Research Article Clonal Heterogeneity in the Neuronal and...

Research ArticleClonal Heterogeneity in the Neuronal and Glial Differentiationof Dental Pulp StemProgenitor Cells

Fraser I Young12 Vsevolod Telezhkin34 Sarah J Youde1 Martin S Langley1

Maria Stack1 Paul J Kemp3 Rachel J Waddington1 Alastair J Sloan1 and Bing Song1

1Oral and Biomedical Sciences School of Dentistry Cardiff University Heath Park Cardiff CF14 4XY UK2Neuroscience and Mental Health Research Institute School of Medicine Cardiff University Hadyn Ellis BuildingMaindy Road Cardiff CF24 4HQ UK3Cardiff School of Biosciences Cardiff University Sir Martin Evans Building Museum Avenue Cardiff CF10 3AX UK4Department of Neuroscience Physiology amp Pharmacology University College London London WC1E 6BT UK

Correspondence should be addressed to Fraser I Young youngf1cardiffacuk and Bing Song songb3cardiffacuk

Received 14 January 2016 Revised 24 March 2016 Accepted 8 May 2016

Academic Editor George Huang

Copyright copy 2016 Fraser I Young et alThis 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

Cellular heterogeneity presents an important challenge to the development of cell-based therapies where there is a fundamentalrequirement for predictable and reproducible outcomes Transplanted Dental Pulp StemProgenitor Cells (DPSCs) havedemonstrated early promise in experimental models of spinal cord injury and stroke despite limited evidence of neuronal and glial-like differentiation after transplantation Here we report for the first time on the ability of single cell-derived clonal cultures ofmurine DPSCs to differentiate in vitro into immature neuronal-like and oligodendrocyte-like cells Importantly only DPSC cloneswith high nestin mRNA expression levels were found to successfully differentiate into Map2 and NF-positive neuronal-like cellsNeuronally differentiated DPSCs possessed a membrane capacitance comparable with primary cultured striatal neurons and smallinward voltage-activated K+ but not outward Na+ currents were recorded suggesting a functionally immature phenotype Similarlyonly high nestin-expressing clones demonstrated the ability to adopt Olig1 Olig2 and MBP-positive immature oligodendrocyte-like phenotype Together these results demonstrate that appropriate markers may be used to provide an early indication of thesuitability of a cell population for purposes where differentiation into a specific lineage may be beneficial and highlight that furtherunderstanding of heterogeneity within mixed cellular populations is required

1 Introduction

Stem cell heterogeneity poses a significant obstacle to theclinical implementation of cell-based therapies Mixed cul-tures may contain a combination of stem cells with a broadrange of differentiation potentials and long term proliferativeabilities as well as more lineage-committed progenitor cellsSuch variability amongst cells in the same transplantablepopulation can lead to the adoption of adverse phenotypespotentially limiting improvements in outcome

Dental Pulp StemProgenitor Cells (DPSCs) possess typi-cal mesenchymal progenitor properties in vitro demonstrat-ing the ability to differentiate into odontoblastsosteoblastsadipocytes and chondrocytes [1ndash3] Furthermore endothe-lial myogenic hepatocytic and melanocytic differentiation

capabilities have also been reported suggesting a diverserange of potential therapeutic applications [4ndash8] HoweverDPSCs represent a highly heterogeneous population of cellswith distinct clonal differences in proliferation and min-eralisation capabilities [1 9] Such variability could poten-tially hinder progress in the development of DPSC-basedtreatments Nevertheless transplanted mixed populations ofDPSCs have demonstrated promise at improving functionaloutcome in experimentalmodels of spinal cord injury strokeand Parkinsonrsquos disease [10ndash15] These effects are mostlyprotective and mediated through the release of supportivegrowth factors It is as yet unclear whether DPSCs candifferentiate into and functionally compensate for neuronaland glial cell types after transplantation In vitro studieshave indicated potential for rodent and human DPSCs

Hindawi Publishing CorporationStem Cells InternationalVolume 2016 Article ID 1290561 10 pageshttpdxdoiorg10115520161290561

2 Stem Cells International

to differentiate into neuronal-like cells [16] Furthermoreindependent studies have described a degree of functionalityof such neuronally differentiated DPSCs [17ndash21] Howeveronly a small fraction of cells within such cultures developa functional phenotype [18] Similarly outcomes may differbetween patient samples subjected to the same differen-tiation protocol [22] Understanding the cellular biologybehind such heterogeneity poses a substantial challenge toresearchers The identification of an appropriate marker ofneural differentiation capabilities for which to screen DPSCcultures beforehand could help to minimise such variabilitybetween studies resulting in more defined and predictableoutcomes

In this study single cell-derived clonal populations ofmurine DPSCs (mDPSCs) were isolated and differences inthe expression of early stage neural markers identified Onlyclones with high levels of nestin expression were found to dif-ferentiate into immature neuronal-like cells displayingmini-mal electrical activity Similarly a novel differentiation proto-col was developed for the derivation of oligodendrocyte-likecells from high nestin-expressing mDPSC clones Togetherthese findings suggest that nestinmay act as a suitablemarkerfor use in assessing the ability of mDPSCs to differentiate intoneuronal-like and glial-like cells in vitro

2 Experimental Procedures

21 Isolation of mDPSCs All procedures were approvedby the Cardiff University Biological Standards Office Foreach DPSC isolation the incisor pulpal tissue of 4-5 times21ndash28-day-old C57Bl6 mice sacrificed by CO

2asphyxia-

tion in accordance with Schedule 1 of the UK Animals(Scientific Procedures) Act 1986 was pooled Followingcollagenasedispase digestion to a cellular suspension thepreferential adherence to fibronectin selection technique wasused to select for progenitor cells by isolating cells of moreimmature phenotypes based on 1205731 integrin functionality [2324] After 12 days of primary culture individual colonies offibronectin-adherent cells displaying typical DPSC bipolarfibroblastic-like morphology and numbering greater than32 cells were selected for clonal isolation and expansionas described previously using cloning rings [25] ClonalDPSCswere subsequently expanded in120572MEMsupplementedwith 100 unitsmL penicillin 100120583gmL streptomycin and20 (vv) heat-inactivated foetal bovine serum (all fromLife Technologies) and 100 120583M l-ascorbic acid 2-phosphate(Sigma-Aldrich) Cell counts were performed at every pas-sage and used to track population doublings over time inculture

Population doublings =log10(cell count at passage) minus log

10(no of cells initially seeded)

log10(2)

(1)

Cells of between 20 and 40 population doublings wereused for all experiments Four individual mDPSC clonesthat expanded sufficiently to allow multiple reproducibledifferentiation experiments were used in this study eachderived from a separate pulpal extraction (119899 = 4)

