously would enable aseparation of a homogenous MSC population Understand-ing of molecular mechanisms controlling self-replication anddifferentiation is becoming a fundamental issue for clinicalapplication of MSC In this respect the activity of signalingpathways is important yet the interpretation of the pathwaysespecially their interdependence constitutes a great challengefor the scientific world Moreover the answer to the questionregarding MSC innidation and its survival ability in hosttissues after transplantation is yet to be found Explanationof the abovementioned issues will require a more globalunderstanding of biological activity of these cells especiallyon the basis of the significance of the mesenchymal nicheenvironment of stem cells and its impact on the gene andepigenetic activity It should be assumed that continuedprogress in the understanding of MSCs will finally lead totheir common therapeutic and preventive application andconsequently to the improvement of the quality and lengthof human life

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] R J Deans andA BMoseley ldquoMesenchymal stem cells biologyand potential clinical usesrdquo Experimental Hematology vol 28no 8 pp 875ndash884 2000

[2] M F Pittenger A M Mackay S C Beck et al ldquoMultilineagepotential of adult human mesenchymal stem cellsrdquo Science vol284 no 5411 pp 143ndash147 1999

[3] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006

[4] Y-J Chang D T-B Shih C-P Tseng T-B Hsieh D-C Leeand S-M Hwang ldquoDisparate mesenchyme-lineage tendenciesin mesenchymal stem cells from human bone marrow andumbilical cord bloodrdquo Stem Cells vol 24 no 3 pp 679ndash6852006

[5] R A Panepucci J L C Siufi W A Silva Jr et al ldquoComparisonof gene expression of umbilical cord vein and bone marrowndashderived mesenchymal stem cellsrdquo STEM CELLS vol 22 no 7pp 1263ndash1278 2004

[6] C K Rebelatto A M Aguiar M P Moretao et al ldquoDissimilardifferentiation of mesenchymal stem cells from bone marrowumbilical cord blood and adipose tissuerdquo Experimental Biologyand Medicine vol 233 no 7 pp 901ndash913 2008

[7] D Baksh R Yao and R S Tuan ldquoComparison of proliferativeand multilineage differentiation potential of human mesenchy-mal stem cells derived from umbilical cord and bone marrowrdquoStem Cells vol 25 no 6 pp 1384ndash1392 2007

[8] D L Clarke C B Johansson J Wilbertz et al ldquoGeneralizedpotential of adult neural stem cellsrdquo Science vol 288 no 5471pp 1660ndash1663 2000

[9] D J Anderson F H Gage and I L Weissman ldquoCan stem cellscross lineage boundariesrdquo Nature Medicine vol 7 no 4 pp393ndash395 2001

[10] D L Stemple and D J Anderson ldquoIsolation of a stem cell forneurons and glia from the mammalian neural crestrdquo Cell vol71 no 6 pp 973ndash985 1992

[11] R L Rietze H Valcanis G F Brooker T Thomas A K Vossand P F Bartlett ldquoPurification of a pluripotent neural stem cellfrom the adult mouse brainrdquoNature vol 412 no 6848 pp 736ndash739 2001

[12] R M Seaberg S R Smukler T J Kieffer et al ldquoClonalidentification of multipotent precursors from adult mousepancreas that generate neural and pancreatic lineagesrdquo NatureBiotechnology vol 22 no 9 pp 1115ndash1124 2004

[13] A P Beltrami L Barlucchi D Torella et al ldquoAdult cardiac stemcells are multipotent and support myocardial regenerationrdquoCell vol 114 no 6 pp 763ndash776 2003

[14] L Yang S Li H Hatch et al ldquoIn vitro trans-differentiation ofadult hepatic stem cells into pancreatic endocrine hormone-producing cellsrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 99 no 12 pp 8078ndash80832002

[15] I Chambers D Colby M Robertson et al ldquoFunctional expres-sion cloning of Nanog a pluripotency sustaining factor inembryonic stem cellsrdquo Cell vol 113 no 5 pp 643ndash655 2003

12 Stem Cells International

[16] M Dai P Wang A D Boyd et al ldquoEvolving genetranscriptdefinitions significantly alter the interpretation of GeneChipdatardquo Nucleic Acids Research vol 33 pp 175ndash179 2005

[17] RA Irizarry BHobbs F Collin et al ldquoExploration normaliza-tion and summaries of high density oligonucleotide array probelevel datardquo Biostatistics vol 4 no 2 pp 249ndash264 2003

[18] R B Dean and W J Dixon ldquoSimplified statistics for smallnumbers of observationsrdquo Analytical Chemistry vol 23 no 4pp 636ndash638 1951

[19] G J McLachlan and D Peel ldquoOn computational aspectsof clustering via mixtures of normal and t-componentsrdquo inProceedings of the American Statistical Association (BayesianStatistical Science Section) American Statistical AssociationIndianapolis Ind USA 2000

[20] J Polanska P Widlak J Rzeszowska-Wolny M Kimmel andA Polanski ldquoGaussian mixture decomposition of time-courseDNA microarray datardquo in Mathematical Modeling of BiologicalSystems Volume I pp 351ndash359 Birkhauser Boston Mass USA2007

[21] A J Friedenstein U F Gorskaja and N N Kulagina ldquoFibrob-last precursors in normal and irradiated mouse hematopoieticorgansrdquo Experimental Hematology vol 4 no 5 pp 267ndash2741976

[22] S Wakitani T Saito and A I Caplan ldquoMyogenic cells derivedfrom rat bone marrow mesenchymal stem cells exposed to 5-azacytidinerdquoMuscle ampNerve vol 18 no 12 pp 1417ndash1426 1995

[23] D Woodbury E J Schwarz D J Prockop and I B BlackldquoAdult rat and human bone marrow stromal cells differentiateinto neuronsrdquo Journal of Neuroscience Research vol 61 no 4pp 364ndash370 2000

[24] M Reyes T Lund T Lenvik D Aguiar L Koodie and CM Verfaillie ldquoPurification and ex vivo expansion of postnatalhumanmarrowmesodermal progenitor cellsrdquoBlood vol 98 no9 pp 2615ndash2625 2001

[25] M Ramalho-Santos S Yoon Y Matsuzaki R C Mulliganand D A Melton ldquolsquoStemnessrsquo transcriptional profiling ofembryonic and adult stem cellsrdquo Science vol 298 no 5593 pp597ndash600 2002

[26] KMitsui Y Tokuzawa H Itoh et al ldquoThe homeoprotein nanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003

[27] S Okumura-Nakanishi M Saito H Niwa and F IshikawaldquoOct-34 and Sox2 regulate Oct-34 gene in embryonic stemcellsrdquo The Journal of Biological Chemistry vol 280 no 7 pp5307ndash5317 2005

[28] F Ulloa-Montoya B L Kidder K A Pauwelyn et al ldquoCompar-ative transcriptome analysis of embryonic and adult stem cellswith extended and limited differentiation capacityrdquo GenomeBiology vol 8 no 8 article R163 2007

[29] L Song N EWebb Y Song and R S Tuan ldquoIdentification andfunctional analysis of candidate genes regulating mesenchymalstem cell self-renewal and multipotencyrdquo STEMCELLS vol 24no 7 pp 1707ndash1718 2006

[30] A H H Van Boxel-Dezaire M R S Rani and G R StarkldquoComplexmodulation of cell type-specific signaling in responseto type I interferonsrdquo Immunity vol 25 no 3 pp 361ndash372 2006

[31] C G Kim J J Lee D Y Jung et al ldquoProfiling of differentiallyexpressed genes in human stem cells by cDNA microarrayrdquoMolecules and Cells vol 21 no 3 pp 343ndash355 2006

[32] T Bowman R Garcia J Turkson and R Jove ldquoSTATs inoncogenesisrdquo Oncogene vol 19 no 21 pp 2474ndash2488 2000

[33] A Stephanou andD S Latchman ldquoOpposing actions of STAT-1and STAT-3rdquo Growth Factors vol 23 no 3 pp 177ndash182 2005

[34] A Martino J H Holmes J D Lord J J Moon and B HNelson ldquoStat5 and Sp1 regulate transcription of the cyclin D2gene in response to IL-2rdquo The Journal of Immunology vol 166no 3 pp 1723ndash1729 2001

[35] I Matsumura T Kitamura H Wakao et al ldquoTranscriptionalregulation of the cyclin D1 promoter by STAT5 its involvementin cytokine-dependent growth of hematopoietic cellsrdquo TheEMBO Journal vol 18 no 5 pp 1367ndash1377 1999

[36] V Calo M Migliavacca V Bazan et al ldquoSTAT proteins fromnormal control of cellular events to tumorigenesisrdquo Journal ofCellular Physiology vol 197 no 2 pp 157ndash168 2003

[37] M Socolovsky A E J Fallon S Wang C Brugnara and HF Lodish ldquoFetal anemia and apoptosis of red cell progenitorsin Stat5aminusminus5bminusminus mice a direct role for stat5 in Bcl-X(L)inductionrdquo Cell vol 98 no 2 pp 181ndash191 1999

