Neurogenesis and Increase in Differentiated Neural Cell Survival via

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 582526 9 pageshttpdxdoiorg1011552013582526

Research ArticleNeurogenesis and Increase in Differentiated NeuralCell Survival via Phosphorylation of Akt1 after FluoxetineTreatment of Stem Cells

Anahita Rahmani1 Danial Kheradmand2 Peyman Keyhanvar34

Alireza Shoae-Hassani1 and Amir Darbandi-Azar4

1 Stem Cell and Tissue Engineering Department Research Center for Science and Technology in Medicine (RCSTiM)Tehran University of Medical Sciences Tehran Iran

2 Faculty of Medicine Islamic Azad University Mashhad Branch Mashhad 19988-96953 Iran3Medical Nanotechnology Department School of Advanced Technologies in Medicine Tehran University of Medical SciencesTehran Iran

4Rajaei Cardiovascular Medical and Research Centre Iran University of Medical Sciences PO Box 14185-615 Tehran Iran

Correspondence should be addressed to Amir Darbandi-Azar nanobiotechnologyymailcom

Received 10 April 2013 Revised 15 July 2013 Accepted 15 July 2013

Academic Editor Paul Higgins

Copyright copy 2013 Anahita Rahmani et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Fluoxetine (FLX) is a selective serotonin reuptake inhibitor (SSRI) Its action is possibly through an increase in neural cell survivalThe mechanism of improved survival rate of neurons by FLXmay relate to the overexpression of some kinases such as Akt proteinAkt1 (a serinethreonine kinase) plays a key role in the modulation of cell proliferation and survival Our study evaluated theeffects of FLX on mesenchymal stem cell (MSC) fate and Akt1 phosphorylation levels in MSCs Evaluation tests included reversetranscriptase polymerase chain reaction western blot and immunocytochemistry assays Nestin MAP-2 and 120573-tubulin weredetected after neurogenesis as neural markers Ten 120583M of FLX upregulated phosphorylation of Akt1 protein in induced hEnSCsignificantly Also FLX did increase viability of these MSCs Continuous FLX treatment after neurogenesis elevated the survivalrate of differentiated neural cells probably by enhanced induction of Akt1 phosphorylation This study addresses a novel role ofFLX in neurogenesis and differentiated neural cell survival that may contribute to explaining the therapeutic action of fluoxetinein regenerative pharmacology

1 Introduction

Recent works in stem cell biology have opened up new waysin therapeutic strategies to replace lost cells with stem cells ininjuries Cell therapy will be an effective strategy for treatingneurodegenerative diseases and spinal cord injuries [1] How-ever the important part of the successful therapy depends ondifferentiation and promoting the survival of implanted cellsThe survival rate of stem cells after differentiation and trans-plantation is important for the efficacy of cell therapy [2]Some factors are involved in the regulation of neurogenesisand their survival Corticosteroids were the first hormonesfound to have a neurogenesis effect [3] Neurotransmitters

and growth factors also can affect neurogenesis [4] Basedon the fact that fluoxetine (FLX) improves brain functionresearchers have explored the effect of antidepressants on theneurogenesis FLX was the first selective serotonin reuptakeinhibitor (SSRI) approved for the treatment of depressionOn the molecular basis of its clinical efficacy FLX increasesserotonin synaptic availability [5] Increased neurogenesisstarted by antidepressants in some manner contributes totheir therapeutic effects [6]These results provide some initialinsights but more work is needed to fully elucidate the SSRIactions on stem cell neurogenesis

To promote extended survival of transplanted stem cellswe must modulate the properties of the cells This purpose

2 BioMed Research International

might be accomplished by overexpressing Akt1 protein whichis a general mediator of cell survival signal Akt is a ser-inethreonine kinase that plays a key role in the modulationof cell proliferation and survival It is wellknown for itsantiapoptotic effects against a variety of situations includingoxidative and osmotic stress irradiation and ischemic shock[7ndash10]

This study aimed to investigate whether fluoxetine couldinduce neurogenesis in mesenchymal stem cells and to fur-ther examine the putative role ofAkt1 and its phosphorylationin FLX-mediated effects

2 Materials and Methods

21 Isolation and Culture of Human Endometrial Stem CellsThis study was carried out in accordance with the TehranUniversity ofMedical Sciences Ethical Committee LawTherewas a consent form for each donor that is included in thesupplementary data Here we were using human endometrialstem cell (hEnSC) as a source ofmesenchymal stem cell Stemcell was obtained from 30 donors between 25 and 35 yearsold in the gynecology department as described previously[11] Briefly the biopsies from endometrium were dissectedand then treated with collagenase (Sigma USA) for 45minIsolated cells were suspended in phosphate buffered saline(PBS Sigma USA) supplemented with 10 fetal bovineserum (FBS Gibco UK) 02mM EDTA (Sigma USA)100UmL penicillin and 01mgmL streptomycin (GibcoUK)The cells were layered on Ficoll Paque (SigmaUSA) andcentrifuged at 400 g for 35min at 4∘C EnSCs were retrievedfrom the buffy coat layer washed in PBS and then kept inDMEMF12 medium (Gibco UK) In the passage two theCD146+ CD105+ and CD90+ cells were isolated from totalcells [12] by the fluorescent analyzer cell sorter (FACS)

22 Mesenchymal Stem Cells Differentiation Human EnSCswere placed on collagen precoated plates with DMEM low-glucose mediumThe cultures were kept in a humidified 10CO2atmosphere at 37∘C for 10 daysThemedia were replaced

with freshmedia every 3 d Stem cells that have grown to 70confluence were pretreated with 1120583M dimethyl-sulfoxide(DMSO Sigma USA) and then were treated with fluoxetine(1 2 5 and 10 120583MSigmaUSA) Treatmentwith 1120583Mretinoicacid (RA Sigma USA) and dH