22 Neuronal Differentiation mDPSC clones were seededat 10000 cellscm2 on poly-d-lysinelaminin-coated culturesurfaces in DMEMF12 (1 1) containing L-glutamine andHEPES buffer 100 unitsmL penicillin 100 120583gmL strepto-mycin and 1 times N2 supplement (all from Life Technologies)1 times NEAA (Sigma-Aldrich) and 20 ngmL basic fibrob-last growth factor (bFGF) and 20 ngmL epidermal growthfactor (both from Peprotech) After 5 days cultures werewashed with PBS and changed to neurobasal medium sup-plemented with 100 unitsmL penicillin 100120583gmL strepto-mycin and 2mM L-glutamine (all from Life Technologies) 1times NEAA (Sigma-Aldrich) and 10 ngmL brain-derived neu-rotrophic factor 10 ngmL nerve growth factor and 10 ngmLneurotrophin-3 (all from Peprotech) RNA was extracted ondays 0 5 10 and 15 of differentiation for use in qPCR and cellswere fixed on day 15 for immunocytochemistry

23 Oligodendrocyte Differentiation Clonal mDPSC cultureswere seeded at 10000 cellscm2 on poly-d-lysine-coatedculture surfaces in DMEM containing 100 unitsmL peni-cillin 100 120583gmL streptomycin SATO supplement (16 120583gmL

putrescine 62 ngmL progesterone 5 ngmL sodium seleniteand 100 120583gmL bovine serum albumin (BSA)) 50 120583gmLholo-Transferrin and 5 120583gmL insulin (all from Sigma-Aldrich) and 10 ngmL platelet-derived growth factor-aa and20 ngmL bFGF (both Peprotech) After 10 days differentia-tion cells were fixed for immunocytochemistry

24 Isolation of mSTM Neurons Mouse striatal (mSTM)neuronal tissues were dissected in PBS from P0 micedigested using Accutase and plated on poly-l-lysine-coatedglass coverslips inAdvancedDMEMF-12 supplementedwith2mML-glutamine 100 unitsmL penicillin 100 120583gmL strep-tomycin 18mMCaCl

2 05mML valproic acid and 1 x B27-

supplement (without vitaminA) (all from Life Technologies)

25 Reverse Transcriptase PCR Total RNA was extractedusing an RNeasy Mini Kit with on-column DNase digestion(QIAGEN) according tomanufacturerrsquos directions and cDNAsynthesised using MMLV reverse transcriptase (Promega)PCR reactions were performed using GoTaq Polymerase(Promega) and product-specific primers (Supplemental Table1 in Supplementary Material available online at httpdxdoiorg10115520161290561) RNA extracted from primary cul-tured E145 cortical neural stem cells isolated as describedpreviously [26] was used as a positive control

26 Real Time Quantitative PCR For qPCR readings cDNAsamples generated from three separate experiments per clone

Stem Cells International 3

were used (119899 = 3) and each was measured in triplicateusing an ABI Prism 7000 machine (Advanced Biosystems)Target-specific primers (Supplemental Table 2) were addedto each cDNA sample together with Precision MasterMixwith ROX and SYBRgreen (PrimerDesign) Dissociationcurves were recorded to check for specificity of reactions andproducts were electrophoresed on 14 agarose gels in orderto confirm product size Relative changes in expression werecalculated using the 2minusΔΔCT method [27] Statistical analyseswere performed using Graphpad Prism Software

27 Immunocytochemistry Cells were fixed with 4 (wv)paraformaldehyde for 15min at room temperature andpermeabilised in 01 (vv) Triton X-100 for 10min Nonspe-cific antibody binding was blocked by incubating in 2 (wv)BSA for 30min Cells were incubated overnight with thefollowing primary antibodies nestin (Santa Cruz) musashi(Life Technologies) microtubule-associated protein 2 (Map2(Millipore)) neurofilament light chain NF-l (Abcam) Olig1and Olig2 (both from Millipore) myelin basic protein (MBP(Abcam)) and 120573-actin (Cell Signalling) On the followingday complementary Alexa Fluor 488- and 594-conjugatedsecondary antibodies (Life Technologies) were applied Glasscoverslips were mounted using mounting media supple-mented with DAPI stain (VectorLabs) and preparationsimaged under a fluorescent microscope

28 PatchClampElectrophysiological Recordings Transmem-brane currents of primary culturedmSTMneurons fromdays3 to 21 of culture and mDPSCs neuronally differentiated for15 days were recorded in conventional whole-cell configura-tion The bath solution contained 135mM NaCl 5mM KCl12mMMgCl

2 125mM CaCl

2 10mM d-glucose and 5mM

HEPES pH was adjusted to 74 using NaOH The pipettesolution contained 117mM KCl 10mM NaCl 2mM MgCl

2

1 mM CaCl2 2mM Na

2ATP 1mM Na

2GTP 11mM HEPES

and 11mM ethylene glycol tetra acetic acid free [Ca2+]119894was

adjusted to 100 nM pH was adjusted to 72 with KOH Allrecordings were performed at room temperature (22 plusmn 05∘C)using an Axopatch 200B amplifier and Digidata 1320ADinterface (Axon Instruments) Holding voltages were setto minus70mV and transmembrane currents recorded using avoltage step protocol of 80ms duration in voltage range fromminus120 to +80mV Series resistance and membrane capacitancewere compensated asymp90 Pipette resistances were asymp5ndash10MΩwhen filled with the pipette solution All recordings werefiltered with an 8-pole Bessel filter at 5 kHz and digitized at10 kHz Tetraethylammonium chloride (TEA) was purchasedfrom Sigma

29 Data Analysis The patch clamp data were analyzedusing Clampfit 90MicrosoftOffice Excel 2003 andMicrocalOrigin 60 software Transient inward Na+ currents werepresented as peak values whereas outward steady-state K+currents were presented as means Current densities (pApF)were plotted against command voltage (mV) Statistical com-parisons of the means were performed using independent 119905-test differences were considered significant at 119901 lt 005

3 Results

31 Isolation Expansion and Characterisation of ClonalmDPSC Cultures Dental pulp cells were successfully iso-lated and cultured from murine incisors One day follow-ing isolation sparsely distributed fibroblastic-like cells wereidentified growing on fibronectin-coated culture surfaces Anumber of these rapidly expanded clonally to form discreteindividual colonies Clones were isolated at day 12 andfound to expand over extended periods some reaching50+ population doublings confirming a highly proliferativephenotype (Figure 1(a)) Presence of the neural progenitormarkers nestin and musashi was identified within expandingclones using immunocytochemistry (Figure 1(b)) RNA wasextracted fromeach clone between 20 and 40population dou-blings and used in RT-PCR to identify further similarities inmarker expression between four mDPSC clones and primarycultured murine neural stem cells (mNSCs) (Figure 1(c))mDPSC clones were found to express a range of transcriptsalso expressed byNSCs includingCD90 SCA1 GLAST Sox2Pax6 Myt1l P75 BLBP musashi and NF-l However markedclonal differences were observed with only the general stemcell marker SCA1 the neural crest-associated marker P75and the radial glial protein BLBP being expressed by allmDPSC clones tested No expression of CD133 was foundin any mDPSC clone Furthermore clonal differences wereidentified in themRNAexpression levels of nestin Based on asemiquantitative analysis 8 out of 11 isolated clones appearedto express nestin mRNA transcripts at higher levels than theremaining 3 clonesHowever only 4 of these clones continuedto expand to allow the extraction of further RNA samples fora more accurate quantification of nestin expression and thusare subsequently described in this study qPCR identified thatclone 1 and clone 2 each expressed significantly higher levels(119901 lt 001) of nestin transcripts than both clone 3 and clone4 (Figure 1(d)) Subsequently clones 1 and 2 were definedas high nestin-expressing clones and clones 3 and 4 as lownestin-expressing clones