[38] G Li Z Wang Y Zhang et al ldquoSTAT5 requires the N-domainto maintain hematopoietic stem cell repopulating function andappropriate lymphoid-myeloid lineage outputrdquo ExperimentalHematology vol 35 no 11 pp 1684ndash1694 2007

[39] S Negoro K Kunisada E Tone et al ldquoActivation of JAKSTATpathway transduces cytoprotective signal in rat acute myocar-dial infarctionrdquo Cardiovascular Research vol 47 no 4 pp 797ndash805 2000

[40] H C Blair L J Robinson and M Zaidi ldquoOsteoclast signallingpathwaysrdquo Biochemical and Biophysical Research Communica-tions vol 328 no 3 pp 728ndash738 2005

[41] C J Malemud ldquoProtein kinases in chondrocyte signaling andosteoarthritisrdquo Clinical Orthopaedics and Related Research no427 supplement pp S145ndashS151 2004

[42] E M Pinheiro and D J Montell ldquoRequirement for Par-6 andBazooka inDrosophila border cell migrationrdquoDevelopment vol131 no 21 pp 5243ndash5251 2004

[43] D Strutt ldquoFrizzled signalling and cell polarisation inDrosophilaand vertebratesrdquo Development vol 130 no 19 pp 4501ndash45132003

[44] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[45] J Guo J Jin and L F Cooper ldquoDissection of sets of genes thatcontrol the character of wnt5a-deficient mouse calvarial cellsrdquoBone vol 43 no 5 pp 961ndash971 2008

[46] M Nishizuka A Koyanagi S Osada and M Imagawa ldquoWnt4and Wnt5a promote adipocyte differentiationrdquo FEBS Lettersvol 582 no 21-22 pp 3201ndash3205 2008

[47] Y Yang L Topol H Lee and J Wu ldquoWnt5a andWnt5b exhibitdistinct activities in coordinating chondrocyte proliferation anddifferentiationrdquo Development vol 130 no 5 pp 1003ndash10152003

[48] D J Van Den Berg A K Sharma E Bruno and R Hoff-man ldquoRole of members of the Wnt gene family in humanhematopoiesisrdquo Blood vol 92 no 9 pp 3189ndash3202 1998

[49] D J Prockop C A Gregory and J L Spees ldquoOne strategy forcell and gene therapy harnessing the power of adult stemcells torepair tissuesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 1 pp 11917ndash119232003

[50] X He J-P Saint-Jeannet Y Wang J Nathans I Dawid and HVarmus ldquoA member of the Frizzled protein family mediatingaxis induction byWnt-5Ardquo Science vol 275 no 5306 pp 1652ndash1654 1997

Stem Cells International 13

[51] D G Phinney G Kopen R L Isaacson and D J ProckopldquoPlastic adherent stromal cells from the bone marrow ofcommonly used strains of inbred mice variations in yieldgrowth and differentiationrdquo Journal of Cellular Biochemistryvol 72 no 4 pp 570ndash585 1999

[52] N Sasaki T Sasamura H O Ishikawa et al ldquoPolarized exo-cytosis and transcytosis of Notch during its apical localizationin Drosophila epithelial cellsrdquo Genes to Cells vol 12 no 1 pp89ndash103 2007

[53] M Handler X Yang and J Shen ldquoPresenilin-1 regulatesneuronal differentiation during neurogenesisrdquo Developmentvol 127 no 12 pp 2593ndash2606 2000

[54] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006

[55] A W Duncan F M Rattis L N DiMascio et al ldquoIntegrationof Notch andWnt signaling in hematopoietic stem cell mainte-nancerdquo Nature Immunology vol 6 no 3 pp 314ndash322 2005

[56] N Carlesso J C Aster J Sklar and D T Scadden ldquoNotch1-induced delay of human hematopoietic progenitor cell differen-tiation is associated with altered cell cycle kineticsrdquo Blood vol93 no 3 pp 838ndash848 1999

[57] I Maillard U Koch A Dumortier et al ldquoCanonical notch sig-naling is dispensable for the maintenance of adult hematopoi-etic stem cellsrdquo Cell Stem Cell vol 2 no 4 pp 356ndash366 2008

[58] G Pearson F Robinson T B Gibson et al ldquoMitogen-activatedprotein (MAP) kinase pathways regulation and physiologicalfunctionsrdquo Endocrine Reviews vol 22 no 2 pp 153ndash183 2001

[59] R E Dolmetsch R S Lewis C C Goodnow and J I HealyldquoDifferential activation of transcription factors induced by Ca2+response amplitude and durationrdquoNature vol 386 no 6627 pp855ndash858 1997

[60] S Kawano S Shoji S Ichinose K Yamagata M Tagami andM Hiraoka ldquoCharacterization of Ca2+ signaling pathways inhuman mesenchymal stem cellsrdquo Cell Calcium vol 32 no 4pp 165ndash174 2002

[61] B Ye ldquoCa2+ oscillations and its transporters in mesenchymalstem cellsrdquo Physiological Research vol 59 no 3 pp 323ndash3292010

[62] J E Ip Y Wu J Huang L Zhang R E Pratt and V J DzauldquoMesenchymal stem cells use integrin 1205731 not CXC chemokinereceptor 4 for myocardial migration and engraftmentrdquoMolecu-lar Biology of the Cell vol 18 no 8 pp 2873ndash2882 2007

[63] C A Opitz U M Litzenburger C Lutz et al ldquoToll-like recep-tor engagement enhances the immunosuppressive propertiesof human bone marrow-derived mesenchymal stem cells byinducing indoleamine-23-dioxygenase-1 via Interferon-120573 andprotein kinase Rrdquo Stem Cells vol 27 no 4 pp 909ndash919 2009

[64] B Kulterer G Friedl A Jandrositz et al ldquoGene expressionprofiling of human mesenchymal stem cells derived from bonemarrow during expansion and osteoblast differentiationrdquo BMCGenomics vol 8 article 70 2007

[65] W G Li and X X Xu ldquoThe expression of N-cadherinfibronectin during chondrogenic differentiation of MSCinduced by TGF-1205731rdquo Chinese Journal of Traumatology vol 8no 6 pp 349ndash351 2005

[66] F Ng S Boucher S Koh et al ldquoPDGFTGF-120573 and FGF signal-ing is important for differentiation and growth of mesenchymalstem cells (mscs) transcriptional profiling can identify markersand signaling pathways important in differentiation of MSCsinto adipogenic chondrogenic and osteogenic lineagesrdquo Bloodvol 112 no 2 pp 295ndash307 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

10 Stem Cells International

[51] Currently available data suggest differences which maybe characteristic not only for proliferation and differentiationprograms but also for the phenotype of MSCs from umbilicalcord blood and bone marrow which is in agreement withthe results published by Baksh et al [7] The remaining genestake part in theWnt signaling cascade of increased activity inCD34minus from umbilical cord blood such as MMP7 belongingto metalloproteinases which play a role in the extracellulararray remodeling and wound healing [5]

The Notch signaling pathway belongs to the processes ofintercellular communication which regulate gene expressionpatterns defining a dualistic character of stem cells reflectedby the asymmetric cell division [52] Additionally the Notchsignaling cascade controls various cells biological processesnot only proliferation and differentiation but also apoptosisand regeneration The signaling activity of Notch pathwaygenes determined in the study of 38 genes showed theirsimilar expression as defined by the KEGG classification(Table 4) A similar activity level was noted for all four Notch1ndash4 receptors as well as for JAG1 and JAG2 ligands The dif-ferences were related to two genes DTX4 and PSEN1 whichwere overactive in CD34minus cells from umbilical cord bloodThe function of the PSEN1 gene is mainly developmentalprocesses regarding cell differentiation during neurogenesisAnd the lack of this gene leads to a premature directeddifferentiation of neural progenitor cells [53] The activity ofthe Notch gene cascade plays an important role at all levelsof stem cell development starting from the embryonic stemcell through fetal cells to adult organism stem cells Notchis significantly involved in the asymmetric segregation offactors determining cell purpose during replication divisionsof the stem cell poolThe significance of receptors and ligandsis best described in the hematopoietic stem cells Expressionof the constitutively active Notch1 gene in hematopoieticstem cells induces the immortalized cell lineages which canreproduce the myeloid and lymphoid cell lineage in a long-term repopulation in animal studies [54 55] Apart from thatit delays differentiation of human hematopoietic progenitorstem cells in vitro [56] The studies suggest that Notch1 is animportant factor inHSC self-renewal and can also participatein the inhibition of stem cell differentiation The scientificfindings reveal that a suppression of the Notch signalingpathway via deletion of key genes in HSC did not cause cellsto lose their ability of long-term hematopoiesis renewal inanimal models [57] Therefore the role of ligands and Notchreceptors in HSC has not been precisely defined yet andcurrent publications do not provide precise data regardingthe role played by particular Notch signaling pathway genesin mesenchymal stem cells The only phenomenon whichappears to be superior and characteristic for all stem cellsincluding MSCs is the significance of the Notch cascadein asymmetrical cell divisions This is a significant com-ponent of stem cells population self-renewal as well asthe functionality of the cascade in the stem niche Thisphenomenon constitutes an integral part of the hypothesisabout stem cell population aging However further inves-tigation will be required to determine if the differentiallyexpressed genes described in the studies above could serve asstemness genes