2O was done as positive

and negative controls in order Also hEnSCs cultured inFLX treated media containing 10 120583Mphosphatidylinositol-3-kinase (PI3-K) inhibitor (LY294002 Promega USA) servedas control cells to determine the Akt role After treatment for10 d cells were subjected to examining the Akt1 phosphoryla-tion bywestern blot and for specificmarkers of neural cells viareverse transcriptase PCR and immunocytochemistry assaysIn each experiment a nontreated group was tested

23 Reverse Transcriptase PCR Analysis for Neural SpecificMarkers For collection of total RNA from treated hEnSCswe used an Isogen kit according to the manufacturerrsquosinstructions (Nippon Gene Tokyo Japan) RNA quantityandpuritywere determined by spectrophotometry (Beckman

DU-65) Standard reverse transcription was performed usingthe AMV kit (Takara Biomedicals Ohtsu Japan) with 1 120583gRNA and 05 120583g oligo-dT per reaction according to the man-ufacturerrsquos instructions Reaction mixtures included 25120583LcDNA 1x PCR buffer (AMS TM Cinnagen Iran) 200120583MdNTPs 05 120583M of each of forward and reverse primers(Table 1) and 1U Taq DNA polymerase Polymerase chainreactions were performed at 94∘C for 1min 30 cycles 94∘Cfor 30 s 55ndash63∘C for 30 s and 72∘C for 30 s and 72∘C for10min Amplified DNA fragments were electrophoresed on15 agarose gelThegelswere stainedwith ethidiumbromide(10 120583gmL) and photographed on a UV transilluminator(Uvidoc UK)

24 Neural Markers Immunostaining The hEnSCs treatedwith FLX and FLX + 10 120583M PI3-K inhibitor (LY294002Promega USA) were fixed by incubation in 9 paraformal-dehyde for 20min and permeabilized with 05 Triton X-100for 10min as described previously [1] The cells were thenreacted with primary antibodies for Nestin (Sigma USA)MAP-2 (Sigma USA) 120573-tubulin-III (Chemicon USA) andCD11b as a glial marker (Millipore USA) at 4∘C for 12 hwashed with PBS and reacted with the fluorescent isothio-cyanate (FITC) conjugated secondary antibody (SigmaUSA)at room temperature for 2 h Finally the cells were washedwith PBS three times and DAPI was used for DNA staining

25 Detection of Akt1 Phosphorylation via Western BlottingAfter FLX treatment the cell lysates were collected andprotein concentration was determined by using a proteinassay kit (Bio-Rad USA) Total cell extracts containingequal amounts of protein in sample buffer were subjectedto WB analysis as described previously [13] Briefly thesamples were boiled for 5min and then separated by 12sodium dodecyl sulfate polyacrylamide gel electrophore-sis (SDS-PAGE) After that the gel was transferred ontopolyvinylidene difluoride (PVDF) membrane for blottingThe membrane was first blocked by incubation in bovinealbumin at room temperature for 2 h and then incubatedwith anti-Akt1 antibody (Cell Signaling Technology USA)and anti-phospho-Akt1 antibody (Cell Signaling TechnologyUSA) for 2 h at room temperature washed for 3 timeswith tris buffer containing Tween-20 and incubated at roomtemperature with HRP conjugated secondary antibody for2 h The membrane was washed 5 times with tris buffer andthen specific bands were quantified using a ChemiImagerSystem (Alpha Innotech Corporation) Anti-120573 actin antibody(Abcam UK) was used as an internal control

26 Viability Test (MTT) in Neural Differentiated CellsMTT (3-[45-dimethylthiazol-2-yl]-25-diphenyltetrazoliumbromide) assay is a useful colorimetric test for detectionof cell viability MTT is for measuring the activity of cel-lular enzymes that reduce the yellow tetrazolium dye toits insoluble formazan giving a purple color Differentiatedmesenchymal stem cells were tested for their survival time inthe presence or absence of FLX The EnSCs were plated into96 well enzyme linked immunosorbent assay (ELISA) plates

BioMed Research International 3

Table 1 Primers used for specific neuronal genes expression

Target genes Primer sequences Accession number

Nestin F 51015840-GCCCTGACCACTCCAGTTTA-31015840 NM051373R 51015840-GGAGTCCTGGATTTCCTTCC-31015840

Map-2 F 51015840-CCATTTGCAACAGGAAGACAC-31015840 NM0023743R 51015840-CAGCTCAAATGCTTTGCAACTAT-31015840

Β-tubulin F 51015840-ATGTACGAAGACGACGAGGAG-31015840 NMBC003021R 51015840-GTATCCCCGAAAATATAAACACA-31015840

120573-actin F 51015840-AAGAGAGGCATCCTGACCCT-31015840

R 51015840-ACATGGCTGGGGTGTTGAAGC-31015840

and the differentiation process was repeated as describedpreviously in Section 22 After the differentiation for replac-ing the culture media FLX was added to three of the wellsand PBS was used as a negative control in three otherwells Culture plates were incubated for one week Duringincubation (7 d) culture media were replaced with mediumcontaining FLX FLX + LY294002 and PBS Cell viability wasassessed by MTT assay kit (Biotium Hayward USA) TheMTT reagent (10 120583L) was added to the wells and incubatedfor 3 h At the end of the incubation period the medium wasremoved and 100 120583L DMSO was added into each well Todissolve the formazan crystals the supernatant was pipettedseveral times Absorbance was measured on an ELISA platereader at a wavelength of 540 nm