32 Neuronal-Like Differentiation mDPSCs were typicallybitripolar and fibroblastic-like inmorphology prior to differ-entiation Following 15 days of differentiation clones initiallyidentified as having high levels of nestin mRNA expres-sion adopted a more neuronal-like phenotype with multipleneurite-like extensions Conversely no significant changesin morphology were observed in low nestin-expressingclones (Figure 2(a)) Immunocytochemical staining of themature neuronal proteins Map2 and NF-l was identified inhigh but not low nestin-expressing mDPSC clones after15 days of differentiation (Figure 2(b)) Changes in themRNA expression of early and mature neuronal markersby high nestin-expressing clones over 15 days of differen-tiation were analyzed using qPCR (Figure 2(c)) By day 5of differentiation mRNA expression of SCA1 was stronglydownregulated suggesting a transition from the default mes-enchymal phenotype associated with mDPSCs From day5 onwards after transferring to the neutrophin-containingmaturation medium expression of nestin and Map2 wasseen to increase indicating a more neuronal-like phenotype


0 50 100 150 200 250

Days in culture

Clone 1Clone 2

Clone 3Clone 4

0

10

20

30

40

50

Num

ber o

f pop

ulat

ion

doub

lings

(a)

Clone 1

Nes

tinM

usas

hi

Clone 2 Clone 3 Clone 4

(b)

Clone 1

Clone 2

Clone 3

Clone 4

RT-ve

mNSC

GA

PDH

NF-

l

GLA

ST

BLBP

P75

My1

tl

Pax6

CD133

Sox2

SCA1

CD90

Radi

al g

lia

Hou

se-

keep

ing

Mat

ure

neur

ons

Neu

ral

cres

t

Neu

ral

deve

lopm

ent

Neu

ral

stem

cells

Mes

ench

ymal

stem

cells

(c)

Clone 1 Clone 2 Clone 3 Clone 4

lowastlowast

ns ns

0

2

4

6

8

10

Relat

ive n

estin

mRN

A ex

pres

sion

( o

f GA

PDH

)

(d)

Figure 1 In vitro expansion and heterogeneity in the expression of developmental markers by clonal mDPSC cultures (a) Single cell-derived clones each expanded from a separate pulpal extraction proliferated steadily for up to 240 days of culture reaching 50+ populationdoublings (119899 = 4 clones) Traces represent continuous culture growth from day of primary isolation and cryopreserved cells continued toproliferate beyond the population doublings indicated (b) Double immunostaining of clonal cultures for neural progenitor markers nestinand musashi (c) RNA extracted from each clone between 20 and 40 population doublings was used in RT-PCR to identify clonal differencesin the expression of RNA transcripts for CD90 stem cell antigen 1 (SCA1) glutamate aspartate transporter (GLAST) Sox2 Pax6 myelintranscription factor 1-like (Myt1l) P75 musashi neurofilament light chain (NF-l) and CD133 (d) qPCR analysis of nestin mRNA expressionby four mDPSC clones Clones 1 and 2 were each individually found to express significantly higher levels of nestin than both clones 3 and 4DPSC cultures were subsequently divided into strongly nestin-positive clones (clone 1 and clone 2) and weakly nestin-positive clones (clone3 and clone 4) Nestin expression was calculated as a relative percentage of GAPDH plusmn SEM using the 2minusΔΔCT method (119899 = 3 RNA samplesextracted from three separate passages per clone between 16 and 40 population doublings) One-way ANOVA with Tukey-Kramer posttestns = not significant lowastlowast119901 lt 001 Scale bars = 100120583m

mRNAexpression levels forNF-lwere found to be unchangedduring the 15 days of differentiation

Patch clamp recordings were taken to characterise theelectrophysiological properties of high nestin-expressingmDPSC clones prior to and after 15 days of differentiationPrimary cultured mSTM neurons provided a positive controlfor comparison purposes Only outward K+ currents weredetected in mDPSCs both before and after differentiationThese currents were effectively inhibited with 1mM TEAa nonselective blocker of K+ channels (Figures 3(a) and3(b)) At the same time both voltage-activated K+ and Na+currents were recorded in P0 mSTM neurons (Figure 3(c))

At a voltage of +80mV significantly higher K+ currentdensities were recorded in P0 mSTM neurons (1554 plusmn101 pApF) compared to undifferentiated (64 plusmn 15 pApF)and neuronally differentiated mDPSCs (73 plusmn 14 pApF)119901 lt 30119864 minus 13 and 119901 lt 60119864 minus 15 respectively Nosignificant differencewas found between the current densitiesof undifferentiated and neuronally differentiated mDPSCshowever membrane capacitance varied dramatically (Fig-ure 3(d)) Differentiated mDPSCs (307 plusmn 40) possessedstatistically significant lower membrane capacitances thanundifferentiated mDPSCs (620 plusmn 102) 119901 lt 0005 Thislower capacitance was directly comparable with p0 mSTM


PredifferentiationH

igh

nesti

n clo

neLo

w n

estin

clon

eDifferentiation d15

(a)

NF-l Map2

Hig

h ne

stin

clone

Low

nes

tin cl

one

(b)

SCA1 Nestin Map2 NF-1

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

Day 5Day 10

Day 15

001

01

1

10

Fold

chan

ge in

mRN

A ex

pres

sion

leve

ls

(c)

Figure 2 Only high nestin-expressing mDPSC clones possess the ability to differentiate into neuronal-like cells (a) Representative phasecontrast images of high (clone 2) and low (clone 3) nestin-expressing clones prior to and following 15 days of neuronal differentiationdemonstrating a more neuronal-like morphology in high nestin-expressing clones with small refractive cell somas extending multipleinterconnecting processes (b) Immunocytochemical staining identified the presence of microtubule-associated protein 2 (Map2) and NF-l in high but not low nestin-expressing mDPSC clones following 15 days of neuronal differentiation (c) Changes in mRNA expressionof mesenchymal and neural markers during neuronal differentiation of high nestin-expressing mDPSC clones (clone 2) Expression levelsof target genes were normalized against GAPDH and the 2minusΔΔCT method for qPCR analysis used to calculate fold change in expressionrelative to predifferentiation cells on day 0 plusmn SEM (119899 = 3 independent differentiation experiments) One-way ANOVAwith Dunnett multiplecomparisons posttest to identify significant increasesdecreases in expression compared to day 0 cells lowast119901 lt 005 and lowastlowast119901 lt 001 Scale bars= 100 120583m

neurons (209 plusmn 66) with no significant difference observed(119901 gt 02) Together these results suggest that despiteappropriate morphology the presence of mature neuronalproteins after 15 days of differentiation and a comparablecell capacitance to primary cultured neurons high nestin-expressingmDPSC clonesmaintain an electrophysiologicallyimmature phenotype

33 Oligodendrocyte-Like Differentiation Following 10 daysof differentiation clones with initially high levels of nestinwere seen to adopt a highly branched oligodendrocyte-like morphology Although some branching was observedlow nestin-expressing clones largely failed to survive 10days of differentiation (Figure 4(a)) Immunocytochemicalstaining of myelin basic protein (MBP) Olig1 and Olig2