Remaining signaling pathways which revealed increasedactivity in the studied CD34minus cells of umbilical cord bloodincludedMAPK (Mitogen-Activated Protein Kinase Figure 2)which regulates a wide range of cell biological activities suchas gene expression mitosis proliferation differentiationmigration and apoptosis processes [58]TheMAPKpathwaystake part in regulation of early embryonic development andin defining the role of embryonic stem cells from the earlydevelopmental phases until the moment of formation ofdifferentiated adult cells However this precise role played bythe MAPK pathways in various stem cell types has not yetbeen defined

Transmission via calcium ions was also singled out inthe group of the overactive signaling pathways of the studiedCD34minus stem cells of umbilical cord blood versus CD34minus stemcells of bone marrow (Figure 2) The ions are a vital andcommonly present mediator in the process of extracellularsignals transmission and conversion into a cellular responseVariations of calcium ions concentrations regulate geneexpression through an impact on the activity of numeroussignaling pathways Intracellular increase of calcium ionsconcentration also regulates cell activities including adhe-sion motility gene expression and proliferation [59] Thesignificance of transmission via calcium ions and the ways oftheir concentration changes in mesenchymal stem cells stillhave not been studied Kawano et al were the first to reportthat in the nondifferentiated MSCs there are spontaneouschanges of calcium ions concentrations despite the lack ofany stimulating factors [60] As reported the calcium iontransmission through the activation of different gene groupsmay direct the cells onto specific developmental pathways[61]

The integrin-mediated signaling pathway takes part inextracellular array remodeling which means that it con-trols cell behavior and tissue organization Its increasedactivity was observed in our study in CD34minus stem cells ofumbilical cord blood (Figure 2) In recent years the role ofintegrins and selectins has been stressed in the adhesion andtransendothelial migration which has a significant meaningin the processes of repopulation of tissues damaged bydiseases and their regeneration via the circulating MSCs Forinstance Ip et al showed that blocking of beta 1-integrinwhich constitutes a component of the adhesive moleculeVLA-4 in MSC weakened the ability of mesenchymal stemcells to engraft into the infarction-changedmyocardium [62]The above results may suggest a better migration abilityof CD34minus stem cells of umbilical cord blood into the areaof pathologic tissue MSCs own cytokinetic activity thathas similar significance in terms of tissue regenerationThe study of cytokinetic activity of CD34minus stem cells ofumbilical cord blood showed increased activity of signalingpathways activated via cytokines (Figure 2 Table 2) On theother hand the TLR signaling pathway (toll-like receptorsignaling) which in our own results revealed a significantincreased activity in CD34minus stem cells of umbilical cordblood (Figure 2) increases the immunosuppressive charac-teristics of MSCs Weakening of the immunological responsegenerated by the TLR pathway depends on the produc-tion of immunosuppressive kynurenines by the indoleamine

Stem Cells International 11

23-dioxygenase enzyme which degrades tryptophan Induc-tion of indoleamine 23-dioxygenase by TLR entails autocrinesignaling loop of interferon-beta which plays a role in MSCsimmunomodulative effects [63]

According to the GO and KEGG classifications carriedout for cluster 13 with the gene pool of a decreased expressiona statistically significant decrease of gene activity was shownfor the TGF-beta and BMP pathways (Figure 3 Table 3) Itis believed that the family of TGF-beta members whichalso includes BMP plays an important role in initiating theprocess of a directed MSCs differentiation TGF-beta sig-naling transmission runs through specific serine-threoninekinase receptors and their nuclear receptors called the Smadproteins TGF-beta signaling transmission promotes theprocess of early differentiation stages into the chondrogenicand osteogenic lineages [64 65] However cell differentiatinginto the adipogenic lineage results in inhibition of the TGF-beta signaling pathway This was confirmed in experimentalstudies developed by Ng et al where the inhibition of TGF-beta transmission resulted in an increase of adipogenesisand a decrease of differentiating ability towards chondrocytes[66] The BMP signaling pathway strongly promotes differ-entiation towards osteoblasts but it also leads to inhibitionof myogenesis and adipogenesis Such a gene expressionprofile may suggest that the MSCs biological characteristicsare strongly influenced by the significance of stem cell nicheTherefore the mesenchymal stem cells from bone marrowmay be more involved in the promotion of the phenomenawhich maintain the stability of bone marrow environment

5 Conclusions

Multipotentiality of the mesenchymal stem cells their highproliferation ability and not too-demanding culturing con-ditions reveal their potential for wide application in variousmedical areas These are the reasons for the common pursuitto understand their biology based onmore andmore sublimebiomolecular procedures Scientists have been searchingfor an unequivocal marker which would characterize themesenchymal stem cells and simultaneously would enable aseparation of a homogenous MSC population Understand-ing of molecular mechanisms controlling self-replication anddifferentiation is becoming a fundamental issue for clinicalapplication of MSC In this respect the activity of signalingpathways is important yet the interpretation of the pathwaysespecially their interdependence constitutes a great challengefor the scientific world Moreover the answer to the questionregarding MSC innidation and its survival ability in hosttissues after transplantation is yet to be found Explanationof the abovementioned issues will require a more globalunderstanding of biological activity of these cells especiallyon the basis of the significance of the mesenchymal nicheenvironment of stem cells and its impact on the gene andepigenetic activity It should be assumed that continuedprogress in the understanding of MSCs will finally lead totheir common therapeutic and preventive application andconsequently to the improvement of the quality and lengthof human life

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] R J Deans andA BMoseley ldquoMesenchymal stem cells biologyand potential clinical usesrdquo Experimental Hematology vol 28no 8 pp 875ndash884 2000

[2] M F Pittenger A M Mackay S C Beck et al ldquoMultilineagepotential of adult human mesenchymal stem cellsrdquo Science vol284 no 5411 pp 143ndash147 1999

[3] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006

[4] Y-J Chang D T-B Shih C-P Tseng T-B Hsieh D-C Leeand S-M Hwang ldquoDisparate mesenchyme-lineage tendenciesin mesenchymal stem cells from human bone marrow andumbilical cord bloodrdquo Stem Cells vol 24 no 3 pp 679ndash6852006

[5] R A Panepucci J L C Siufi W A Silva Jr et al ldquoComparisonof gene expression of umbilical cord vein and bone marrowndashderived mesenchymal stem cellsrdquo STEM CELLS vol 22 no 7pp 1263ndash1278 2004

[6] C K Rebelatto A M Aguiar M P Moretao et al ldquoDissimilardifferentiation of mesenchymal stem cells from bone marrowumbilical cord blood and adipose tissuerdquo Experimental Biologyand Medicine vol 233 no 7 pp 901ndash913 2008

[7] D Baksh R Yao and R S Tuan ldquoComparison of proliferativeand multilineage differentiation potential of human mesenchy-mal stem cells derived from umbilical cord and bone marrowrdquoStem Cells vol 25 no 6 pp 1384ndash1392 2007

[8] D L Clarke C B Johansson J Wilbertz et al ldquoGeneralizedpotential of adult neural stem cellsrdquo Science vol 288 no 5471pp 1660ndash1663 2000

[9] D J Anderson F H Gage and I L Weissman ldquoCan stem cellscross lineage boundariesrdquo Nature Medicine vol 7 no 4 pp393ndash395 2001

[10] D L Stemple and D J Anderson ldquoIsolation of a stem cell forneurons and glia from the mammalian neural crestrdquo Cell vol71 no 6 pp 973ndash985 1992

[11] R L Rietze H Valcanis G F Brooker T Thomas A K Vossand P F Bartlett ldquoPurification of a pluripotent neural stem cellfrom the adult mouse brainrdquoNature vol 412 no 6848 pp 736ndash739 2001

[12] R M Seaberg S R Smukler T J Kieffer et al ldquoClonalidentification of multipotent precursors from adult mousepancreas that generate neural and pancreatic lineagesrdquo NatureBiotechnology vol 22 no 9 pp 1115ndash1124 2004

[13] A P Beltrami L Barlucchi D Torella et al ldquoAdult cardiac stemcells are multipotent and support myocardial regenerationrdquoCell vol 114 no 6 pp 763ndash776 2003

[14] L Yang S Li H Hatch et al ldquoIn vitro trans-differentiation ofadult hepatic stem cells into pancreatic endocrine hormone-producing cellsrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 99 no 12 pp 8078ndash80832002