27 Labeling Cell with 5-Bromo-20-deoxyuridine One 120583MBrdU was added to the cultures for 6 h on the 8th day ofFLX induction The cells were observed on the 9th day ofBrdU labeling and then fixed on the 10th day of developmentand stained for BrdU and Nestin with anti-BrdUmonoclonalantibody (Sigma St Louis) overnight at 4∘C and labeledwith secondary antibody conjugated with Rhodamine for 120minutes at room temperature BrdU and Nestin positive cellswere counted in 10 microscopic fields The percentage andstandard deviation of double stained cells among all Nestinpositive cells were calculated

28 Statistical Analyses Data shownwere expressed asmeansplusmn SD from data obtained in three independent experi-ments MTT and western blot results are obtained from sixindependent experiments ANOVA was used to comparethe effects of all treatments Differences of viability withinexperimental groups were determined by theMann-Whitneytest Differences were considered statistically significant at119875 le 005

3 Results

31 Human Endometrial Stem Cell Culture Human EnSCsanalysis by FACS showed thatmore than 90of the cells wereCD146+ CD105+ and CD90+ (Figure 1) This test confirmsthe true isolation of stem cells from the lining womb Todetermine whether the hEnSCs could respond to FLX or notwe treated the stem cells with 1ndash10 120583g serial concentrations of

FLX over a period of 10 d on collagen precoated and DMSOpretreated plates In this work human EnSCs with spindleshaped morphology were cultured (Figure 2(a)) After 10 dfibroblast-like cells with spindle-shapemorphology appearedon culture dishes (Figure 2(b)) Considering the role of FLXwe investigated its effect on the stem cell differentiationDendrite and axon formation and also neurite outgrowththat are phenotypic changes into neural fate were obvious(Figure 2(c)) and it was comparable with retinoic acid effectsas a positive control (Figure 2(d))

32 Expression of Differential Neural Genes The in vitro studyis conducted to determine that the neurogenesis regulatingeffect could be replicated in cell culture system One weekafter the treatment of hEnSCs we examined the neuralmarkers by RT-PCR RT-PCR analysis was indicative of theexpression of nestinmap-2 and 120573-tubulin genes as shown inFigure 3

33 Immunostaining of Neuronal Induced Specific MarkersFluoxetine treated and differentiatedmesenchymal stem cellswere visualized by the staining of some neuronal markersImmunocytochemistry was used to analyze theNestinMAP-2 and 120573-tubulin proteins The result showed that theseproteins were detected after treatment in 10 d (Figure 4(a))Also a group of cells that were treated with LY294002 asa phosphatidylinositol-3-kinase (PI3-K) inhibitor did notexpress the neural markers even after 10 d treatment by FLX(Figure 4(b))

34 Overexpression of Phosphorylated Akt1 Phosphorylationof Akt1 in FLX-treated mesenchymal stem cells as detectedby western blot was higher than non-FLX-treated stem cellsAlso LY294002 as a PI3-K inhibitor inhibited the phosphory-lation of Akt1 significantly (Figure 5)

35 Viability of FLX Treated Cells The effect of FLX on thesurvival period of the differentiated stem cells was observeddirectly and investigated byMTT assay As shown in Figure 6FLX treatment affects the cell survival rate significantly (119875 lt005) higher than all other groups While the retinoic acidstrongly suppressed cell survival in this assay the suppressiveeffect of FLX on differentiated stem cells was very lowThe continuance of FLX treatment extended the time and


100

80

60

40

20

0

CD90

0 102 103 104 105

NegativePositive

(a)

100

80

60

40

20

0

CD105

0 102 103 104 105

NegativePositive

(b)

100

80

60

40

20

0

CD1460 102 103 104 105

NegativePositive

(c)

Figure 1 Characterization of human endometrial stem cells via fluorescent analyzer cell sorter (FACS) Human EnSCs after the secondpassage were sorted and analyzed by FACS for CD105+ CD146+ and CD90+ markers

percentage of cell viability (Figure 6) The BrdU labeled cellswere observed 24 h after labeling that showed over 90 ofhEnSCs were BrdU positive (Figure 7(a)) The amounts ofNestin-BrdU positive cells were determined on the 10th dayin cultures The percentage of double stained cells among allNestin positive cells and standard deviation were calculatedfromdata of 10microscopic fields Cultures treatedwith BrdU

and fixed on the 10th day contained about 60double stainedpositive cells (Figure 7(b))

4 Discussion

Differentiation of mesenchymal stem cells into a specificlineage their survival and functionality are related to


20120583M

(a)

20120583M

(b)

20120583M

(c)

20120583M

(d)

Figure 2 Morphological characteristics and differentiation of hEnSCs into neural cells (a) Human EnSC (passage 2) in DMEM low glucose(b) after 10 d in DMEM pretreated with DMSO (c) plus 10120583M of FLX and (d) 1 120583gmL of retinoic acid as a positive control (magnificationtimes400)

Control FLX treated

Nestin

MAP-2

120573-Tubulin

120573-Actin

Figure 3 Expression of differential genes Expression of NestinMap-2 and 120573-tubulin neuron markers was analyzed in controlculture and FLX (10120583M) treated and induced differentiation cultureafter 10 d

molecular regulationThemain finding of this research is thatthe fluoxetine antidepressant could induce neurogenesis inmesenchymal stem cells and upregulate the phosphorylation

of serinethreonine kinase Akt1 and therefore it could bea differentiation and proliferation signaling mediator in celltherapy