1

2

0

2

4

6

8

10

Curr

ent d

ensit

y (p

Ap

F)

200minus20 40minus120 minus60 60minus40minus80 80minus100

Voltage (mV)

10ms100pA

(a)

1

2

0

2

4

6

8

10

Curr

ent d

ensit

y (p

Ap

F)

minus100 minus80 minus60 minus40 800 20 40 60minus20minus120

Voltage (mV)

10ms20pA

(b)

Curr

ent d

ensit

y (p

Ap

F)

minus100

minus50

0

50

100

150

Voltage (mV)minus120 minus100 minus80 minus60 minus40 minus20 0 20 40 60 80

10ms1nA

(c)

0

50

100

150

200

Capa

cita

nce (

pF)

UndifferentiatedmDPSCs

Neuronallydifferentiated

mDPSCs

P0 mouseneurons

(d)

Figure 3 Neuronally differentiated high nestin-expressing mDPSCs show immature electrophysiology properties Current density-voltagerelationships of transmembrane K+ currents of undifferentiated (a) and neuronally differentiated (b) high nestin-expressing mDPSCs (clone2) in the absence (I inlet 1 illustrates exemplar trace of currents 119899 = 14 and 17 cells resp) and presence of TEA (1mM) (◼ inlet 2 illustratesexemplar trace of currents 119899 = 9 for each differentiation condition) (c) Current density-voltage relationships of transmembrane K+ current(I) and Na+ currents (998787) of mSTM neurons (119899 = 23 cells) Inlet illustrates exemplar trace of currents The mean plusmn SEM current densitiesat +80mV of undifferentiated (64 plusmn 15 pApF) and neuronally differentiated mDPSCs (73 plusmn 14 pApF) showed a significant difference incomparison with primary cultured mSTM neurons (1554 plusmn 101 pApF) 119901 lt 30119864 minus 13 and 119901 lt 60119864 minus 15 respectively (d) Comparisonof capacitances of all three cell types The mean values plusmn SEM (◻) of undifferentiated mDPSCs (620 plusmn 102) and neuronally differentiatedhigh nestin-expressing mDPSCs (307 plusmn 40) as well as undifferentiated mDPSCs and P0 mSTM neurons (209 plusmn 66) were considered assignificantly different 119901 lt 0005 and 119901 lt 0002 respectively e and I are maximal and minimal values respectively

was only observed in high nestin-expressing mDPSC clones(Figure 4(b)) The expression of oligodendrocyte-associatedproteins togetherwith appropriatemorphology suggests thatthis novel protocolmay be used to derive an oligodendrocyte-like phenotype from mDPSCs with high levels of nestinexpression

4 Discussion

In this study we have identified heterogeneity in the ability ofsingle cell-derived clonal cultures of mDPSCs to differentiateinto neuronal-like and glial-like cellsThose clones possessingthe highest levels of mRNA expression for the neuronal

progenitor-associated intermediate filament protein nestinshowed a greater potential for differentiation down bothneural lineages Although some evidence of variability inthe neural differentiation potential of heterogeneous DPSChas been previously described [18 22] our findings sug-gest that nestin may act as a suitable marker for whichto screen DPSC cultures in vitro prior to use in neuraltissue engineering applicationsThe problems associatedwithcellular heterogeneity are increasingly becoming recognisedin the stem cell research field and gaining a fuller under-standing of the variability within transplantable populationswill help maximise the potential of any stem cell-basedtherapy


Differentiation d10

Hig

h ne

stin

clone

Low

nes

tin cl

one

(a)

Hig

h ne

stin

clone

Low

nes

tin cl

one

120573-actinOlig1 120573-actinOlig2 MBP

(b)

Figure 4 Only high nestin-expressing mDPSC clones display the ability to differentiate into oligodendrocyte-like cells (a) Representativephase contrast images of high (clone 2) and low (clone 3) nestin-expressing clones following 15 days of oligodendrocyte-like differentiationClones with higher levels of nestin mRNAwere found to adopt a more highly branched oligodendrocyte-like morphology compared to lowernestin-expressing clones (b) Immunocytochemical staining identified the presence of myelin basic protein (MBP) and the oligodendrocytetranscription factors Olig1 and Olig2 in high but not low nestin-expressing mDPSC clones following 10 days of differentiation 120573-actinstaining was performed to demonstrate highly branched morphology Scale bars = 100 120583m


Prior to differentiation the expression of a range ofdevelopmental and neural progenitor markers by singlecell-derived mDPSC cultures was extensively analyzed andcompared with primary mNSCs Although mDPSCs werefound to express a number of markers also associated withNSCs including Sox2 Pax6 GLAST BLBP nestin and NF-l expression patterns were highly variable between clonesdemonstrating the degree of heterogeneity that exists withinthe mixed populations of DPSCs typically used for neu-ral transplantation studies [10ndash15] Importantly only thoseclones identified with high levels of nestin mRNA expres-sion displayed the ability to differentiate into a neuronal-like phenotype based on cell morphology and increasedexpression levels of the more mature neuronal marker Map2To test the electrophysiological properties of these cellspatch clamp recordings were made The electrical propertiesof neuronal-like cells derived from murine DPSCs remainlargely uncharacterised in contrast to humanDPSCs inwhichvoltage-activated Na+ and K+ currents and ATP-activatedCa2+ surges have been recorded [17 18 20 21] In theonly previous functional study using rodent DPSCs mixedpopulations of mDPSCs differentiated using an establishedprotocol displayed voltage-activated Ca2+ but not K+ or Na+currents directly contradicting recordings taken when thesame protocol was applied to human hDPSCs [18 19] Singlecell-derived cultures of high nestin-expressing mDPSCs dif-ferentiated using the protocol described in this report on theother hand display TEA-sensitive voltage-gated K+ currentsdemonstrating the presence of functional voltage-activatedK+ channels in neuronally differentiated rodent DPSCs forthe first time Although the amplitude of these currents isreduced when compared to mSTM neurons similar mem-brane capacitances were measured for each cell type Thisreduction in capacitance is indicative of cells with an abilityto store electrical charge directly comparable to primarycultured striatal neurons confirming a more neuronal-likephenotype after differentiation Although a fully functionalphenotype with the ability to fire action potentials has yetto be derived from either human or rodent DPSCs there issufficient evidence here to suggest that high nestin-expressingmDPSCs may be promoted to differentiate at least partiallyalong this lineage However further steps will be required toobtain a more mature neuronal-like phenotype and futurestudies might focus on incorporating a supporting cell typein coculture to provide appropriate trophic factor support forthe development andmaturation of functional properties forexample astrocytes [28 29]

Oligodendrocyte-like differentiation of DPSCs has onlypreviously been described in vivo following mixed pop-ulation transplantation into a rat model of spinal cordinjury [12] Using a novel protocol adapted from thoseused in the culture and differentiation of oligodendro-cyte progenitor cells (OPCs) [30 31] mDPSC clones withhigh levels of nestin mRNA expression adopted a highlybranched oligodendrocyte-like morphology and stained pos-itive for oligodendrocyte markers Olig1 Olig2 and MBPDespite the expression of MBP in differentiated mDPSCsthere was no observation of membranous sheets associated

with mature myelinating oligodendrocytes in vitro [30 32]This suggests that similar to neuronal differentiation highnestin-expressing mDPSCs are able to differentiate partiallyto an immature premyelinating phenotype but furtherdifferentiation steps may be required for full functionalityNevertheless the development of this protocol represents asignificant finding and may provide a useful in vitro researchtool for further studies into mechanisms through whichDPSCs may promote central nervous system repair andregeneration