[15] I Chambers D Colby M Robertson et al ldquoFunctional expres-sion cloning of Nanog a pluripotency sustaining factor inembryonic stem cellsrdquo Cell vol 113 no 5 pp 643ndash655 2003

12 Stem Cells International

[16] M Dai P Wang A D Boyd et al ldquoEvolving genetranscriptdefinitions significantly alter the interpretation of GeneChipdatardquo Nucleic Acids Research vol 33 pp 175ndash179 2005

[17] RA Irizarry BHobbs F Collin et al ldquoExploration normaliza-tion and summaries of high density oligonucleotide array probelevel datardquo Biostatistics vol 4 no 2 pp 249ndash264 2003

[18] R B Dean and W J Dixon ldquoSimplified statistics for smallnumbers of observationsrdquo Analytical Chemistry vol 23 no 4pp 636ndash638 1951

[19] G J McLachlan and D Peel ldquoOn computational aspectsof clustering via mixtures of normal and t-componentsrdquo inProceedings of the American Statistical Association (BayesianStatistical Science Section) American Statistical AssociationIndianapolis Ind USA 2000

[20] J Polanska P Widlak J Rzeszowska-Wolny M Kimmel andA Polanski ldquoGaussian mixture decomposition of time-courseDNA microarray datardquo in Mathematical Modeling of BiologicalSystems Volume I pp 351ndash359 Birkhauser Boston Mass USA2007

[21] A J Friedenstein U F Gorskaja and N N Kulagina ldquoFibrob-last precursors in normal and irradiated mouse hematopoieticorgansrdquo Experimental Hematology vol 4 no 5 pp 267ndash2741976

[22] S Wakitani T Saito and A I Caplan ldquoMyogenic cells derivedfrom rat bone marrow mesenchymal stem cells exposed to 5-azacytidinerdquoMuscle ampNerve vol 18 no 12 pp 1417ndash1426 1995

[23] D Woodbury E J Schwarz D J Prockop and I B BlackldquoAdult rat and human bone marrow stromal cells differentiateinto neuronsrdquo Journal of Neuroscience Research vol 61 no 4pp 364ndash370 2000

[24] M Reyes T Lund T Lenvik D Aguiar L Koodie and CM Verfaillie ldquoPurification and ex vivo expansion of postnatalhumanmarrowmesodermal progenitor cellsrdquoBlood vol 98 no9 pp 2615ndash2625 2001

[25] M Ramalho-Santos S Yoon Y Matsuzaki R C Mulliganand D A Melton ldquolsquoStemnessrsquo transcriptional profiling ofembryonic and adult stem cellsrdquo Science vol 298 no 5593 pp597ndash600 2002

[26] KMitsui Y Tokuzawa H Itoh et al ldquoThe homeoprotein nanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003

[27] S Okumura-Nakanishi M Saito H Niwa and F IshikawaldquoOct-34 and Sox2 regulate Oct-34 gene in embryonic stemcellsrdquo The Journal of Biological Chemistry vol 280 no 7 pp5307ndash5317 2005

[28] F Ulloa-Montoya B L Kidder K A Pauwelyn et al ldquoCompar-ative transcriptome analysis of embryonic and adult stem cellswith extended and limited differentiation capacityrdquo GenomeBiology vol 8 no 8 article R163 2007

[29] L Song N EWebb Y Song and R S Tuan ldquoIdentification andfunctional analysis of candidate genes regulating mesenchymalstem cell self-renewal and multipotencyrdquo STEMCELLS vol 24no 7 pp 1707ndash1718 2006

[30] A H H Van Boxel-Dezaire M R S Rani and G R StarkldquoComplexmodulation of cell type-specific signaling in responseto type I interferonsrdquo Immunity vol 25 no 3 pp 361ndash372 2006

[31] C G Kim J J Lee D Y Jung et al ldquoProfiling of differentiallyexpressed genes in human stem cells by cDNA microarrayrdquoMolecules and Cells vol 21 no 3 pp 343ndash355 2006

[32] T Bowman R Garcia J Turkson and R Jove ldquoSTATs inoncogenesisrdquo Oncogene vol 19 no 21 pp 2474ndash2488 2000

[33] A Stephanou andD S Latchman ldquoOpposing actions of STAT-1and STAT-3rdquo Growth Factors vol 23 no 3 pp 177ndash182 2005

[34] A Martino J H Holmes J D Lord J J Moon and B HNelson ldquoStat5 and Sp1 regulate transcription of the cyclin D2gene in response to IL-2rdquo The Journal of Immunology vol 166no 3 pp 1723ndash1729 2001

[35] I Matsumura T Kitamura H Wakao et al ldquoTranscriptionalregulation of the cyclin D1 promoter by STAT5 its involvementin cytokine-dependent growth of hematopoietic cellsrdquo TheEMBO Journal vol 18 no 5 pp 1367ndash1377 1999

[36] V Calo M Migliavacca V Bazan et al ldquoSTAT proteins fromnormal control of cellular events to tumorigenesisrdquo Journal ofCellular Physiology vol 197 no 2 pp 157ndash168 2003

[37] M Socolovsky A E J Fallon S Wang C Brugnara and HF Lodish ldquoFetal anemia and apoptosis of red cell progenitorsin Stat5aminusminus5bminusminus mice a direct role for stat5 in Bcl-X(L)inductionrdquo Cell vol 98 no 2 pp 181ndash191 1999

[38] G Li Z Wang Y Zhang et al ldquoSTAT5 requires the N-domainto maintain hematopoietic stem cell repopulating function andappropriate lymphoid-myeloid lineage outputrdquo ExperimentalHematology vol 35 no 11 pp 1684ndash1694 2007

[39] S Negoro K Kunisada E Tone et al ldquoActivation of JAKSTATpathway transduces cytoprotective signal in rat acute myocar-dial infarctionrdquo Cardiovascular Research vol 47 no 4 pp 797ndash805 2000

[40] H C Blair L J Robinson and M Zaidi ldquoOsteoclast signallingpathwaysrdquo Biochemical and Biophysical Research Communica-tions vol 328 no 3 pp 728ndash738 2005

[41] C J Malemud ldquoProtein kinases in chondrocyte signaling andosteoarthritisrdquo Clinical Orthopaedics and Related Research no427 supplement pp S145ndashS151 2004

[42] E M Pinheiro and D J Montell ldquoRequirement for Par-6 andBazooka inDrosophila border cell migrationrdquoDevelopment vol131 no 21 pp 5243ndash5251 2004

[43] D Strutt ldquoFrizzled signalling and cell polarisation inDrosophilaand vertebratesrdquo Development vol 130 no 19 pp 4501ndash45132003

[44] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[45] J Guo J Jin and L F Cooper ldquoDissection of sets of genes thatcontrol the character of wnt5a-deficient mouse calvarial cellsrdquoBone vol 43 no 5 pp 961ndash971 2008

[46] M Nishizuka A Koyanagi S Osada and M Imagawa ldquoWnt4and Wnt5a promote adipocyte differentiationrdquo FEBS Lettersvol 582 no 21-22 pp 3201ndash3205 2008

[47] Y Yang L Topol H Lee and J Wu ldquoWnt5a andWnt5b exhibitdistinct activities in coordinating chondrocyte proliferation anddifferentiationrdquo Development vol 130 no 5 pp 1003ndash10152003

[48] D J Van Den Berg A K Sharma E Bruno and R Hoff-man ldquoRole of members of the Wnt gene family in humanhematopoiesisrdquo Blood vol 92 no 9 pp 3189ndash3202 1998

[49] D J Prockop C A Gregory and J L Spees ldquoOne strategy forcell and gene therapy harnessing the power of adult stemcells torepair tissuesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 1 pp 11917ndash119232003

[50] X He J-P Saint-Jeannet Y Wang J Nathans I Dawid and HVarmus ldquoA member of the Frizzled protein family mediatingaxis induction byWnt-5Ardquo Science vol 275 no 5306 pp 1652ndash1654 1997

Stem Cells International 13

[51] D G Phinney G Kopen R L Isaacson and D J ProckopldquoPlastic adherent stromal cells from the bone marrow ofcommonly used strains of inbred mice variations in yieldgrowth and differentiationrdquo Journal of Cellular Biochemistryvol 72 no 4 pp 570ndash585 1999

[52] N Sasaki T Sasamura H O Ishikawa et al ldquoPolarized exo-cytosis and transcytosis of Notch during its apical localizationin Drosophila epithelial cellsrdquo Genes to Cells vol 12 no 1 pp89ndash103 2007

[53] M Handler X Yang and J Shen ldquoPresenilin-1 regulatesneuronal differentiation during neurogenesisrdquo Developmentvol 127 no 12 pp 2593ndash2606 2000

[54] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006

[55] A W Duncan F M Rattis L N DiMascio et al ldquoIntegrationof Notch andWnt signaling in hematopoietic stem cell mainte-nancerdquo Nature Immunology vol 6 no 3 pp 314ndash322 2005