Selective serotonin reuptake inhibitors like FLX facil-itate signaling of serotonin by inhibiting its reuptake Atthe molecular levels FLX increases synaptic availability ofserotonin [14] It has been shown that FLX could inhibitor activate PI3kAkt or ERK12 pathways depending on thecell types and their function [15ndash17] So it could have a dualrole in the cancer therapy and cell therapy in regenerativemedicine For this purpose we selected the Akt signaling todetermine its role in mesenchymal stem cell proliferationdifferentiation and survival

First our in vitro study was conducted to determine thepossibility of neurogenesis by FLX in the mesenchymal stemcell system (Figures 2(a) and 2(b)) Our results demonstratedthat hEnSCs could be induced to differentiate into neurons inthe presence of FLX (Figure 2(c)) as was seen in cells treatedwith retinoic acid (Figure 2(d)) High concentrations of FLXhave nonserotoninergic effects on cells [5] Nonserotonin-ergic targets of fluoxetine that mediate its other effects arelikely to have a lower binding affinity or lower availability forFLX Inhibition of cytochrome P450 is known to be due to anonserotoninergic effect of FLX [18] Lee et al (2008) showedthat liver cytochrome P450-dependent ROS formation isresponsible for cyclin-A downregulation and inhibition of


Nestin

DAPI DAPI DAPI DAPI

120573-TubulinMAP-2 CD11b

(a)

DAPI DAPI

Nestin

DAPI

120573-TubulinMAP-2

(b)

Figure 4 Immunostaining of hEnSCs differential markers Expression of neuron markers including Nestin MAP-2 and 120573-tubulin wasanalyzed in FLX induced differentiation after 10 d The expression of CD11b as a glial marker was not obvious The size bar is 10120583m (a)Immunostaining of LY294002 treated hEnSCs after culture in FLX supplemented medium There is not an expression of neuron markers inFLX induced differentiation after 10 d (b)

FluoxetineLY294002

pAktAkt

120573-Actin

+

+

+

+

minus

minus

minus

minus

Figure 5 Western blot results after treatment of hEnSC with 10 120583Mfluoxetine after 10 d Protein samples (100120583g) were loaded ontoSDS PAGE gels and transferred to PVDF membranes The inducedEnSCs produced an increase expression of phosphorylated Akt1protein 120573-Actin was used as an internal control

neural progenitor cell proliferation [19] so inhibition ofcytochrome P450 may inhibit cyclin-A downregulation andrestoring cyclin-A reverse inhibition of neural progenitorcell proliferation However this induction did not occur inthe brain and suggested a tissue-specific response [20] Ithas been demonstrated that the SSRIs interact with the eagvoltage-gated K

1channel [21] and voltage-gated potassium

channels play a functional role in the development of humanneural progenitor cells and differentiation of neurons [22]Also studies by deOliveira and colleagues in 2012 showed thateag potassium channel expression in the rat hippocampus has


Treatment after 1 week

MTT of FLX treated SCs

Cel

l sur

viva

l rat

e (

)

120

100

80

60

40

20

01 2 3 4 5 6 7 8

lowast

lowast

Figure 6 MTT shows EnSCs differentiated neural cell viability ratein the presence of FLX FLX + LY294002 LY294002 and retinoicacid after one week (119875 le 005) The numbers from the left side areas follows (1) 1 120583M FLX (2) 5 120583M FLX (3) 10 120583M FLX (4) 10 120583MFLX + 10120583M LY294002 (5) 10120583M LY294002 (6) 1120583M retinoicacid (7) neural differentiated cells without FLX supplement and (8)undifferentiated mesenchymal stem cells

been associated with neural cell survival and transient brainischemia [23]

In our experiment fluoxetine-induced hEnSCs displayedneuronal morphology with axon formation and neuriteoutgrowth and expressed Nestin and other neuronal specificmolecular markers by the reverse transcriptase polymerasechain reaction (Figure 3) and immunocytochemistry (Fig-ure 4(a)) assays EnSCs cultured in serum-free DMEMF12medium could not differentiate or even proliferated con-stantly These findings indicate FLX-induced EnSCs mayprovide a stem-cell-based way to generate neuronal cells forthe treatment of neurodegenerative diseases This findingcould determine that the endometrial stem cells express sero-tonin receptors In neuropharmacology it may be beneficialto study the effect of the different antidepressants on theneurogenesis since this provides insights into the regulationof stem cell proliferation

Regeneration of neurons that undergo cell death soonafter injury is the main goal of all cell-based therapies Fromthe point that neurogenesis has the capacity to enhancebrain repair andor spinal cord repair after ischemia andortrauma by replacement of dead cells or seriously injured cellsFLX neurogenesis could lead to recovery of these cells FLXwould appear to be a candidate as a neurogenesis factor Ourexperiment showed that the hEnSCs expressed neural cellmarkers after FLX treatment but there was no expression ofCD11b marker that is a specific glial marker (Figure 4(a))This is an important point because glial scar formation thatoccurs following CNS injuries is an important obstacle ofneuroregeneration FLX could introduce a regenerative prod-uct in neuronal injuries and find its position in regenerativepharmacology

Many groups have examined the potential of neu-ron replacement from embryonic stem cells [24] inducedpluripotent stem cells (iPS) [25 26] and cord blood derived

stem cells Endometrial stem cells have demonstrated supe-rior potential They expand rapidly and they have fewertechnical and ethical problems so they have great potentialas therapeutic agents and autologous grafts [2] Our teamhas previously shown that the endometrial stem cells couldbe differentiated successfully into other lineages of cellsespecially neural cells [1 11] It represents a progress in thedevelopment of a new source of neurons

We further confirmed the protective mechanisms of Akt1phosphorylation as a survival signaling that introduces thispathway as a potential target for stem cell survival after dif-ferentiation of mesenchymal stem cells (Figure 6) PI3kAktis the important cell survival pathways and phosphorylationof Akt mediates anti-apoptosis in many kinds of cells [27] In2003 Kim et al have shown that the Akt activation results inphosphorylation and then inactivation of the pro-apoptoticprotein glycogen synthase kinase 3 (GSK3b) thus extendingthe cell survival period [28] In support of the role of Akt1phosphorylation we found that the PI3K inhibitor LY294002in the concentration of 10 120583M decreased the FLX mediatedAkt phosphorylation (Figure 5) differentiation (Figure 4(b))and the viability of neuronal differentiated cells (Figure 6)This suggests a key role for activating Akt1 in the protectionof mesenchymal stem cell from the precocious death Alsowe have labeled the hEnSCs with BrdU to obtain more exactcell proliferation rate (Figure 7(a)) The FLX treated stemcells that were labeled with BrdU showed double-staining forboth BrdU and Nestin on the 10th day Cells acquiring Nestinpositive phenotype seemed to go through cell cycles enoughto dilute the BrdU label to the non-detectable levels In ourexperiment BrdUwas added on the 8th day of induction andabout half of nestin positive cells carried BrdU-label on the10th day (Figure 7(b)) In cultures that FLX was absent about70 of Nestin positive cells carried BrdU-label

In 2006 Frebel and Wiese confirmed the neuronal sur-vival and differentiation implicated PI3-KAKT in synapticplasticity learning and memory in the mammalian brain[29]

Although this finding supports the neurogenesis role ofSSRIs it has been suggested that excessive neurogenesis isnot beneficial and could result in inappropriate migrationinto existing neural networks Scharfman and Hen believethat this manner could cause some pathological conditionsas epilepsy [30] but previously Rush et al reporting thatSSRIs could lose their effect after long-term use [31] Thishas been called relapse during maintenance treatment Apossible mechanism underlying this effect is the weaknessof neurogenesis because of excessive stimulation So it ispossible that long-term SSRI therapy might affect PI3Aktpathway and its activation that leads to cognitive functionsby disrupting the normal regulation of neurogenesis

5 Conclusion

In conclusion our data argue with fluoxetine effect onmesenchymal stem cell neurogenesis and its role in thephosphorylation of Akt1 in the differentiated cells to increasethe survival rate of differentiated cells


(a)

Nestin BrdU

(b)

Figure 7 Endometrial stem cells labeled by BrdU (a) Cultures were treated with FLX and labeled with BrdU They were then stained withantibodies against BrdU and nestin followed by either rhodamine- (for BrdU red) or FITC- (nestin green) labeled secondary antibodies (b)Data are representative of 3 independent experiments

Conflict of Interests

The authors have no conflict of interests

References

[1] M Noureddini J Verdi S A Mortazavi Tabatabaei S Sharifand A Shoae Hassani ldquoHuman endometrial stem cell neuroge-nesis in response to NGF and bFGFrdquo Cell Biology Internationalvol 36 no 1 pp 961ndash966 2012

[2] A Shoae-Hassani S A Mortazavi-Tabatabaei S Sharif HRezaei-Khaligh and J Verdi ldquoDHEA provides a microen-vironment for endometrial stem cells neurogenesisrdquo MedicalHypotheses vol 76 no 6 pp 843ndash846 2011

[3] H A Cameron ldquoAdult neurogenesis is regulated by adrenalsteroids in the dentate gyrusrdquo Neuroscience vol 61 no 2 pp203ndash209 1994

[4] P-M Lledo M Alonso and M S Grubb ldquoAdult neurogenesisand functional plasticity in neuronal circuitsrdquo Nature ReviewsNeuroscience vol 7 no 3 pp 179ndash193 2006

[5] R Ranganathan E R Sawin C Trent and H R HorvitzldquoMutations in the Caenorhabditis elegansserotonin reuptaketransporter MOD-5 reveal serotonin-dependent and -independent activities of fluoxetinerdquo Journal of Neurosciencevol 21 no 16 pp 5871ndash5884 2001

[6] A Abdipranoto S Wu S Stayte and B Vissel ldquoThe role ofneurogenesis in neurodegenerative diseases and its implicationsfor therapeutic developmentrdquo CNS and Neurological Disorders-Drug Targets vol 7 no 2 pp 187ndash210 2008

[7] HDudek S RDatta T F Franke et al ldquoRegulation of neuronalsurvival by the serine-threonine protein kinase Aktrdquo Sciencevol 275 no 5300 pp 661ndash665 1997

[8] S G Kennedy E S Kandel T K Cross and N HayldquoAktprotein kinase B inhibits cell death by preventing therelease of cytochrome C from mitochondriardquo Molecular andCellular Biology vol 19 no 8 pp 5800ndash5810 1999

[9] A Brunet S R Datta and M E Greenberg ldquoTranscription-dependent andmdashindependent control of neuronal survival bythe PI3K-Akt signaling pathwayrdquo Current Opinion in Neurobi-ology vol 11 no 3 pp 297ndash305 2001

[10] T F Franke C P Hornik L Segev G A Shostak and CSugimoto ldquoPI3KAkt and apoptosis size mattersrdquo Oncogenevol 22 no 56 pp 8983ndash8998 2003

[11] A Shoae-Hassani A M Seifalian S A Mortazavi-TabatabaeiS Sharif A Azimi and J Verdi ldquoDifferentiation of humanendometrial stem cells into urothelial cells on a three-dimensional nanofibrous silk-collagen scaffold an autologouscell resource for reconstruction of the urinary bladder wallrdquoJournal of Tissue Engineering and Regenerative Medicine 2013

[12] C E Gargett ldquoUterine stem cells what is the evidencerdquoHumanReproduction Update vol 13 no 1 pp 87ndash101 2007

[13] A Shoae-Hassani S A Mortazavi-Tabatabaei S Sharif SMadadi H Rezaei-Khaligh and J Verdi ldquoRecombinant lambdabacteriophage displaying nanobody towards third domain ofHer-II epitope inhibits proliferation of breast carcinoma SKBR-3 cell linerdquo Archivum Immunologiae et Therapiae Experimen-talis vol 61 no 1 pp 75ndash83 2013

[14] F Karege M Schwald and M Cisse ldquoPostnatal developmentalprofile of brain-derived neurotrophic factor in rat brain andplateletsrdquoNeuroscience Letters vol 328 no 3 pp 261ndash264 2002


[15] G Mercier A M Lennon B Renouf et al ldquoMAP kinaseactivation by fluoxetine and its relation to gene expression incultured rat astrocytesrdquo Journal of Molecular Neuroscience vol24 no 2 pp 207ndash216 2004

[16] F Fumagalli R Molteni F Calabrese A Frasca G Racagniand M A Riva ldquoChronic fluoxetine administration inhibitsextracellular signal-regulated kinase 12 phosphorylation in ratbrainrdquo Journal of Neurochemistry vol 93 no 6 pp 1551ndash15602005

[17] E Ha K H Jung B-K Choe et al ldquoFluoxetine increases thenitric oxide production via nuclear factor kappa B-mediatedpathway in BV2 murine microglial cellsrdquo Neuroscience Lettersvol 397 no 3 pp 185ndash189 2006

[18] R K Choy and J H Thomas ldquoFluoxetine-resistant mutants inC elegans define a novel family of transmembrane proteinsrdquoMolecular Cell vol 4 no 2 pp 143ndash152 1999

[19] C-T Lee J Chen T Hayashi et al ldquoA mechanism for theinhibition of neural progenitor cell proliferation by cocainerdquoPLoS Medicine vol 5 no 6 article e117 2008

[20] W J Ray G Bain M Yao and D I Gottlieb ldquoCYP26 a novelmammalian cytochrome P450 is induced by retinoic acid anddefines a new familyrdquo Journal of Biological Chemistry vol 272no 30 pp 18702ndash18708 1997

[21] D Weinshenker G Garriga and J H Thomas ldquoGenetic andpharmacological analysis of neurotransmitters controlling egglaying in C elegansrdquo Journal of Neuroscience vol 15 no 10 pp6975ndash6985 1995

[22] G Schaarschmidt F Wegner S C Schwarz H Schmidtand J Schwarz ldquoCharacterization of voltage-gated potassiumchannels in human neural progenitor cellsrdquo PLoS ONE vol 4no 7 Article ID e6168 2009

[23] R M W de Oliveira S Martin C L de Oliveira et alldquoEag1 Eag2 and SK3 potassium channel expression in the rathippocampus after global transient brain ischemiardquo Journal ofNeuroscience Research vol 90 no 3 pp 632ndash640 2012

[24] M S Cho Y-E Lee J Y Kim et al ldquoHighly efficient and large-scale generation of functional dopamine neurons from humanembryonic stem cellsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 9 pp 3392ndash3397 2008

[25] Y-H Rhee J-Y Ko M-Y Chang et al ldquoProtein-based humaniPS cells efficiently generate functional dopamine neurons andcan treat a rat model of Parkinson diseaserdquo Journal of ClinicalInvestigation vol 121 no 6 pp 2326ndash2335 2011

[26] A Shoae-Hassani S Sharif and J Verdi ldquoThe neurosteroiddehydroepiandrosterone could improve somatic cell repro-grammingrdquo Cell Biology International vol 35 no 1 pp 1037ndash1041 2011

[27] D S Kwon C H Kwon J H Kim J S Woo J S Jung andY K Kim ldquoSignal transduction of MEKERK and PI3KAktactivation by hypoxiareoxygenation in renal epithelial cellsrdquoEuropean Journal of Cell Biology vol 85 no 11 pp 1189ndash11992006

[28] J W Kim J E Lee M J Kim E-G Cho S-G Cho and E-J Choi ldquoGlycogen synthase kinase 3120573 is a natural activator ofmitogen-activated protein kinaseextracellular signal-regulatedkinase kinase kinase 1 (MEKK1)rdquo Journal of Biological Chem-istry vol 278 no 16 pp 13995ndash14001 2003

[29] K Frebel and S Wiese ldquoSignalling molecules essential forneuronal survival and differentiationrdquo Biochemical SocietyTransactions vol 34 no 6 pp 1287ndash1290 2006

[30] H E Scharfman and R Hen ldquoIs more neurogenesis alwaysbetterrdquo Science vol 315 no 5810 pp 336ndash338 2007

[31] A J Rush M H Trivedi S R Wisniewski et al ldquoAcute andlonger-term outcomes in depressed outpatients requiring oneor several treatment steps a STARlowastD reportrdquoAmerican Journalof Psychiatry vol 163 no 11 pp 1905ndash1917 2006

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


BioMed Research International


Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


Neural Plasticity


Parkinsonrsquos Disease


Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Psychiatry Journal


Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases


Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


might be accomplished by overexpressing Akt1 protein whichis a general mediator of cell survival signal Akt is a ser-inethreonine kinase that plays a key role in the modulationof cell proliferation and survival It is wellknown for itsantiapoptotic effects against a variety of situations includingoxidative and osmotic stress irradiation and ischemic shock[7ndash10]

This study aimed to investigate whether fluoxetine couldinduce neurogenesis in mesenchymal stem cells and to fur-ther examine the putative role ofAkt1 and its phosphorylationin FLX-mediated effects

2 Materials and Methods

21 Isolation and Culture of Human Endometrial Stem CellsThis study was carried out in accordance with the TehranUniversity ofMedical Sciences Ethical Committee LawTherewas a consent form for each donor that is included in thesupplementary data Here we were using human endometrialstem cell (hEnSC) as a source ofmesenchymal stem cell Stemcell was obtained from 30 donors between 25 and 35 yearsold in the gynecology department as described previously[11] Briefly the biopsies from endometrium were dissectedand then treated with collagenase (Sigma USA) for 45minIsolated cells were suspended in phosphate buffered saline(PBS Sigma USA) supplemented with 10 fetal bovineserum (FBS Gibco UK) 02mM EDTA (Sigma USA)100UmL penicillin and 01mgmL streptomycin (GibcoUK)The cells were layered on Ficoll Paque (SigmaUSA) andcentrifuged at 400 g for 35min at 4∘C EnSCs were retrievedfrom the buffy coat layer washed in PBS and then kept inDMEMF12 medium (Gibco UK) In the passage two theCD146+ CD105+ and CD90+ cells were isolated from totalcells [12] by the fluorescent analyzer cell sorter (FACS)

22 Mesenchymal Stem Cells Differentiation Human EnSCswere placed on collagen precoated plates with DMEM low-glucose mediumThe cultures were kept in a humidified 10CO2atmosphere at 37∘C for 10 daysThemedia were replaced

with freshmedia every 3 d Stem cells that have grown to 70confluence were pretreated with 1120583M dimethyl-sulfoxide(DMSO Sigma USA) and then were treated with fluoxetine(1 2 5 and 10 120583MSigmaUSA) Treatmentwith 1120583Mretinoicacid (RA Sigma USA) and dH

2O was done as positive

and negative controls in order Also hEnSCs cultured inFLX treated media containing 10 120583Mphosphatidylinositol-3-kinase (PI3-K) inhibitor (LY294002 Promega USA) servedas control cells to determine the Akt role After treatment for10 d cells were subjected to examining the Akt1 phosphoryla-tion bywestern blot and for specificmarkers of neural cells viareverse transcriptase PCR and immunocytochemistry assaysIn each experiment a nontreated group was tested

23 Reverse Transcriptase PCR Analysis for Neural SpecificMarkers For collection of total RNA from treated hEnSCswe used an Isogen kit according to the manufacturerrsquosinstructions (Nippon Gene Tokyo Japan) RNA quantityandpuritywere determined by spectrophotometry (Beckman

DU-65) Standard reverse transcription was performed usingthe AMV kit (Takara Biomedicals Ohtsu Japan) with 1 120583gRNA and 05 120583g oligo-dT per reaction according to the man-ufacturerrsquos instructions Reaction mixtures included 25120583LcDNA 1x PCR buffer (AMS TM Cinnagen Iran) 200120583MdNTPs 05 120583M of each of forward and reverse primers(Table 1) and 1U Taq DNA polymerase Polymerase chainreactions were performed at 94∘C for 1min 30 cycles 94∘Cfor 30 s 55ndash63∘C for 30 s and 72∘C for 30 s and 72∘C for10min Amplified DNA fragments were electrophoresed on15 agarose gelThegelswere stainedwith ethidiumbromide(10 120583gmL) and photographed on a UV transilluminator(Uvidoc UK)

24 Neural Markers Immunostaining The hEnSCs treatedwith FLX and FLX + 10 120583M PI3-K inhibitor (LY294002Promega USA) were fixed by incubation in 9 paraformal-dehyde for 20min and permeabilized with 05 Triton X-100for 10min as described previously [1] The cells were thenreacted with primary antibodies for Nestin (Sigma USA)MAP-2 (Sigma USA) 120573-tubulin-III (Chemicon USA) andCD11b as a glial marker (Millipore USA) at 4∘C for 12 hwashed with PBS and reacted with the fluorescent isothio-cyanate (FITC) conjugated secondary antibody (SigmaUSA)at room temperature for 2 h Finally the cells were washedwith PBS three times and DAPI was used for DNA staining

25 Detection of Akt1 Phosphorylation via Western BlottingAfter FLX treatment the cell lysates were collected andprotein concentration was determined by using a proteinassay kit (Bio-Rad USA) Total cell extracts containingequal amounts of protein in sample buffer were subjectedto WB analysis as described previously [13] Briefly thesamples were boiled for 5min and then separated by 12sodium dodecyl sulfate polyacrylamide gel electrophore-sis (SDS-PAGE) After that the gel was transferred ontopolyvinylidene difluoride (PVDF) membrane for blottingThe membrane was first blocked by incubation in bovinealbumin at room temperature for 2 h and then incubatedwith anti-Akt1 antibody (Cell Signaling Technology USA)and anti-phospho-Akt1 antibody (Cell Signaling TechnologyUSA) for 2 h at room temperature washed for 3 timeswith tris buffer containing Tween-20 and incubated at roomtemperature with HRP conjugated secondary antibody for2 h The membrane was washed 5 times with tris buffer andthen specific bands were quantified using a ChemiImagerSystem (Alpha Innotech Corporation) Anti-120573 actin antibody(Abcam UK) was used as an internal control

26 Viability Test (MTT) in Neural Differentiated CellsMTT (3-[45-dimethylthiazol-2-yl]-25-diphenyltetrazoliumbromide) assay is a useful colorimetric test for detectionof cell viability MTT is for measuring the activity of cel-lular enzymes that reduce the yellow tetrazolium dye toits insoluble formazan giving a purple color Differentiatedmesenchymal stem cells were tested for their survival time inthe presence or absence of FLX The EnSCs were plated into96 well enzyme linked immunosorbent assay (ELISA) plates












3 Results








100

80

60

40

20

0

CD90

0 102 103 104 105

NegativePositive

(a)

100

80

60

40

20

0

CD105

0 102 103 104 105

NegativePositive

(b)

100

80

60

40

20

0

CD1460 102 103 104 105

NegativePositive

(c)




4 Discussion



20120583M

(a)

20120583M

(b)

20120583M

(c)

20120583M

(d)


Control FLX treated

Nestin

MAP-2

120573-Tubulin

120573-Actin







Nestin

DAPI DAPI DAPI DAPI


(a)

DAPI DAPI

Nestin

DAPI

120573-TubulinMAP-2

(b)


FluoxetineLY294002

pAktAkt

120573-Actin

+

+

+

+

minus

minus

minus

minus








Cel

l sur

viva

l rat

e (

)

120

100

80

60

40

20

01 2 3 4 5 6 7 8

lowast

lowast










5 Conclusion



(a)

Nestin BrdU

(b)




References













































Neural Plasticity










Psychiatry Journal









Journal of













3 Results








100

80

60

40

20

0

CD90

0 102 103 104 105

NegativePositive

(a)

100

80

60

40

20

0

CD105

0 102 103 104 105

NegativePositive

(b)

100

80

60

40

20

0

CD1460 102 103 104 105

NegativePositive

(c)




4 Discussion



20120583M

(a)

20120583M

(b)

20120583M

(c)

20120583M

(d)


Control FLX treated

Nestin

MAP-2

120573-Tubulin

120573-Actin







Nestin

DAPI DAPI DAPI DAPI


(a)

DAPI DAPI

Nestin

DAPI

120573-TubulinMAP-2

(b)


FluoxetineLY294002

pAktAkt

120573-Actin

+

+

+

+

minus

minus

minus

minus








Cel

l sur

viva

l rat

e (

)

120

100

80

60

40

20

01 2 3 4 5 6 7 8

lowast

lowast










5 Conclusion



(a)

Nestin BrdU

(b)




References













































Neural Plasticity










Psychiatry Journal









Journal of



100

80

60

40

20

0

CD90

0 102 103 104 105

NegativePositive

(a)

100

80

60

40

20

0

CD105

0 102 103 104 105

NegativePositive

(b)

100

80

60

40

20

0

CD1460 102 103 104 105

NegativePositive

(c)




4 Discussion



20120583M

(a)

20120583M

(b)

20120583M

(c)

20120583M

(d)


Control FLX treated

Nestin

MAP-2

120573-Tubulin

120573-Actin







Nestin

DAPI DAPI DAPI DAPI


(a)

DAPI DAPI

Nestin

DAPI

120573-TubulinMAP-2

(b)


FluoxetineLY294002

pAktAkt

120573-Actin

+

+

+

+

minus

minus

minus

minus








Cel

l sur

viva

l rat

e (

)

120

100

80

60

40

20

01 2 3 4 5 6 7 8

lowast

lowast










5 Conclusion



(a)

Nestin BrdU

(b)




References













































Neural Plasticity










Psychiatry Journal









Journal of



20120583M

(a)

20120583M

(b)

20120583M

(c)

20120583M

(d)


Control FLX treated

Nestin

MAP-2

120573-Tubulin

120573-Actin







Nestin

DAPI DAPI DAPI DAPI


(a)

DAPI DAPI

Nestin

DAPI

120573-TubulinMAP-2

(b)


FluoxetineLY294002

pAktAkt

120573-Actin

+

+

+

+

minus

minus

minus

minus








Cel

l sur

viva

l rat

e (

)

120

100

80

60

40

20

01 2 3 4 5 6 7 8

lowast

lowast










5 Conclusion



(a)

Nestin BrdU

(b)




References













































Neural Plasticity










Psychiatry Journal









Journal of



Nestin

DAPI DAPI DAPI DAPI


(a)

DAPI DAPI

Nestin

DAPI

120573-TubulinMAP-2

(b)


FluoxetineLY294002

pAktAkt

120573-Actin

+

+

+

+

minus

minus

minus

minus








Cel

l sur

viva

l rat

e (

)

120

100

80

60

40

20

01 2 3 4 5 6 7 8

lowast

lowast










5 Conclusion



(a)

Nestin BrdU

(b)




References













































Neural Plasticity










Psychiatry Journal









Journal of





Cel

l sur

viva

l rat

e (

)

120

100

80

60

40

20

01 2 3 4 5 6 7 8

lowast

lowast










5 Conclusion



(a)

Nestin BrdU

(b)




References













































Neural Plasticity










Psychiatry Journal









Journal of



(a)

Nestin BrdU

(b)




References













































Neural Plasticity










Psychiatry Journal









Journal of
































Neural Plasticity










Psychiatry Journal









Journal of


Neurogenesis and Increase in Differentiated Neural Cell Survival via

Documents

Transcript of Neurogenesis and Increase in Differentiated Neural Cell Survival via