Unlike bone marrow another common source of mes-enchymal stem cells dental pulp is a nonhaematopoetic tissueand clonal DPSC cultures may be more lineage-restricted innature [9] Their highly heterogeneous nature is purportedto be attributable to multiple populations of progenitor cellsresiding in different locations of the pulp which may possessdifferent proliferative and differentiation capabilities Differ-ent niches have been identified in situ associated with thevasculature within the pulpal stroma in the subodontoblastlayer and amongst peripheral nerve-associated glial cells [33ndash38]During development the dental pulp and central nervoussystem both derive from the embryonic ectoderm Followingneurulationmultipotent neural crest cells migrate away fromthe neural tube into developing craniofacial tissues At theinitiation of tooth morphogenesis these cells populate theunderlyingmesenchyme eventually giving rise to the cellularcomponents of pulpal tissue [39] Multipotent adult DPSCsthat maintain neural crest stem cell characteristics and mayrepresent a source of cells with greater potential for neuronaland glial differentiation given their developmental originhave been isolated from different niches within the pulp[25 38 40 41] A recent study compared the proliferativeand differentiation potentials of human DPSCs based onthe expression of the pericyte-associated cell surface antigenCD34 [42] Only CD34+ hDPSCs were found to expressnestin and possess the ability to differentiate down neu-ronal lineages similar to the high nestin-expressing mDPSCdescribed here suggesting that they may be neural crest inorigin and derived from a perivascular-associated niche Itmay prove beneficial to select for suchDPSCs in future neuraltissue engineering studies

Together the results presented herein suggest that mRNAlevels of nestin may be indicative of the potential of mDP-SCs for neuronal-like and oligodendrocyte-like differenti-ation Nestin expression is associated with stem cells inthe developing neural tube as well as specific subtypes ofOPCs [43 44] As such its link to mDPSCs with neu-ronal and oligodendrocyte-like differentiation capabilitiesfits However nestin-positive cells make up only a smallfraction of the total cellular component of dental pulpless than 35 reported in isolates from rat incisors [45]Most published studies utilise such mixed populations ofcells and so likely contain a significant proportion of othercell types perhaps explaining previous inconsistencies inresponse to neuronal differentiation cues [18 22] The use ofclonally derived cultures allows investigations to be carriedout at the single cell level and the subsequent identificationof differences between individual clonal cell lines Largedifferences in the proliferation and mineralisation potential


of clonal DPSC cultures have been previously reported inthis manner [1 9] Similarly differences in the neuronal-likeand oligodendrocyte-like differentiation potential of mDPSCclones are reported hereThe use of single cell-derived clonesis unlikely to be therapeutically applicable due to scalabilityissues within short time frames However clonal culturesserve as an extremely useful research tool to identify desir-able properties of cells within mixed populations In futurestudies the screening of single cell-derived clones on a largerscale to that described in this report will serve to further ourunderstanding of cellular heterogeneity and its implicationsfor the development of stem cell-based therapies

5 Conclusions

Significant heterogeneity exists between clonal cultures ofmDPSCs and clones with comparatively higher levels ofnestin expression possess a greater capacity for differentiationinto neural lineages These findings help explain previousreports of only small numbers of transplanted DPSCs adopt-ing neuronal-like and glial-like phenotypes after transplan-tation as well as inconsistencies in in vitro differentiationstudies In conclusion high nestin-expressing DPSCs mayrepresent a more desirable cell source for promoting centralnervous system repair and regeneration

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This study was funded by European Research Council StGGrant 243261 Wellcome Trust Grant WT082887 and theRoyal Society URF Award UF051616 to Bing Song

References

[1] S Gronthos J Brahim W Li et al ldquoStem cell properties ofhuman dental pulp stem cellsrdquo Journal of Dental Research vol81 no 8 pp 531ndash535 2002

[2] A Balic H L Aguila M J Caimano V P Francone and MMina ldquoCharacterization of stem and progenitor cells in thedental pulp of erupted and unerupted murine molarsrdquo Bonevol 46 no 6 pp 1639ndash1651 2010

[3] V Govindasamy A N Abdullah V S Ronald et al ldquoInherentdifferential propensity of dental pulp stem cells derived fromhuman deciduous and permanent teethrdquo Journal of Endodon-tics vol 36 no 9 pp 1504ndash1515 2010

[4] K Iohara L Zheng HWake et al ldquoA novel stem cell source forvasculogenesis in ischemia subfraction of side population cellsfrom dental pulprdquo STEM CELLS vol 26 no 9 pp 2408ndash24182008

[5] I Kerkis C E Ambrosio A Kerkis et al ldquoEarly transplantationof human immature dental pulp stem cells from baby teeth togolden retriever muscular dystrophy (GRMD) dogs local orsystemicrdquo Journal of Translational Medicine vol 6 article 352008

[6] F Paino G Ricci A De Rosa et al ldquoEcto-mesenchymal stemcells from dental pulp are committed to differentiate into activemelanocytesrdquo European Cells amp Materials vol 20 pp 295ndash3052010

[7] R Nakatsuka T Nozaki Y Uemura et al ldquo5-Aza-21015840-deoxycy-tidine treatment induces skeletal myogenic differentiation ofmouse dental pulp stem cellsrdquo Archives of Oral Biology vol 55no 5 pp 350ndash357 2010

[8] N Ishkitiev K Yaegaki T Imai et al ldquoHigh-purity hepaticlineage differentiated from dental pulp stem cells in serum-freemediumrdquo Journal of Endodontics vol 38 no 4 pp 475ndash4802012

[9] J Harrington A J Sloan and R JWaddington ldquoQuantificationof clonal heterogeneity of mesenchymal progenitor cells indental pulp and bone marrowrdquo Connective Tissue Research vol55 no 1 pp 62ndash67 2014

[10] F M De Almeida S A Marques B D S Ramalho et alldquoHuman dental pulp cells a new source of cell therapy in amouse model of compressive spinal cord injuryrdquo Journal ofNeurotrauma vol 28 no 9 pp 1939ndash1949 2011

[11] M Sugiyama K Iohara H Wakita et al ldquoDental pulp-derivedCD31minusCD146minus side population stemprogenitor cells enhancerecovery of focal cerebral ischemia in ratsrdquoTissue EngineeringmdashPart A vol 17 no 9-10 pp 1303ndash1311 2011

[12] K Sakai A Yamamoto K Matsubara et al ldquoHuman dentalpulp-derived stem cells promote locomotor recovery aftercomplete transection of the rat spinal cord by multiple neuro-regenerative mechanismsrdquoThe Journal of Clinical Investigationvol 122 no 1 pp 80ndash90 2012

[13] C-Z Fang Y-J Yang Q-H Wang Y Yao X-Y Zhang andX-HHe ldquoIntraventricular injection of humandental pulp stemcells improves hypoxic-ischemic brain damage in neonatal ratsrdquoPLoS ONE vol 8 no 6 article e66748 2013