[56] N Carlesso J C Aster J Sklar and D T Scadden ldquoNotch1-induced delay of human hematopoietic progenitor cell differen-tiation is associated with altered cell cycle kineticsrdquo Blood vol93 no 3 pp 838ndash848 1999

[57] I Maillard U Koch A Dumortier et al ldquoCanonical notch sig-naling is dispensable for the maintenance of adult hematopoi-etic stem cellsrdquo Cell Stem Cell vol 2 no 4 pp 356ndash366 2008

[58] G Pearson F Robinson T B Gibson et al ldquoMitogen-activatedprotein (MAP) kinase pathways regulation and physiologicalfunctionsrdquo Endocrine Reviews vol 22 no 2 pp 153ndash183 2001

[59] R E Dolmetsch R S Lewis C C Goodnow and J I HealyldquoDifferential activation of transcription factors induced by Ca2+response amplitude and durationrdquoNature vol 386 no 6627 pp855ndash858 1997

[60] S Kawano S Shoji S Ichinose K Yamagata M Tagami andM Hiraoka ldquoCharacterization of Ca2+ signaling pathways inhuman mesenchymal stem cellsrdquo Cell Calcium vol 32 no 4pp 165ndash174 2002

[61] B Ye ldquoCa2+ oscillations and its transporters in mesenchymalstem cellsrdquo Physiological Research vol 59 no 3 pp 323ndash3292010

[62] J E Ip Y Wu J Huang L Zhang R E Pratt and V J DzauldquoMesenchymal stem cells use integrin 1205731 not CXC chemokinereceptor 4 for myocardial migration and engraftmentrdquoMolecu-lar Biology of the Cell vol 18 no 8 pp 2873ndash2882 2007

[63] C A Opitz U M Litzenburger C Lutz et al ldquoToll-like recep-tor engagement enhances the immunosuppressive propertiesof human bone marrow-derived mesenchymal stem cells byinducing indoleamine-23-dioxygenase-1 via Interferon-120573 andprotein kinase Rrdquo Stem Cells vol 27 no 4 pp 909ndash919 2009

[64] B Kulterer G Friedl A Jandrositz et al ldquoGene expressionprofiling of human mesenchymal stem cells derived from bonemarrow during expansion and osteoblast differentiationrdquo BMCGenomics vol 8 article 70 2007

[65] W G Li and X X Xu ldquoThe expression of N-cadherinfibronectin during chondrogenic differentiation of MSCinduced by TGF-1205731rdquo Chinese Journal of Traumatology vol 8no 6 pp 349ndash351 2005

[66] F Ng S Boucher S Koh et al ldquoPDGFTGF-120573 and FGF signal-ing is important for differentiation and growth of mesenchymalstem cells (mscs) transcriptional profiling can identify markersand signaling pathways important in differentiation of MSCsinto adipogenic chondrogenic and osteogenic lineagesrdquo Bloodvol 112 no 2 pp 295ndash307 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Stem Cells International 11

23-dioxygenase enzyme which degrades tryptophan Induc-tion of indoleamine 23-dioxygenase by TLR entails autocrinesignaling loop of interferon-beta which plays a role in MSCsimmunomodulative effects [63]

According to the GO and KEGG classifications carriedout for cluster 13 with the gene pool of a decreased expressiona statistically significant decrease of gene activity was shownfor the TGF-beta and BMP pathways (Figure 3 Table 3) Itis believed that the family of TGF-beta members whichalso includes BMP plays an important role in initiating theprocess of a directed MSCs differentiation TGF-beta sig-naling transmission runs through specific serine-threoninekinase receptors and their nuclear receptors called the Smadproteins TGF-beta signaling transmission promotes theprocess of early differentiation stages into the chondrogenicand osteogenic lineages [64 65] However cell differentiatinginto the adipogenic lineage results in inhibition of the TGF-beta signaling pathway This was confirmed in experimentalstudies developed by Ng et al where the inhibition of TGF-beta transmission resulted in an increase of adipogenesisand a decrease of differentiating ability towards chondrocytes[66] The BMP signaling pathway strongly promotes differ-entiation towards osteoblasts but it also leads to inhibitionof myogenesis and adipogenesis Such a gene expressionprofile may suggest that the MSCs biological characteristicsare strongly influenced by the significance of stem cell nicheTherefore the mesenchymal stem cells from bone marrowmay be more involved in the promotion of the phenomenawhich maintain the stability of bone marrow environment

5 Conclusions

Multipotentiality of the mesenchymal stem cells their highproliferation ability and not too-demanding culturing con-ditions reveal their potential for wide application in variousmedical areas These are the reasons for the common pursuitto understand their biology based onmore andmore sublimebiomolecular procedures Scientists have been searchingfor an unequivocal marker which would characterize themesenchymal stem cells and simultaneously would enable aseparation of a homogenous MSC population Understand-ing of molecular mechanisms controlling self-replication anddifferentiation is becoming a fundamental issue for clinicalapplication of MSC In this respect the activity of signalingpathways is important yet the interpretation of the pathwaysespecially their interdependence constitutes a great challengefor the scientific world Moreover the answer to the questionregarding MSC innidation and its survival ability in hosttissues after transplantation is yet to be found Explanationof the abovementioned issues will require a more globalunderstanding of biological activity of these cells especiallyon the basis of the significance of the mesenchymal nicheenvironment of stem cells and its impact on the gene andepigenetic activity It should be assumed that continuedprogress in the understanding of MSCs will finally lead totheir common therapeutic and preventive application andconsequently to the improvement of the quality and lengthof human life

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] R J Deans andA BMoseley ldquoMesenchymal stem cells biologyand potential clinical usesrdquo Experimental Hematology vol 28no 8 pp 875ndash884 2000

[2] M F Pittenger A M Mackay S C Beck et al ldquoMultilineagepotential of adult human mesenchymal stem cellsrdquo Science vol284 no 5411 pp 143ndash147 1999

[3] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006

[4] Y-J Chang D T-B Shih C-P Tseng T-B Hsieh D-C Leeand S-M Hwang ldquoDisparate mesenchyme-lineage tendenciesin mesenchymal stem cells from human bone marrow andumbilical cord bloodrdquo Stem Cells vol 24 no 3 pp 679ndash6852006

[5] R A Panepucci J L C Siufi W A Silva Jr et al ldquoComparisonof gene expression of umbilical cord vein and bone marrowndashderived mesenchymal stem cellsrdquo STEM CELLS vol 22 no 7pp 1263ndash1278 2004

[6] C K Rebelatto A M Aguiar M P Moretao et al ldquoDissimilardifferentiation of mesenchymal stem cells from bone marrowumbilical cord blood and adipose tissuerdquo Experimental Biologyand Medicine vol 233 no 7 pp 901ndash913 2008

[7] D Baksh R Yao and R S Tuan ldquoComparison of proliferativeand multilineage differentiation potential of human mesenchy-mal stem cells derived from umbilical cord and bone marrowrdquoStem Cells vol 25 no 6 pp 1384ndash1392 2007

[8] D L Clarke C B Johansson J Wilbertz et al ldquoGeneralizedpotential of adult neural stem cellsrdquo Science vol 288 no 5471pp 1660ndash1663 2000

[9] D J Anderson F H Gage and I L Weissman ldquoCan stem cellscross lineage boundariesrdquo Nature Medicine vol 7 no 4 pp393ndash395 2001

[10] D L Stemple and D J Anderson ldquoIsolation of a stem cell forneurons and glia from the mammalian neural crestrdquo Cell vol71 no 6 pp 973ndash985 1992

[11] R L Rietze H Valcanis G F Brooker T Thomas A K Vossand P F Bartlett ldquoPurification of a pluripotent neural stem cellfrom the adult mouse brainrdquoNature vol 412 no 6848 pp 736ndash739 2001

[12] R M Seaberg S R Smukler T J Kieffer et al ldquoClonalidentification of multipotent precursors from adult mousepancreas that generate neural and pancreatic lineagesrdquo NatureBiotechnology vol 22 no 9 pp 1115ndash1124 2004

[13] A P Beltrami L Barlucchi D Torella et al ldquoAdult cardiac stemcells are multipotent and support myocardial regenerationrdquoCell vol 114 no 6 pp 763ndash776 2003

[14] L Yang S Li H Hatch et al ldquoIn vitro trans-differentiation ofadult hepatic stem cells into pancreatic endocrine hormone-producing cellsrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 99 no 12 pp 8078ndash80832002

[15] I Chambers D Colby M Robertson et al ldquoFunctional expres-sion cloning of Nanog a pluripotency sustaining factor inembryonic stem cellsrdquo Cell vol 113 no 5 pp 643ndash655 2003

12 Stem Cells International

[16] M Dai P Wang A D Boyd et al ldquoEvolving genetranscriptdefinitions significantly alter the interpretation of GeneChipdatardquo Nucleic Acids Research vol 33 pp 175ndash179 2005

[17] RA Irizarry BHobbs F Collin et al ldquoExploration normaliza-tion and summaries of high density oligonucleotide array probelevel datardquo Biostatistics vol 4 no 2 pp 249ndash264 2003

[18] R B Dean and W J Dixon ldquoSimplified statistics for smallnumbers of observationsrdquo Analytical Chemistry vol 23 no 4pp 636ndash638 1951

[19] G J McLachlan and D Peel ldquoOn computational aspectsof clustering via mixtures of normal and t-componentsrdquo inProceedings of the American Statistical Association (BayesianStatistical Science Section) American Statistical AssociationIndianapolis Ind USA 2000

[20] J Polanska P Widlak J Rzeszowska-Wolny M Kimmel andA Polanski ldquoGaussian mixture decomposition of time-courseDNA microarray datardquo in Mathematical Modeling of BiologicalSystems Volume I pp 351ndash359 Birkhauser Boston Mass USA2007

[21] A J Friedenstein U F Gorskaja and N N Kulagina ldquoFibrob-last precursors in normal and irradiated mouse hematopoieticorgansrdquo Experimental Hematology vol 4 no 5 pp 267ndash2741976

[22] S Wakitani T Saito and A I Caplan ldquoMyogenic cells derivedfrom rat bone marrow mesenchymal stem cells exposed to 5-azacytidinerdquoMuscle ampNerve vol 18 no 12 pp 1417ndash1426 1995

[23] D Woodbury E J Schwarz D J Prockop and I B BlackldquoAdult rat and human bone marrow stromal cells differentiateinto neuronsrdquo Journal of Neuroscience Research vol 61 no 4pp 364ndash370 2000

[24] M Reyes T Lund T Lenvik D Aguiar L Koodie and CM Verfaillie ldquoPurification and ex vivo expansion of postnatalhumanmarrowmesodermal progenitor cellsrdquoBlood vol 98 no9 pp 2615ndash2625 2001

[25] M Ramalho-Santos S Yoon Y Matsuzaki R C Mulliganand D A Melton ldquolsquoStemnessrsquo transcriptional profiling ofembryonic and adult stem cellsrdquo Science vol 298 no 5593 pp597ndash600 2002

[26] KMitsui Y Tokuzawa H Itoh et al ldquoThe homeoprotein nanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003

[27] S Okumura-Nakanishi M Saito H Niwa and F IshikawaldquoOct-34 and Sox2 regulate Oct-34 gene in embryonic stemcellsrdquo The Journal of Biological Chemistry vol 280 no 7 pp5307ndash5317 2005

[28] F Ulloa-Montoya B L Kidder K A Pauwelyn et al ldquoCompar-ative transcriptome analysis of embryonic and adult stem cellswith extended and limited differentiation capacityrdquo GenomeBiology vol 8 no 8 article R163 2007

[29] L Song N EWebb Y Song and R S Tuan ldquoIdentification andfunctional analysis of candidate genes regulating mesenchymalstem cell self-renewal and multipotencyrdquo STEMCELLS vol 24no 7 pp 1707ndash1718 2006

[30] A H H Van Boxel-Dezaire M R S Rani and G R StarkldquoComplexmodulation of cell type-specific signaling in responseto type I interferonsrdquo Immunity vol 25 no 3 pp 361ndash372 2006

[31] C G Kim J J Lee D Y Jung et al ldquoProfiling of differentiallyexpressed genes in human stem cells by cDNA microarrayrdquoMolecules and Cells vol 21 no 3 pp 343ndash355 2006

[32] T Bowman R Garcia J Turkson and R Jove ldquoSTATs inoncogenesisrdquo Oncogene vol 19 no 21 pp 2474ndash2488 2000

[33] A Stephanou andD S Latchman ldquoOpposing actions of STAT-1and STAT-3rdquo Growth Factors vol 23 no 3 pp 177ndash182 2005

[34] A Martino J H Holmes J D Lord J J Moon and B HNelson ldquoStat5 and Sp1 regulate transcription of the cyclin D2gene in response to IL-2rdquo The Journal of Immunology vol 166no 3 pp 1723ndash1729 2001

[35] I Matsumura T Kitamura H Wakao et al ldquoTranscriptionalregulation of the cyclin D1 promoter by STAT5 its involvementin cytokine-dependent growth of hematopoietic cellsrdquo TheEMBO Journal vol 18 no 5 pp 1367ndash1377 1999

[36] V Calo M Migliavacca V Bazan et al ldquoSTAT proteins fromnormal control of cellular events to tumorigenesisrdquo Journal ofCellular Physiology vol 197 no 2 pp 157ndash168 2003

[37] M Socolovsky A E J Fallon S Wang C Brugnara and HF Lodish ldquoFetal anemia and apoptosis of red cell progenitorsin Stat5aminusminus5bminusminus mice a direct role for stat5 in Bcl-X(L)inductionrdquo Cell vol 98 no 2 pp 181ndash191 1999

[38] G Li Z Wang Y Zhang et al ldquoSTAT5 requires the N-domainto maintain hematopoietic stem cell repopulating function andappropriate lymphoid-myeloid lineage outputrdquo ExperimentalHematology vol 35 no 11 pp 1684ndash1694 2007

[39] S Negoro K Kunisada E Tone et al ldquoActivation of JAKSTATpathway transduces cytoprotective signal in rat acute myocar-dial infarctionrdquo Cardiovascular Research vol 47 no 4 pp 797ndash805 2000

[40] H C Blair L J Robinson and M Zaidi ldquoOsteoclast signallingpathwaysrdquo Biochemical and Biophysical Research Communica-tions vol 328 no 3 pp 728ndash738 2005

[41] C J Malemud ldquoProtein kinases in chondrocyte signaling andosteoarthritisrdquo Clinical Orthopaedics and Related Research no427 supplement pp S145ndashS151 2004

[42] E M Pinheiro and D J Montell ldquoRequirement for Par-6 andBazooka inDrosophila border cell migrationrdquoDevelopment vol131 no 21 pp 5243ndash5251 2004

[43] D Strutt ldquoFrizzled signalling and cell polarisation inDrosophilaand vertebratesrdquo Development vol 130 no 19 pp 4501ndash45132003

[44] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[45] J Guo J Jin and L F Cooper ldquoDissection of sets of genes thatcontrol the character of wnt5a-deficient mouse calvarial cellsrdquoBone vol 43 no 5 pp 961ndash971 2008

[46] M Nishizuka A Koyanagi S Osada and M Imagawa ldquoWnt4and Wnt5a promote adipocyte differentiationrdquo FEBS Lettersvol 582 no 21-22 pp 3201ndash3205 2008

[47] Y Yang L Topol H Lee and J Wu ldquoWnt5a andWnt5b exhibitdistinct activities in coordinating chondrocyte proliferation anddifferentiationrdquo Development vol 130 no 5 pp 1003ndash10152003

[48] D J Van Den Berg A K Sharma E Bruno and R Hoff-man ldquoRole of members of the Wnt gene family in humanhematopoiesisrdquo Blood vol 92 no 9 pp 3189ndash3202 1998

[49] D J Prockop C A Gregory and J L Spees ldquoOne strategy forcell and gene therapy harnessing the power of adult stemcells torepair tissuesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 1 pp 11917ndash119232003

[50] X He J-P Saint-Jeannet Y Wang J Nathans I Dawid and HVarmus ldquoA member of the Frizzled protein family mediatingaxis induction byWnt-5Ardquo Science vol 275 no 5306 pp 1652ndash1654 1997

Stem Cells International 13

[51] D G Phinney G Kopen R L Isaacson and D J ProckopldquoPlastic adherent stromal cells from the bone marrow ofcommonly used strains of inbred mice variations in yieldgrowth and differentiationrdquo Journal of Cellular Biochemistryvol 72 no 4 pp 570ndash585 1999

[52] N Sasaki T Sasamura H O Ishikawa et al ldquoPolarized exo-cytosis and transcytosis of Notch during its apical localizationin Drosophila epithelial cellsrdquo Genes to Cells vol 12 no 1 pp89ndash103 2007

[53] M Handler X Yang and J Shen ldquoPresenilin-1 regulatesneuronal differentiation during neurogenesisrdquo Developmentvol 127 no 12 pp 2593ndash2606 2000

[54] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006

[55] A W Duncan F M Rattis L N DiMascio et al ldquoIntegrationof Notch andWnt signaling in hematopoietic stem cell mainte-nancerdquo Nature Immunology vol 6 no 3 pp 314ndash322 2005

[56] N Carlesso J C Aster J Sklar and D T Scadden ldquoNotch1-induced delay of human hematopoietic progenitor cell differen-tiation is associated with altered cell cycle kineticsrdquo Blood vol93 no 3 pp 838ndash848 1999

[57] I Maillard U Koch A Dumortier et al ldquoCanonical notch sig-naling is dispensable for the maintenance of adult hematopoi-etic stem cellsrdquo Cell Stem Cell vol 2 no 4 pp 356ndash366 2008