[14] A Yamamoto K Sakai K Matsubara F Kano and M UedaldquoMultifaceted neuro-regenerative activities of human dentalpulp stem cells for functional recovery after spinal cord injuryrdquoNeuroscience Research vol 78 no 1 pp 16ndash20 2014

[15] H Fujii K Matsubara K Sakai et al ldquoDopaminergic differ-entiation of stem cells from human deciduous teeth and theirtherapeutic benefits for Parkinsonian ratsrdquo Brain Research vol1613 pp 59ndash72 2015

[16] F Young A Sloan and B Song ldquoDental pulp stem cells andtheir potential roles in central nervous system regeneration andrepairrdquo Journal of Neuroscience Research vol 91 no 11 pp 1383ndash1393 2013

[17] A Arthur G Rychkov S Shi S A Koblar and S GronthoseldquoAdult human dental pulp stem cells differentiate toward func-tionally active neurons under appropriate environmental cuesrdquoStem Cells vol 26 no 7 pp 1787ndash1795 2008

[18] M Kiraly B Porcsalmy A Pataki et al ldquoSimultaneous PKC andcAMP activation induces differentiation of human dental pulpstem cells into functionally active neuronsrdquo NeurochemistryInternational vol 55 no 5 pp 323ndash332 2009

[19] KM Ellis D COrsquoCarrollMD Lewis G Y Rychkov and S AKoblar ldquoNeurogenic potential of dental pulp stem cells isolatedfrom murine incisorsrdquo Stem Cell Research and Therapy vol 5article 30 2014

[20] M Kanafi D Majumdar R Bhonde P Gupta and I DattaldquoMidbrain cues dictate differentiation of human dental pulpstem cells towards functional dopaminergic neuronsrdquo Journalof Cellular Physiology vol 229 no 10 pp 1369ndash1377 2014


[21] PGervois T Struys PHilkens et al ldquoNeurogenicmaturation ofhuman dental pulp stem cells following neurosphere generationinduces morphological and electrophysiological characteristicsof functional neuronsrdquo Stem Cells and Development vol 24 no3 pp 296ndash311 2015

[22] R Aanismaa J Hautala A Vuorinen S Miettinen and SNarkilahti ldquoHuman dental pulp stem cells differentiate intoneural precursors but not intomature functional neuronsrdquo StemCell Discovery vol 2 no 3 pp 85ndash91 2012

[23] P H Jones and F M Watt ldquoSeparation of human epidermalstem cells from transit amplifying cells on the basis of differ-ences in integrin function and expressionrdquo Cell vol 73 no 4pp 713ndash724 1993

[24] G P Dowthwaite J C Bishop S N Redman et al ldquoThe surfaceof articular cartilage contains a progenitor cell populationsrdquoJournal of Cell Science vol 117 no 6 pp 889ndash897 2004

[25] R JWaddington S J Youde C P Lee andA J Sloan ldquoIsolationof distinct progenitor stem cell populations from dental pulprdquoCells Tissues Organs vol 189 no 1ndash4 pp 268ndash274 2009

[26] X Meng W Li F Young et al ldquoElectric field-controlleddirected migration of neural progenitor cells in 2D and 3Denvironmentsrdquo Journal of Visualized Experiments no 60 2012

[27] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCRand the 2minusΔΔ119862TmethodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[28] X Tang L Zhou A M Wagner et al ldquoAstroglial cells regulatethe developmental timeline of human neurons differentiatedfrom induced pluripotent stem cellsrdquo Stem Cell Research vol11 no 2 pp 743ndash757 2013

[29] D Pre MW Nestor A A Sproul et al ldquoA time course analysisof the electrophysiological properties of neurons differentiatedfrom human induced Pluripotent Stem Cells (iPSCs)rdquo PLoSONE vol 9 no 7 Article ID e103418 2014

[30] A Jagielska A L Norman G Whyte K J V Vliet J Guckand R J M Franklin ldquoMechanical environment modulatesbiological properties of oligodendrocyte progenitor cellsrdquo StemCells and Development vol 21 no 16 pp 2905ndash2914 2012

[31] B Zhu C Zhao F I Young R J M Franklin and B SongldquoIsolation and long-term expansion of functional myelinatingoligodendrocyte progenitor cells from neonatal rat brainrdquoCurrent Protocols in Stem Cell Biology 2014

[32] C A Dyer and J-M Matthieu ldquoAntibodies to myelinoligodendrocyte-specific protein and myelinoligodendrocyteglycoprotein signal distinct changes in the organization ofcultured oligodendroglial membrane sheetsrdquo Journal of Neuro-chemistry vol 62 no 2 pp 777ndash787 1994

[33] S Shi and S Gronthos ldquoPerivascular niche of postnatal mes-enchymal stem cells in human bone marrow and dental pulprdquoJournal of Bone andMineral Research vol 18 no 4 pp 696ndash7042003

[34] H LoslashvschallM Tummers IThesleff E-M Fuchtbauer andKPoulsen ldquoActivation of theNotch signaling pathway in responseto pulp capping of ratmolarsrdquoEuropean Journal ofOral Sciencesvol 113 no 4 pp 312ndash317 2005

[35] O Tecles P Laurent S Zygouritsas et al ldquoActivation of humandental pulp progenitorstem cells in response to odontoblastinjuryrdquoArchives of Oral Biology vol 50 no 2 pp 103ndash108 2005

[36] J Feng A Mantesso C De Bari A Nishiyama and P T SharpldquoDual origin of mesenchymal stem cells contributing to organgrowth and repairrdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 108 no 16 pp 6503ndash6508 2011

[37] N F Lizier A Kerkis C M Gomes et al ldquoScaling-up of dentalpulp stem cells isolated from multiple nichesrdquo PLoS ONE vol7 no 6 Article ID e39885 2012

[38] N KaukuaM K Shahidi C Konstantinidou et al ldquoGlial originofmesenchymal stem cells in a toothmodel systemrdquoNature vol513 no 7519 pp 551ndash554 2014

[39] Y Chai X Jiang Y Ito et al ldquoFate of the mammalian cranialneural crest during tooth and mandibular morphogenesisrdquoDevelopment vol 127 no 8 pp 1671ndash1679 2000

[40] K Janebodin O V Horst N Ieronimakis et al ldquoIsolation andcharacterization of neural crest-derived stem cells from dentalpulp of neonatal micerdquo PLoS ONE vol 6 no 11 Article IDe27526 2011

[41] S Abe K Hamada M Miura and S Yamaguchi ldquoNeural creststem cell property of apical pulp cells derived from humandeveloping toothrdquo Cell Biology International vol 36 no 10 pp927ndash936 2012

[42] A Pisciotta G Carnevale S Meloni et al ldquoHumanDental pulpstem cells (hDPSCs) isolation enrichment and comparativedifferentiation of two sub-populations Integrative control ofdevelopmentrdquo BMCDevelopmental Biology vol 15 no 1 article14 2015

[43] U Lendahl L B Zimmerman and R D G McKay ldquoCNS stemcells express a new class of intermediate filament proteinrdquo Cellvol 60 no 4 pp 585ndash595 1990

[44] G Almazan J M Vela E Molina-Holgado and C Guaza ldquoRe-evaluation of nestin as a marker of oligodendrocyte lineagecellsrdquoMicroscopy Research andTechnique vol 52 no 6 pp 753ndash765 2001

[45] M Takeyasu T Nozaki andMDaito ldquoDifferentiation of dentalpulp stem cells into a neural lineagerdquo Pediatric Dental Journalvol 16 no 2 pp 154ndash162 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


to differentiate into neuronal-like cells [16] Furthermoreindependent studies have described a degree of functionalityof such neuronally differentiated DPSCs [17ndash21] Howeveronly a small fraction of cells within such cultures developa functional phenotype [18] Similarly outcomes may differbetween patient samples subjected to the same differen-tiation protocol [22] Understanding the cellular biologybehind such heterogeneity poses a substantial challenge toresearchers The identification of an appropriate marker ofneural differentiation capabilities for which to screen DPSCcultures beforehand could help to minimise such variabilitybetween studies resulting in more defined and predictableoutcomes

In this study single cell-derived clonal populations ofmurine DPSCs (mDPSCs) were isolated and differences inthe expression of early stage neural markers identified Onlyclones with high levels of nestin expression were found to dif-ferentiate into immature neuronal-like cells displayingmini-mal electrical activity Similarly a novel differentiation proto-col was developed for the derivation of oligodendrocyte-likecells from high nestin-expressing mDPSC clones Togetherthese findings suggest that nestinmay act as a suitablemarkerfor use in assessing the ability of mDPSCs to differentiate intoneuronal-like and glial-like cells in vitro

2 Experimental Procedures

21 Isolation of mDPSCs All procedures were approvedby the Cardiff University Biological Standards Office Foreach DPSC isolation the incisor pulpal tissue of 4-5 times21ndash28-day-old C57Bl6 mice sacrificed by CO

2asphyxia-

tion in accordance with Schedule 1 of the UK Animals(Scientific Procedures) Act 1986 was pooled Followingcollagenasedispase digestion to a cellular suspension thepreferential adherence to fibronectin selection technique wasused to select for progenitor cells by isolating cells of moreimmature phenotypes based on 1205731 integrin functionality [2324] After 12 days of primary culture individual colonies offibronectin-adherent cells displaying typical DPSC bipolarfibroblastic-like morphology and numbering greater than32 cells were selected for clonal isolation and expansionas described previously using cloning rings [25] ClonalDPSCswere subsequently expanded in120572MEMsupplementedwith 100 unitsmL penicillin 100120583gmL streptomycin and20 (vv) heat-inactivated foetal bovine serum (all fromLife Technologies) and 100 120583M l-ascorbic acid 2-phosphate(Sigma-Aldrich) Cell counts were performed at every pas-sage and used to track population doublings over time inculture

Population doublings =log10(cell count at passage) minus log

10(no of cells initially seeded)

log10(2)

(1)

Cells of between 20 and 40 population doublings wereused for all experiments Four individual mDPSC clonesthat expanded sufficiently to allow multiple reproducibledifferentiation experiments were used in this study eachderived from a separate pulpal extraction (119899 = 4)

22 Neuronal Differentiation mDPSC clones were seededat 10000 cellscm2 on poly-d-lysinelaminin-coated culturesurfaces in DMEMF12 (1 1) containing L-glutamine andHEPES buffer 100 unitsmL penicillin 100 120583gmL strepto-mycin and 1 times N2 supplement (all from Life Technologies)1 times NEAA (Sigma-Aldrich) and 20 ngmL basic fibrob-last growth factor (bFGF) and 20 ngmL epidermal growthfactor (both from Peprotech) After 5 days cultures werewashed with PBS and changed to neurobasal medium sup-plemented with 100 unitsmL penicillin 100120583gmL strepto-mycin and 2mM L-glutamine (all from Life Technologies) 1times NEAA (Sigma-Aldrich) and 10 ngmL brain-derived neu-rotrophic factor 10 ngmL nerve growth factor and 10 ngmLneurotrophin-3 (all from Peprotech) RNA was extracted ondays 0 5 10 and 15 of differentiation for use in qPCR and cellswere fixed on day 15 for immunocytochemistry

23 Oligodendrocyte Differentiation Clonal mDPSC cultureswere seeded at 10000 cellscm2 on poly-d-lysine-coatedculture surfaces in DMEM containing 100 unitsmL peni-cillin 100 120583gmL streptomycin SATO supplement (16 120583gmL

putrescine 62 ngmL progesterone 5 ngmL sodium seleniteand 100 120583gmL bovine serum albumin (BSA)) 50 120583gmLholo-Transferrin and 5 120583gmL insulin (all from Sigma-Aldrich) and 10 ngmL platelet-derived growth factor-aa and20 ngmL bFGF (both Peprotech) After 10 days differentia-tion cells were fixed for immunocytochemistry

24 Isolation of mSTM Neurons Mouse striatal (mSTM)neuronal tissues were dissected in PBS from P0 micedigested using Accutase and plated on poly-l-lysine-coatedglass coverslips inAdvancedDMEMF-12 supplementedwith2mML-glutamine 100 unitsmL penicillin 100 120583gmL strep-tomycin 18mMCaCl

2 05mML valproic acid and 1 x B27-

supplement (without vitaminA) (all from Life Technologies)

25 Reverse Transcriptase PCR Total RNA was extractedusing an RNeasy Mini Kit with on-column DNase digestion(QIAGEN) according tomanufacturerrsquos directions and cDNAsynthesised using MMLV reverse transcriptase (Promega)PCR reactions were performed using GoTaq Polymerase(Promega) and product-specific primers (Supplemental Table1 in Supplementary Material available online at httpdxdoiorg10115520161290561) RNA extracted from primary cul-tured E145 cortical neural stem cells isolated as describedpreviously [26] was used as a positive control

26 Real Time Quantitative PCR For qPCR readings cDNAsamples generated from three separate experiments per clone





2 125mM CaCl



2

1 mM CaCl2 2mM Na

2ATP 1mM Na

2GTP 11mM HEPES




3 Results




0 50 100 150 200 250

Days in culture

Clone 1Clone 2

Clone 3Clone 4

0

10

20

30

40

50

Num

ber o

f pop

ulat

ion

doub

lings

(a)

Clone 1

Nes

tinM

usas

hi


(b)

Clone 1

Clone 2

Clone 3

Clone 4

RT-ve

mNSC

GA

PDH

NF-

l

GLA

ST

BLBP

P75

My1

tl

Pax6

CD133

Sox2

SCA1

CD90

Radi

al g

lia

Hou

se-

keep

ing

Mat

ure

neur

ons

Neu

ral

cres

t

Neu

ral

deve

lopm

ent

Neu

ral

stem

cells

Mes

ench

ymal

stem

cells

(c)


lowastlowast

ns ns

0

2

4

6

8

10

Relat

ive n

estin

mRN

A ex

pres

sion

( o

f GA

PDH

)

(d)






PredifferentiationH

igh

nesti

n clo

neLo

w n

estin

clon


(a)

NF-l Map2

Hig

h ne

stin

clone

Low

nes

tin cl

one

(b)


lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

Day 5Day 10

Day 15

001

01

1

10

Fold

chan

ge in

mRN

A ex

pres

sion

leve

ls

(c)





1

2

0

2

4

6

8

10

Curr

ent d

ensit

y (p

Ap

F)


Voltage (mV)

10ms100pA

(a)

1

2

0

2

4

6

8

10

Curr

ent d

ensit

y (p

Ap

F)


Voltage (mV)

10ms20pA

(b)

Curr

ent d

ensit

y (p

Ap

F)

minus100

minus50

0

50

100

150


10ms1nA

(c)

0

50

100

150

200

Capa

cita

nce (

pF)



mDPSCs

P0 mouseneurons

(d)



4 Discussion




Differentiation d10

Hig

h ne

stin

clone

Low

nes

tin cl

one

(a)

Hig

h ne

stin

clone

Low

nes

tin cl

one


(b)










5 Conclusions


Competing Interests


Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





2 125mM CaCl



2

1 mM CaCl2 2mM Na

2ATP 1mM Na

2GTP 11mM HEPES




3 Results




0 50 100 150 200 250

Days in culture

Clone 1Clone 2

Clone 3Clone 4

0

10

20

30

40

50

Num

ber o

f pop

ulat

ion

doub

lings

(a)

Clone 1

Nes

tinM

usas

hi


(b)

Clone 1

Clone 2

Clone 3

Clone 4

RT-ve

mNSC

GA

PDH

NF-

l

GLA

ST

BLBP

P75

My1

tl

Pax6

CD133

Sox2

SCA1

CD90

Radi

al g

lia

Hou

se-

keep

ing

Mat

ure

neur

ons

Neu

ral

cres

t

Neu

ral

deve

lopm

ent

Neu

ral

stem

cells

Mes

ench

ymal

stem

cells

(c)


lowastlowast

ns ns

0

2

4

6

8

10

Relat

ive n

estin

mRN

A ex

pres

sion

( o

f GA

PDH

)

(d)






PredifferentiationH

igh

nesti

n clo

neLo

w n

estin

clon


(a)

NF-l Map2

Hig

h ne

stin

clone

Low

nes

tin cl

one

(b)


lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

Day 5Day 10

Day 15

001

01

1

10

Fold

chan

ge in

mRN

A ex

pres

sion

leve

ls

(c)





1

2

0

2

4

6

8

10

Curr

ent d

ensit

y (p

Ap

F)


Voltage (mV)

10ms100pA

(a)

1

2

0

2

4

6

8

10

Curr

ent d

ensit

y (p

Ap

F)


Voltage (mV)

10ms20pA

(b)

Curr

ent d

ensit

y (p

Ap

F)

minus100

minus50

0

50

100

150


10ms1nA

(c)

0

50

100

150

200

Capa

cita

nce (

pF)



mDPSCs

P0 mouseneurons

(d)



4 Discussion




Differentiation d10

Hig

h ne

stin

clone

Low

nes

tin cl

one

(a)

Hig

h ne

stin

clone

Low

nes

tin cl

one


(b)










5 Conclusions


Competing Interests


Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 50 100 150 200 250

Days in culture

Clone 1Clone 2

Clone 3Clone 4

0

10

20

30

40

50

Num

ber o

f pop

ulat

ion

doub

lings

(a)

Clone 1

Nes

tinM

usas

hi


(b)

Clone 1

Clone 2

Clone 3

Clone 4

RT-ve

mNSC

GA

PDH

NF-

l

GLA

ST

BLBP

P75

My1

tl

Pax6

CD133

Sox2

SCA1

CD90

Radi

al g

lia

Hou

se-

keep

ing

Mat

ure

neur

ons

Neu

ral

cres

t

Neu

ral

deve

lopm

ent

Neu

ral

stem

cells

Mes

ench

ymal

stem

cells

(c)


lowastlowast

ns ns

0

2

4

6

8

10

Relat

ive n

estin

mRN

A ex

pres

sion

( o

f GA

PDH

)

(d)






PredifferentiationH

igh

nesti

n clo

neLo

w n

estin

clon


(a)

NF-l Map2

Hig

h ne

stin

clone

Low

nes

tin cl

one

(b)


lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

Day 5Day 10

Day 15

001

01

1

10

Fold

chan

ge in

mRN

A ex

pres

sion

leve

ls

(c)





1

2

0

2

4

6

8

10

Curr

ent d

ensit

y (p

Ap

F)


Voltage (mV)

10ms100pA

(a)

1

2

0

2

4

6

8

10

Curr

ent d

ensit

y (p

Ap

F)


Voltage (mV)

10ms20pA

(b)

Curr

ent d

ensit

y (p

Ap

F)

minus100

minus50

0

50

100

150


10ms1nA

(c)

0

50

100

150

200

Capa

cita

nce (

pF)



mDPSCs

P0 mouseneurons

(d)



4 Discussion




Differentiation d10

Hig

h ne

stin

clone

Low

nes

tin cl

one

(a)

Hig

h ne

stin

clone

Low

nes

tin cl

one


(b)










5 Conclusions


Competing Interests


Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


PredifferentiationH

igh

nesti

n clo

neLo

w n

estin

clon


(a)

NF-l Map2

Hig

h ne

stin

clone

Low

nes

tin cl

one

(b)


lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

Day 5Day 10

Day 15

001

01

1

10

Fold

chan

ge in

mRN

A ex

pres

sion

leve

ls

(c)





1

2

0

2

4

6

8

10

Curr

ent d

ensit

y (p

Ap

F)


Voltage (mV)

10ms100pA

(a)

1

2

0

2

4

6

8

10

Curr

ent d

ensit

y (p

Ap

F)


Voltage (mV)

10ms20pA

(b)

Curr

ent d

ensit

y (p

Ap

F)

minus100

minus50

0

50

100

150


10ms1nA

(c)

0

50

100

150

200

Capa

cita

nce (

pF)



mDPSCs

P0 mouseneurons

(d)



4 Discussion




Differentiation d10

Hig

h ne

stin

clone

Low

nes

tin cl

one

(a)

Hig

h ne

stin

clone

Low

nes

tin cl

one


(b)










5 Conclusions


Competing Interests


Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1

2

0

2

4

6

8

10

Curr

ent d

ensit

y (p

Ap

F)


Voltage (mV)

10ms100pA

(a)

1

2

0

2

4

6

8

10

Curr

ent d

ensit

y (p

Ap

F)


Voltage (mV)

10ms20pA

(b)

Curr

ent d

ensit

y (p

Ap

F)

minus100

minus50

0

50

100

150


10ms1nA

(c)

0

50

100

150

200

Capa

cita

nce (

pF)



mDPSCs

P0 mouseneurons

(d)



4 Discussion




Differentiation d10

Hig

h ne

stin

clone

Low

nes

tin cl

one

(a)

Hig

h ne

stin

clone

Low

nes

tin cl

one


(b)










5 Conclusions


Competing Interests


Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Differentiation d10

Hig

h ne

stin

clone

Low

nes

tin cl

one

(a)

Hig

h ne

stin

clone

Low

nes

tin cl

one


(b)










5 Conclusions


Competing Interests


Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









5 Conclusions


Competing Interests


Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Clonal Heterogeneity in the Neuronal and...

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Transcript of Research Article Clonal Heterogeneity in the Neuronal and...