[58] G Pearson F Robinson T B Gibson et al ldquoMitogen-activatedprotein (MAP) kinase pathways regulation and physiologicalfunctionsrdquo Endocrine Reviews vol 22 no 2 pp 153ndash183 2001

[59] R E Dolmetsch R S Lewis C C Goodnow and J I HealyldquoDifferential activation of transcription factors induced by Ca2+response amplitude and durationrdquoNature vol 386 no 6627 pp855ndash858 1997

[60] S Kawano S Shoji S Ichinose K Yamagata M Tagami andM Hiraoka ldquoCharacterization of Ca2+ signaling pathways inhuman mesenchymal stem cellsrdquo Cell Calcium vol 32 no 4pp 165ndash174 2002

[61] B Ye ldquoCa2+ oscillations and its transporters in mesenchymalstem cellsrdquo Physiological Research vol 59 no 3 pp 323ndash3292010

[62] J E Ip Y Wu J Huang L Zhang R E Pratt and V J DzauldquoMesenchymal stem cells use integrin 1205731 not CXC chemokinereceptor 4 for myocardial migration and engraftmentrdquoMolecu-lar Biology of the Cell vol 18 no 8 pp 2873ndash2882 2007

[63] C A Opitz U M Litzenburger C Lutz et al ldquoToll-like recep-tor engagement enhances the immunosuppressive propertiesof human bone marrow-derived mesenchymal stem cells byinducing indoleamine-23-dioxygenase-1 via Interferon-120573 andprotein kinase Rrdquo Stem Cells vol 27 no 4 pp 909ndash919 2009

[64] B Kulterer G Friedl A Jandrositz et al ldquoGene expressionprofiling of human mesenchymal stem cells derived from bonemarrow during expansion and osteoblast differentiationrdquo BMCGenomics vol 8 article 70 2007

[65] W G Li and X X Xu ldquoThe expression of N-cadherinfibronectin during chondrogenic differentiation of MSCinduced by TGF-1205731rdquo Chinese Journal of Traumatology vol 8no 6 pp 349ndash351 2005

[66] F Ng S Boucher S Koh et al ldquoPDGFTGF-120573 and FGF signal-ing is important for differentiation and growth of mesenchymalstem cells (mscs) transcriptional profiling can identify markersand signaling pathways important in differentiation of MSCsinto adipogenic chondrogenic and osteogenic lineagesrdquo Bloodvol 112 no 2 pp 295ndash307 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

12 Stem Cells International

[16] M Dai P Wang A D Boyd et al ldquoEvolving genetranscriptdefinitions significantly alter the interpretation of GeneChipdatardquo Nucleic Acids Research vol 33 pp 175ndash179 2005

[17] RA Irizarry BHobbs F Collin et al ldquoExploration normaliza-tion and summaries of high density oligonucleotide array probelevel datardquo Biostatistics vol 4 no 2 pp 249ndash264 2003

[18] R B Dean and W J Dixon ldquoSimplified statistics for smallnumbers of observationsrdquo Analytical Chemistry vol 23 no 4pp 636ndash638 1951

[19] G J McLachlan and D Peel ldquoOn computational aspectsof clustering via mixtures of normal and t-componentsrdquo inProceedings of the American Statistical Association (BayesianStatistical Science Section) American Statistical AssociationIndianapolis Ind USA 2000

[20] J Polanska P Widlak J Rzeszowska-Wolny M Kimmel andA Polanski ldquoGaussian mixture decomposition of time-courseDNA microarray datardquo in Mathematical Modeling of BiologicalSystems Volume I pp 351ndash359 Birkhauser Boston Mass USA2007

[21] A J Friedenstein U F Gorskaja and N N Kulagina ldquoFibrob-last precursors in normal and irradiated mouse hematopoieticorgansrdquo Experimental Hematology vol 4 no 5 pp 267ndash2741976

[22] S Wakitani T Saito and A I Caplan ldquoMyogenic cells derivedfrom rat bone marrow mesenchymal stem cells exposed to 5-azacytidinerdquoMuscle ampNerve vol 18 no 12 pp 1417ndash1426 1995

[23] D Woodbury E J Schwarz D J Prockop and I B BlackldquoAdult rat and human bone marrow stromal cells differentiateinto neuronsrdquo Journal of Neuroscience Research vol 61 no 4pp 364ndash370 2000

[24] M Reyes T Lund T Lenvik D Aguiar L Koodie and CM Verfaillie ldquoPurification and ex vivo expansion of postnatalhumanmarrowmesodermal progenitor cellsrdquoBlood vol 98 no9 pp 2615ndash2625 2001

[25] M Ramalho-Santos S Yoon Y Matsuzaki R C Mulliganand D A Melton ldquolsquoStemnessrsquo transcriptional profiling ofembryonic and adult stem cellsrdquo Science vol 298 no 5593 pp597ndash600 2002

[26] KMitsui Y Tokuzawa H Itoh et al ldquoThe homeoprotein nanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003

[27] S Okumura-Nakanishi M Saito H Niwa and F IshikawaldquoOct-34 and Sox2 regulate Oct-34 gene in embryonic stemcellsrdquo The Journal of Biological Chemistry vol 280 no 7 pp5307ndash5317 2005

[28] F Ulloa-Montoya B L Kidder K A Pauwelyn et al ldquoCompar-ative transcriptome analysis of embryonic and adult stem cellswith extended and limited differentiation capacityrdquo GenomeBiology vol 8 no 8 article R163 2007

[29] L Song N EWebb Y Song and R S Tuan ldquoIdentification andfunctional analysis of candidate genes regulating mesenchymalstem cell self-renewal and multipotencyrdquo STEMCELLS vol 24no 7 pp 1707ndash1718 2006

[30] A H H Van Boxel-Dezaire M R S Rani and G R StarkldquoComplexmodulation of cell type-specific signaling in responseto type I interferonsrdquo Immunity vol 25 no 3 pp 361ndash372 2006

[31] C G Kim J J Lee D Y Jung et al ldquoProfiling of differentiallyexpressed genes in human stem cells by cDNA microarrayrdquoMolecules and Cells vol 21 no 3 pp 343ndash355 2006

[32] T Bowman R Garcia J Turkson and R Jove ldquoSTATs inoncogenesisrdquo Oncogene vol 19 no 21 pp 2474ndash2488 2000

[33] A Stephanou andD S Latchman ldquoOpposing actions of STAT-1and STAT-3rdquo Growth Factors vol 23 no 3 pp 177ndash182 2005

[34] A Martino J H Holmes J D Lord J J Moon and B HNelson ldquoStat5 and Sp1 regulate transcription of the cyclin D2gene in response to IL-2rdquo The Journal of Immunology vol 166no 3 pp 1723ndash1729 2001

[35] I Matsumura T Kitamura H Wakao et al ldquoTranscriptionalregulation of the cyclin D1 promoter by STAT5 its involvementin cytokine-dependent growth of hematopoietic cellsrdquo TheEMBO Journal vol 18 no 5 pp 1367ndash1377 1999

[36] V Calo M Migliavacca V Bazan et al ldquoSTAT proteins fromnormal control of cellular events to tumorigenesisrdquo Journal ofCellular Physiology vol 197 no 2 pp 157ndash168 2003

[37] M Socolovsky A E J Fallon S Wang C Brugnara and HF Lodish ldquoFetal anemia and apoptosis of red cell progenitorsin Stat5aminusminus5bminusminus mice a direct role for stat5 in Bcl-X(L)inductionrdquo Cell vol 98 no 2 pp 181ndash191 1999

[38] G Li Z Wang Y Zhang et al ldquoSTAT5 requires the N-domainto maintain hematopoietic stem cell repopulating function andappropriate lymphoid-myeloid lineage outputrdquo ExperimentalHematology vol 35 no 11 pp 1684ndash1694 2007

[39] S Negoro K Kunisada E Tone et al ldquoActivation of JAKSTATpathway transduces cytoprotective signal in rat acute myocar-dial infarctionrdquo Cardiovascular Research vol 47 no 4 pp 797ndash805 2000

[40] H C Blair L J Robinson and M Zaidi ldquoOsteoclast signallingpathwaysrdquo Biochemical and Biophysical Research Communica-tions vol 328 no 3 pp 728ndash738 2005

[41] C J Malemud ldquoProtein kinases in chondrocyte signaling andosteoarthritisrdquo Clinical Orthopaedics and Related Research no427 supplement pp S145ndashS151 2004

[42] E M Pinheiro and D J Montell ldquoRequirement for Par-6 andBazooka inDrosophila border cell migrationrdquoDevelopment vol131 no 21 pp 5243ndash5251 2004

[43] D Strutt ldquoFrizzled signalling and cell polarisation inDrosophilaand vertebratesrdquo Development vol 130 no 19 pp 4501ndash45132003

[44] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[45] J Guo J Jin and L F Cooper ldquoDissection of sets of genes thatcontrol the character of wnt5a-deficient mouse calvarial cellsrdquoBone vol 43 no 5 pp 961ndash971 2008

[46] M Nishizuka A Koyanagi S Osada and M Imagawa ldquoWnt4and Wnt5a promote adipocyte differentiationrdquo FEBS Lettersvol 582 no 21-22 pp 3201ndash3205 2008

[47] Y Yang L Topol H Lee and J Wu ldquoWnt5a andWnt5b exhibitdistinct activities in coordinating chondrocyte proliferation anddifferentiationrdquo Development vol 130 no 5 pp 1003ndash10152003

[48] D J Van Den Berg A K Sharma E Bruno and R Hoff-man ldquoRole of members of the Wnt gene family in humanhematopoiesisrdquo Blood vol 92 no 9 pp 3189ndash3202 1998

[49] D J Prockop C A Gregory and J L Spees ldquoOne strategy forcell and gene therapy harnessing the power of adult stemcells torepair tissuesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 1 pp 11917ndash119232003

[50] X He J-P Saint-Jeannet Y Wang J Nathans I Dawid and HVarmus ldquoA member of the Frizzled protein family mediatingaxis induction byWnt-5Ardquo Science vol 275 no 5306 pp 1652ndash1654 1997

Stem Cells International 13

[51] D G Phinney G Kopen R L Isaacson and D J ProckopldquoPlastic adherent stromal cells from the bone marrow ofcommonly used strains of inbred mice variations in yieldgrowth and differentiationrdquo Journal of Cellular Biochemistryvol 72 no 4 pp 570ndash585 1999

[52] N Sasaki T Sasamura H O Ishikawa et al ldquoPolarized exo-cytosis and transcytosis of Notch during its apical localizationin Drosophila epithelial cellsrdquo Genes to Cells vol 12 no 1 pp89ndash103 2007

[53] M Handler X Yang and J Shen ldquoPresenilin-1 regulatesneuronal differentiation during neurogenesisrdquo Developmentvol 127 no 12 pp 2593ndash2606 2000

[54] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006

[55] A W Duncan F M Rattis L N DiMascio et al ldquoIntegrationof Notch andWnt signaling in hematopoietic stem cell mainte-nancerdquo Nature Immunology vol 6 no 3 pp 314ndash322 2005

[56] N Carlesso J C Aster J Sklar and D T Scadden ldquoNotch1-induced delay of human hematopoietic progenitor cell differen-tiation is associated with altered cell cycle kineticsrdquo Blood vol93 no 3 pp 838ndash848 1999

[57] I Maillard U Koch A Dumortier et al ldquoCanonical notch sig-naling is dispensable for the maintenance of adult hematopoi-etic stem cellsrdquo Cell Stem Cell vol 2 no 4 pp 356ndash366 2008

[58] G Pearson F Robinson T B Gibson et al ldquoMitogen-activatedprotein (MAP) kinase pathways regulation and physiologicalfunctionsrdquo Endocrine Reviews vol 22 no 2 pp 153ndash183 2001

[59] R E Dolmetsch R S Lewis C C Goodnow and J I HealyldquoDifferential activation of transcription factors induced by Ca2+response amplitude and durationrdquoNature vol 386 no 6627 pp855ndash858 1997

[60] S Kawano S Shoji S Ichinose K Yamagata M Tagami andM Hiraoka ldquoCharacterization of Ca2+ signaling pathways inhuman mesenchymal stem cellsrdquo Cell Calcium vol 32 no 4pp 165ndash174 2002

[61] B Ye ldquoCa2+ oscillations and its transporters in mesenchymalstem cellsrdquo Physiological Research vol 59 no 3 pp 323ndash3292010

[62] J E Ip Y Wu J Huang L Zhang R E Pratt and V J DzauldquoMesenchymal stem cells use integrin 1205731 not CXC chemokinereceptor 4 for myocardial migration and engraftmentrdquoMolecu-lar Biology of the Cell vol 18 no 8 pp 2873ndash2882 2007

[63] C A Opitz U M Litzenburger C Lutz et al ldquoToll-like recep-tor engagement enhances the immunosuppressive propertiesof human bone marrow-derived mesenchymal stem cells byinducing indoleamine-23-dioxygenase-1 via Interferon-120573 andprotein kinase Rrdquo Stem Cells vol 27 no 4 pp 909ndash919 2009

[64] B Kulterer G Friedl A Jandrositz et al ldquoGene expressionprofiling of human mesenchymal stem cells derived from bonemarrow during expansion and osteoblast differentiationrdquo BMCGenomics vol 8 article 70 2007

[65] W G Li and X X Xu ldquoThe expression of N-cadherinfibronectin during chondrogenic differentiation of MSCinduced by TGF-1205731rdquo Chinese Journal of Traumatology vol 8no 6 pp 349ndash351 2005

[66] F Ng S Boucher S Koh et al ldquoPDGFTGF-120573 and FGF signal-ing is important for differentiation and growth of mesenchymalstem cells (mscs) transcriptional profiling can identify markersand signaling pathways important in differentiation of MSCsinto adipogenic chondrogenic and osteogenic lineagesrdquo Bloodvol 112 no 2 pp 295ndash307 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Stem Cells International 13

[51] D G Phinney G Kopen R L Isaacson and D J ProckopldquoPlastic adherent stromal cells from the bone marrow ofcommonly used strains of inbred mice variations in yieldgrowth and differentiationrdquo Journal of Cellular Biochemistryvol 72 no 4 pp 570ndash585 1999

[52] N Sasaki T Sasamura H O Ishikawa et al ldquoPolarized exo-cytosis and transcytosis of Notch during its apical localizationin Drosophila epithelial cellsrdquo Genes to Cells vol 12 no 1 pp89ndash103 2007

[53] M Handler X Yang and J Shen ldquoPresenilin-1 regulatesneuronal differentiation during neurogenesisrdquo Developmentvol 127 no 12 pp 2593ndash2606 2000

[54] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006

[55] A W Duncan F M Rattis L N DiMascio et al ldquoIntegrationof Notch andWnt signaling in hematopoietic stem cell mainte-nancerdquo Nature Immunology vol 6 no 3 pp 314ndash322 2005

[56] N Carlesso J C Aster J Sklar and D T Scadden ldquoNotch1-induced delay of human hematopoietic progenitor cell differen-tiation is associated with altered cell cycle kineticsrdquo Blood vol93 no 3 pp 838ndash848 1999

[57] I Maillard U Koch A Dumortier et al ldquoCanonical notch sig-naling is dispensable for the maintenance of adult hematopoi-etic stem cellsrdquo Cell Stem Cell vol 2 no 4 pp 356ndash366 2008

[58] G Pearson F Robinson T B Gibson et al ldquoMitogen-activatedprotein (MAP) kinase pathways regulation and physiologicalfunctionsrdquo Endocrine Reviews vol 22 no 2 pp 153ndash183 2001

[59] R E Dolmetsch R S Lewis C C Goodnow and J I HealyldquoDifferential activation of transcription factors induced by Ca2+response amplitude and durationrdquoNature vol 386 no 6627 pp855ndash858 1997

[60] S Kawano S Shoji S Ichinose K Yamagata M Tagami andM Hiraoka ldquoCharacterization of Ca2+ signaling pathways inhuman mesenchymal stem cellsrdquo Cell Calcium vol 32 no 4pp 165ndash174 2002

[61] B Ye ldquoCa2+ oscillations and its transporters in mesenchymalstem cellsrdquo Physiological Research vol 59 no 3 pp 323ndash3292010

[62] J E Ip Y Wu J Huang L Zhang R E Pratt and V J DzauldquoMesenchymal stem cells use integrin 1205731 not CXC chemokinereceptor 4 for myocardial migration and engraftmentrdquoMolecu-lar Biology of the Cell vol 18 no 8 pp 2873ndash2882 2007

[63] C A Opitz U M Litzenburger C Lutz et al ldquoToll-like recep-tor engagement enhances the immunosuppressive propertiesof human bone marrow-derived mesenchymal stem cells byinducing indoleamine-23-dioxygenase-1 via Interferon-120573 andprotein kinase Rrdquo Stem Cells vol 27 no 4 pp 909ndash919 2009

[64] B Kulterer G Friedl A Jandrositz et al ldquoGene expressionprofiling of human mesenchymal stem cells derived from bonemarrow during expansion and osteoblast differentiationrdquo BMCGenomics vol 8 article 70 2007

[65] W G Li and X X Xu ldquoThe expression of N-cadherinfibronectin during chondrogenic differentiation of MSCinduced by TGF-1205731rdquo Chinese Journal of Traumatology vol 8no 6 pp 349ndash351 2005

[66] F Ng S Boucher S Koh et al ldquoPDGFTGF-120573 and FGF signal-ing is important for differentiation and growth of mesenchymalstem cells (mscs) transcriptional profiling can identify markersand signaling pathways important in differentiation of MSCsinto adipogenic chondrogenic and osteogenic lineagesrdquo Bloodvol 112 no 2 pp 295ndash307 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology