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Isolation of human mesenchymal stem cells from the skin and their neurogenic differentiation in vitro

Jun-Ho Byun1, Eun-Ju Kang2, Seong-Cheol Park1, Dong-Ho Kang3,

Mun-Jeong Choi1, Gyu-Jin Rho2, Bong-Wook Park1

1Department of Oral and Maxillofacial Surgery, School of Medicine and Institute of Health Science, Gyeongsang National University, 2OBS/Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University,

3Department of Neurosurgery, School of Medicine, Gyeongsang National University, Jinju, Korea

Abstract (J Korean Assoc Oral Maxillofac Surg 2012;38:343-53)

Objectives: This aim of this study was to effectively isolate mesenchymal stem cells (hSMSCs) from human submandibular skin tissues (termed hSMSCs) and evaluate their characteristics. These hSMSCs were then chemically induced to the neuronal lineage and analyzed for their neurogenic characteristics in vitro. Materials and Methods: Submandibular skin tissues were harvested from four adult patients and cultured in stem cell media. Isolated hSMSCs were evaluated for their multipotency and other stem cell characteristics. These cells were differentiated into neuronal cells with a chemical induction protocol. During the neuronal induction of hSMSCs, morphological changes and the expression of neuron-specific proteins (by fluorescence-activated cell sorting [FACS]) were evaluated. Results: The hSMSCs showed plate-adherence, fibroblast-like growth, expression of the stem-cell transcription factors Oct 4 and Nanog, and positive staining for mesenchymal stem cell (MSC) marker proteins (CD29, CD44, CD90, CD105, and vimentin) and a neural precursor marker (nestin). Moreover, the hSMSCs in this study were successfully differentiated into multiple mesenchymal lineages, including osteocytes, adipocytes, and chondrocytes. Neuron-like cell morphology and various neural markers were highly visible six hours after the neuronal induction of hSMSCs, but their neuron-like characteristics disappeared over time (24-48 hrs). Interestingly, when the chemical induction medium was changed to Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS), the differentiated cells returned to their hSMSC morphology, and their cell number increased. These results indicate that chemically induced neuron-like cells should not be considered true nerve cells.Conclusion: Isolated hSMSCs have MSC characteristics and express a neural precursor marker, suggesting that human skin is a source of stem cells. However, the in vitro chemical neuronal induction of hSMSC does not produce long-lasting nerve cells and more studies are required before their use in nerve-tissue transplants.

Key words: Skin, Mesenchymal stem cell, In vitro neuronal differentiation[paper submitted 2012. 8. 10 / revised 2012. 11. 15 / accepted 2012. 11. 22]

neuralstemcellshavebeentransplantedintonervedefect

sites to improveperipheralandcentralnervefunctions1-3.

Amongthem,mesenchymalstemcells(MSCs)havebeen

thefocusinimprovingnerveregenerationbecauseoftheir

capabilitytoprovidemulti-lineagedifferentiationsandself-

renewalpotential4.Bonemarrow-derivedMSCs(BMSCs)

cantrans-differentiateinvitrointoSchwanncell-likecells,whichproduceremarkableinvivonerveregenerationwhentransplantedintoaperipheralnervedefect4-7.Note,however,

thatbonemarrowaspirationsometimesrequires invasive

procedures,possibly inducinga rangeofcomplications

inpatientssuchaspain,hemorrhage,andfear.Therefore,

moreaccessibletissuessuchasskinorfatareinvestigated

I. Introduction

Recently,manyresearchershavetriedtoregeneratenerve

tissuewith tissueengineering techniques.Multipotentor

pluripotentstemcells,culturedSchwanncells,andisolated

Bong-Wook ParkDepartment of Oral and Maxillofacial Surgery, School of Medicine and Institute of Health Science, Gyeongsang National University, 79, Gangnam-ro, Jinju 660-702, KoreaTEL: +82-55-750-8263 FAX: +82-55-761-7024E-mail: parkbw@gnu.ac.kr

*ThisresearchwassupportedbytheBasicResearchProgramthroughtheNationalResearchFoundationofKorea(NRF)fundedbytheMinistryofEducation,Science,andTechnology(2012-0472).

This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

CC

ORIGINAL ARTICLEhttp://dx.doi.org/10.5125/jkaoms.2012.38.6.343

pISSN 2234-7550·eISSN 2234-5930

J Korean Assoc Oral Maxillofac Surg 2012;38:343-53

344

toevaluate theefficiencyof theirneuraldifferentiation,

hSMSCsweredifferentiated intoneuralcellsunder the

chemicallyneural inductionprotocolofMSCs16,17, and

variousneurogenicandangiogenicproteinswereevaluated

byimmunocytochemistry(ICC).

II. Materials and Methods

1. Isolation and culture of hSMSCs

Human facial skin sampleswereobtained from four

patients(2malesand2females;24-45yearsold,averageof

33.3years)whohadundergoneheadandnecksurgeryvia

thesubmandibularapproach.(Fig.1.A)Allexperimentswere

authorizedbytheGyeongsangNationalUniversityHospital

EthicsCommittee,and thepatientsgave their informed

consenttotissuedonation.Freshhumanskinsampleswere

transportedtothelaboratory,andhSMSCswereisolatedas

previousprotocol15.Briefly,allhairsandsubcutaneousfat

tissueswereremoved,andthesampleswerethencut into

1-3mm2explantscontainingtheepidermisanddermis.Skin

explantswereattachedto thecultureplates,and2mLof

Dulbecco’sModifiedEagleMedium(DMEM)/F12(1:1)

(Invitrogen,Carlsbad,CA,USA)supplementedwith10%

fetalbovineserum(FBS;Invitrogen),10ng/mLepidermal

as alternative sourcesof adult stemcells for the tissue

engineeringtechniquenowadays.

Recently,skinhasbeenconsideredapotentialadultstem

cellsource.It ishighlyaccessible,andenoughautologous

tissuecouldbeeasilyobtainedwithminimaldonor site

complications.Moreover,skinisanabundantpluripotent,

multipotentcellsourcewithimmuneprivilegeandpotential

forself-replication8-10.Severalresearchershavedemonstrated

thatthereareseveraldifferenttypesofstemcells-suchas

skin-derivedprecursors(SKPs),skin-derivedmesenchymal

stemcells(SMSCs),andepidermalstemcells--inthedermis

andepidermisofskins10-14.Inthepreviousstudy,weisolated

porcineskin-derivedcellsfromtheearskinofminiaturepigs

andshowed themultipotencyandMSCcharacteristics15.

Thecellswere isolatedfromtheepidermisanddermis in

serum-containingmedium,andtheyproliferatedadherently

onthecultureplateandexpressedMSC-markerproteins.In

thisstudy,humanSMSCs(hSMSCs)fromsubmandibular

skinwereisolatedandcultured,andinvitrodifferentiationintomesenchymalcells suchasosteocytes, adipocytes,

andchondrocyteswasevaluatedunderspecific induction

media.Inaddition,isolatedhSMSCswerecharacterizedby

evaluating theexpressionofvariouscellsurfacemarkers

(CD29,CD44,CD90,CD105,vimentin,andnestin)and

transcriptionfactors (Oct4,Nanog,andSox2).Finally,

Fig. 1. Isolation and primary culture of human skin-derived cells with serum-containing adherent cell culture method (scale bar=100 μm). A. Harvested submandibular skin tissue. B-D. Culturing human skin-derived cells (hSDCs) on the 3rd (B), 7th (C), and 14th (D) day can be observed during primary culture (P0). B. Irregular and heterogeneous hSDCs isolated from a skin fragment (black shadow) in the primary culture plates. C. After 7 days of P0, proliferating irregularly shaped hSDCs were detected in the plates. D. After about 2 weeks of P0, plate-adherent, fibroblast-like homogeneous cells were detected in the culture plates. Jun-Ho Byun et al: Isolation of human mesenchymal stem cells from the skin and their neurogenic differentiation in vitro. J Korean Assoc Oral Maxillofac Surg 2012

Isolation of human mesenchymal stem cells from the skin and their neurogenic differentiation in vitro

345

anti-humanCD29[1:100,BDPharmingen],mouseanti-

humanvimentin[1:100,Sigma-Aldrich],andmouseanti-

humannestin[1:100,BDPharmingen])for45minat37oC,

followedbylabelingwiththeFITC-conjugatedsecondary

goatanti-mouseantibody(1:100,BDPharmingen)foran

hour.

3. In vitro neuronal differentiation

NeuronaldifferentiationofhSMSCswasperformedusing

amodifiedchemicalneuralinductionprotocolforBMSCs

differentiation16,17.Briefly,whenhSMSCsatpassage3reached

70%confluence, thecellswere transferred toaneuronal

preinductionmediumcontainingDMEM (Invitrogen)

with20%FBS(Invitrogen)and10ng/mLbFGF(Sigma-

Aldrich) for 24 hr. The cellswerewashedwith PBS

and cultured inneuronal inductionmediumconsisting

ofDMEM supplementedwith 2%dimethylsulfoxide

(DMSO),200μMbutylatedhydroxyanisole (BHA),25

mMKCl,2mMValproicacid,10μMForskolin,1μM

Hydrocortisol,5μg/mLInsulin,and2mML-glutamine

withoutFBS forup to48hr.The cellswere fixed for

ICC at 0 hr, 6 hr, 24 hr, and 48 hr of induction.All

supplementedchemicals in theneural inductionmedium

weremanufacturedbySigma-AldrichCompany.

Tocomparethemorphologicalchangesoftheneuronally

differentiatedcells, thechemically inductivemediumof

thecontrol cellswaschanged toDMEMsupplemented

with20%FBSafter24hrofchemicalinduction,andtheir

morphologicalchangeswereobservedafteranadditional24

hr.

growthfactor(EGF;Sigma-Aldrich,St.Louis,MO,USA),

10ng/mLbasic fibroblastgrowth factor (bFGF;Sigma-

Aldrich),100U/mLpenicillin(Sigma-Aldrich),and100μg/

mLstreptomycin(Sigma-Aldrich)wereadded.Theculture

plateswereincubatedat37oCinahumidifiedatmosphere

containing5%CO2inairfor3or5days.Afterremovingthe

remainingskinfragments,theattachedcellswereexpanded

invitro,with theculturemediumchanged twiceaweek.Onceconfluent,thecellsweredissociatedusing0.25%(w/v)

trypsin-ethylenediaminetetraaceticacid(EDTA;Invitrogen)

solutionandpelletedat500×gfor5min.Thecellswere

thenre-grownandincubateduntilpassage3.

2. Cell surface and intracellular markers analysis

Thecell surfaceand intracelluarmarkersofhSMSCs

atpassage3wereanalyzedusinga flowcytometer (BD

FACSCalibur;BectonDickinsonandCompany,Franklin

Lakes,NJ,USA) in triplicate.Briefly,cells that reached

90%confluencewereharvestedusing0.25%EDTAand

washed twice inDulbecco’sphosphatebuffered saline

(DPBS; Invitrogen) supplementedwith10%FBS.The

cellsfordetectingCD44,CD90,andCD105werelabeled

directlywithfluoresceinisothiocyanate(FITC)-conjugated

CDmarkers(ratanti-mouseCD44[1:100,BDPharmingen;

BDBiosciences,FranklinLake,NJ,USA],mouseanti-

humanCD90 [1 :100,BDPharmingen],andgoatanti-

mouseCD105[1:100,BDPharmingen]).Thecellswere

fixedin3.7%formaldehydeforanhourtoanalyzethelevels

ofCD29,vimentin,andnestin.AfterwashingwithDPBS,

thesampleswerelabeledwithprimaryantibodies(mouse

Table 1. Primary antibodies for the immunocytochemical study of chemically neural induced cells

Antibody Type Company Catalognumber Dilution

NestinS-100NFNGFp75NGFRtrkAVEGFVEGFR1VEGFR2β-tubulinMBPNeuN

MousemonoclonalRabbitpolyclonalGoatpolyclonalRabbitpolyclonalMousemonoclonalGoatpolyclonalRabbitpolyclonalMousemonoclonalRabbitpolyclonalGoatpolyclonalGoatpolyclonalMousepolyclonal

Sigma-AldrichCo.,St.Louis,MO,USAThermoScientific,Rockford,IL,USASantaCruz,SantaCruz,CA,USASantaCruzSantaCruzSantaCruzSantaCruzAbcam,Cambridge,UKAbcamSantaCruzSantaCruzChemicon,Temecula,CA,USA

61658RB9018sc-16143sc-548sc-13577sc-20537sc-152ab9540ab71772sc-9935sc-13914MAB377

1:2001:3001:1001:1001:1001:1001:1001:1001:1001:1001:1001:100

(NF:neurofilament,NGF:nervegrowthfactor,trkA:tyrosinekinasereceptorA,VEGF:endothelialcellgrowthfactor,MBP:myelinbasicprotein,NeuN:neural-specificnuclearprotein)Jun-Ho Byun et al: Isolation of human mesenchymal stem cells from the skin and their neurogenic differentiation in vitro. J Korean Assoc Oral Maxillofac Surg 2012

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5. Reverse transcription-polymerase chain reaction

(RT-PCR) analysis

ThehSMSCsatpassage3wereevaluatedbyRT-PCR

fortheexpressionoftranscriptionfactorsOct4,Sox2,and

Nanog.Table2lists theRT-PCRprimersfor themarkers

usedinthisstudy.ThetotalRNAwasextractedfromthe

culturedcellsusinganRNeasyMiniKit(Qiagen,Valencia,

CA,USA).cDNAsynthesiswasperformedfor30minat

55oCusinganOmniscriptReverseTranscriptionKit(Qiagen)

witholigo-dTprimers.ThecDNAsproducedwereusedas

templateforPCRamplification.PCRwasperformedusing

MaximePCRPremix(iNtRONBiotechnology,Seongnam,

Korea)underthefollowingconditions:pre-denaturationat

94oCfor3min, followedby34cyclesofdenaturationat

94oCfor45s,annealingat60oCor58oCfor30s,elongation

at72oCfor45s,andfinalextensionat72oCfor10minusing

aThermocycler(PTC-200;GMI,Anoka,MN,USA).

6. Statistical analysis

Allvaluesof thecountedandcalculatedcellnumbers

and intensitiesof immunostainingsof theinvitroneuraldifferentiatedcellswerestatisticallyanalyzedbytheManova

test,and independentgroupingvariableswerecompared

usingBonferroniandSPSSsoftwareversion18.0(SPSS

Inc.,Chicago,IL,USA).Datawereexpressedasmean±SD.

DifferenceswereconsideredtobesignificantwhenP<0.05.

4. Immunocytochemical analysis of hSMSCs and in

vitro neural induced cells

WhenthehSMSCsreachedpassage3, theywererinsed

withPBSandfixedin4%neutralbufferedformaldehydefor

30minatroomtemperature.ICCforthetranscriptionfactors

(Oct4,Nanog,andSox2)wasconducted.A1:200dilution

ofprimarygoatpolyclonalanti-humanOct3/4(sc-8628;

SantaCruz,SantaCruz,CA,USA),a1 :200dilutionof

primarygoatpolyclonalanti-humanNanog(sc-30331,Santa

Cruz),anda1:200dilutionofprimaryrabbitpolyclonal

anti-humanSox2(sc-20088,SantaCruz)wereusedtodetect

theexpressionoftranscriptionfactors.

In theneural inductionmedium, thedifferentiatedcells

werefixedat0hr(immediatelyafterpreinduction),6hr,24

hr,and48hrafterneuralinduction.Table1liststheprimary

antibodiesusedfortheevaluationofneuraldifferentiation.

Dilutions(1 :100)ofFITC-conjugateddonkeyanti-goat

polyclonalIgG(JacksonImmunoResearchLaboratoriesInc.,

WestGrove,PA,USA),FITC-conjugateddonkeyanti-rabbit

polyclonal IgG(711-095-152,JacksonImmunoResearch

LaboratoriesInc.),andFITC-conjugatedgoatanti-mouse

polyclonal IgG(115-096-003,JacksonImmunoResearch

LaboratoriesInc.)wereusedassecondaryantibodies.

Densitometricanalysesofeach immunostainingwere

performedusing analySISTS software (OlympusSoft

ImagingSolution,Münster,Germany).For theevaluation

ofoneantibody’sexpression, at least three slideswere

immunostainedandstatisticallyanalyzedateachtimepoint.

Table 2. RT-PCR primers used for evaluating transcription factors and osteogenic and adipogenic differentiations

Gene Sequenceofprimer(5’–3’) Amplificationsize(bp) Temperature(oC) Locous

GAPDH

Oct4

Nanog

Sox2

Osteonectin

Osteocalcin

PPARγ2

Ap2

F-GAGTCAACGGATTTGGTCGTR-TTGATTTTGGAGGGATCTCGF-GATCCTCGGACCTGGCTAAGR-GACTCCTGCTTCACCCTCAGF-CAAAGGCAAACAACCCACTTR-TCTGGAACCAGGTCTTCACCF-TCACGTACACTGCCCTGAAGR-TGCAACGGATTGTGTTGTTTF-GTGCAGAGGAAACCGAAGAGR-AAGTGGCAGGAAGAGTCGAAF-GGCAGCGAGGTAGTGAAGAGR-CTGGAGAGGAGCAGAACTGGF-ACTGCGCTACAAATGCACACR-TTGATGCCGAGAAAGGAGATF-TACTGGGCCAGGAATTTGACR-ATGCGAACTTCAGTCCAGGT

238

213

218

175

202

230

248

237

60

60

60

60

60

60

60

58

AB062273

AM851115

AB093576

Z31560

NM_003118

X53698

AJ563369

NM_001442

(RT-PCR:reversetranscription-polymerasechainreaction,GAPDH:glyceraldehyde3-phosphatedehydrogenase)Jun-Ho Byun et al: Isolation of human mesenchymal stem cells from the skin and their neurogenic differentiation in vitro. J Korean Assoc Oral Maxillofac Surg 2012

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Sox2washardlyobservedbyICCorRT-PCR.(Figs.2.A,2.

B)ThehSMSCsatpassage3werepositivefortypicalMSC

markers(CD29,CD44,CD90,CD105,andvimentin)based

onFACSanalysis.Inaddition,theneuralprecursormarker,

nestin,wasdetectedinthehSDCs.(Fig.3)Theresultsabove

demonstratethathumanskin-derivedcellsinthisstudywere

multipotentMSCsexhibitingthecharacteristicsofaneural

precursor.Moreover,theculturedhSMSCsinthisstudywere

successfullydifferentiatedintomesenchymallineagecells,

osteocytes,adipocytes,andchondrocytesinspecificinduction

media.(Fig.4.A)ThesedifferentiatedcellsfromhSMSCsalso

showedspecificosteogenicandadipogenicmarkerproteinsby

RT-PCR.(Fig.4.B)

3. In vitro neural induction of hSMSCs

SpecificmorphologicalchangefromhSMSCswasnot

observedafterneuralpreinduction.(Fig.5.A)WhenhSMSCs

wereneuronallydifferentiatedusingvarious chemical

compoundsunderFBS-deprivedconditions,moststrongly

resembledneuronsandincludedtheretractionofcellbody

andprocesselaboration,andtheywereobservedafter6hrof

induction.(Fig.5.B)Astheneuronalinductiontimereached

24hrs and48hrs, however, thedifferentiatedneuron-

likecellsdecreasedinnumber,andtheircellmorphology

III. Results

1. Cell isolation and culture

Both floatedsphere-formingcellsandplate-adherent,

fibroblast-likecellswereco-detectedintheculturemedium

fromthefirstdayofprimaryculture.After3daysofprimary

culture,mostcellshadshownplate-adherentgrowthinthe

gelatin-coatedplates. Initially, theattachedskin-derived

cellsshowedheterogeneouslyirregularshapes,andpartial

colony formationswereobserved.Note, however, that

homogeneouslyshapedandplate-adherent fibroblast-like

cellsweremainlydetectedattheend-about2weekslater-

oftheprimaryculture.(Figs.1.B-D)Thesehomogeneously

shaped,plate-adherentfibroblast-likecellswereallowedto

proliferate,reachingpassage2or3.

2. Expression of transcription factors determined by

RT-PCR and cell surface markers measured by

FACS analysis

Afterhumanskin-derivedcellswereculturedtopassage3,

theexpressionoftranscriptionfactorssuchasOct4,Nanog,

andSox2wasevaluatedbyICCandRT-PCR.Oct4and

NanogwerehighlyvisibleinculturedadulthSMSCs,whereas

Fig. 2. Expression of early transcription factors Oct 4, Nanog, and Sox 2 by immunocytochemistry (A: scale bar=100 μm) and reverse transcription-polymerase chain reaction (B) in human skin-derived cells (hSDCs) at passage 3. Positive expression of Oct 4 and Nanog, even though Sox 2 was hardly expressed, indicates that the hSDCs in this study are multipotential primitive cells. (GAPDH: glyceraldehyde 3-phosphate dehydrogenase)Jun-Ho Byun et al: Isolation of human mesenchymal stem cells from the skin and their neurogenic differentiation in vitro. J Korean Assoc Oral Maxillofac Surg 2012

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Fig. 3. Fluorescence-activated cell sorting analysis of cultured human skin-derived cells. Skin-derived cells at passage 3 were positive for specific mesenchymal stem cell markers (CD29, CD44, CD90, CD105, and vimentin) and neural precursor cell marker (nestin). Open histograms represent staining with negative control, with the black histograms depicting the fluorescence intensity of each of the cell surface antibodies. Jun-Ho Byun et al: Isolation of human mesenchymal stem cells from the skin and their neurogenic differentiation in vitro. J Korean Assoc Oral Maxillofac Surg 2012

Fig. 4. Mesenchymal-lineage differentiations of human skin-derived mesenchymal stem cells (hSMSCs) into ostocytes (a, b), adiopcytes (c), and chondrocytes (d) for 4 weeks (A: scale bar=100 μm). A. In vitro differentiated cells showed positive staining in the specific staining methods. (a, b) Calcium deposits were observed on the cell surface by von Kossa (a) and Alizalin red (b) staining. (c) Lipid droplets were noted in the cytoplasm of cells by Oil red O staining. (d) Proteoglycans were confirmed on the cell surface using Alcian blue. B. Reverse transcription-polymerase chain reaction results for in vitro differentiated osteocytes and adipocytes from hSMSCs. (a) ON and OC were detected in osteogenic differentiated cells. (b) PPARγ2 and aP2 were expressed in adipogenic differentiated cells. (OC: osteocalcin, ON: osteonectin, GAPDH: glyceraldehyde 3-phosphate dehydrogenase, aP2: adipocyte protein 2)Jun-Ho Byun et al: Isolation of human mesenchymal stem cells from the skin and their neurogenic differentiation in vitro. J Korean Assoc Oral Maxillofac Surg 2012

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Fig. 5. The upper graph illustrates the schematic in vitro neural induction protocol used in this study. Cultured hSMSCs at passage 3 were preinduced for 24 hrs. The experimental group was neurally induced by a chemical protocol for 48 hr. In the control group, 24 hr after neural induction, the inductive medium was changed to DMEM supplemented with 20% FBS, and morphologic changes were then observed after an additional 24 hrs of media change. A-E. The microphotographs show the morphologic changes of hSMSCs after chemical neural induction (scale bar=100 μm). A. Immediately after neuronal preinduction (0 hr). There are no remarkable morphological changes compared to the original hSMSCs. B. Six hours (6 hr) after neural induction, the neuron-like cells exhibit peak activity. C, D. After the passage of neural induction time (24 and 48 hrs post-neural induction), the neuron-like cells decreased in number, and their shape deteriorated. E. In the control cells, neural differentiated cells returned to the original hSMSC morphology, and cell number increased 24 hrs after media change as DMEM with 20% FBS. F. The number of cells decreased with the passage of neural induction time, but the number increased after the inductive medium was changed. (hSMSCs: human skin-derived mesenchymal stem cells, bFGF: basic fibroblast growth factor, FBS: fetal bovine serum, DMEM: Dulbecco’s Modified Eagle Medium, DMSO: dimethylsulfoxide, VPA: valporic acid, BHA: butylated hydroxyanisole)Jun-Ho Byun et al: Isolation of human mesenchymal stem cells from the skin and their neurogenic differentiation in vitro. J Korean Assoc Oral Maxillofac Surg 2012

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oftheseproteinswasthenobservedafter24hrand48hrof

neurogenicinduction.(Fig.6)

IV. Discussion

Amongtheskin-derivedstemcells,SKPs-believedtobe

endogenousembryonicneuralcrest-derivedprecursorcells

thatpersistintoadulthood-havebeenstudiedmostwidely.

SKPsoriginatedwith thedermisof theskinandformed

floating spheres in the serum-freeculturingconditions

supplementedwithvariousgrowth factors - including

FGF-2,EGF, andB27 - after enzymaticdigestion and

celldissociationof theskin.Moreover, theyhavedistinct

characteristicsfromMSCs,andtheirfullpotentialisnormally

restrictedbythelocalenvironment,butsuchcanberevealed

undercultureconditions18-21.TheseresultssuggestthatSKPs

havemanycharacteristicsthatwouldbebeneficialinnervous

systemregeneration22,23. In thepreviousstudy,however,

deteriorated (Figs. 5.C,5.D) In the control cells, the

inductionmediumwaschangedtoDMEMwith20%FBS

after24hrsofneuralinduction,andtheneuraldifferentiated

cellsreturnedtotheoriginalshapeofhSMSCs.(Fig.5.E)In

addition,thenumberofcellsincreased24hrsaftermedium

change.(Fig.5.F)

Intheimmunocytochemicalstudiesofdifferentiatedneuron-

likecells,neuron-andangiogenesis-relatedproteinswere

highlyexpressed,peaking6hrsafterneuralinduction.Note,

however,thattheexpressionintensityofallproteinsdecreased

asinductiontimereached24hrsand48hrs.Interestingly,the

neuralprecursormarker,nestin,wassubstantiallyexpressed

in thepre-inducedcells (0hr). Inaddition, theenhanced

co-expressionof thenervegrowth factor (NGF)and its

tworeceptors(p75NGFRandtrkA)aswellasthevascular

endothelialgrowth factor (VEGF)and its two receptors

(VEGFR1andVEGFR2)wasdetectedat theearlystages

ofneurogenicinduction(6hrs).Thedecreasedexpression

Fig. 6. A. Immunocytochemica l studies for various neuronal and angiogenic marker proteins after the in vitro chemical neural induction of hSMSCs (scale bar=100 μm). Most marker proteins were highly visible 6 hrs after neural induction. Nestin was expressed in the 0 hr specimen (before nerve induction), which is similar to the result of FACS analysis. NGF and VEGF were highly visible with their receptors (p75NGFR, trkA, VEGFR1, and VEGFR2) during neuronal diffe-rentiation. B. Immunocytochemical intensities for specific proteins. The expression of most proteins, except NeuN, peaked 6 hrs after neural induction, and then decreased over time (24 hrs and 48 hrs after induction). Data represent the mean±SE of four independent experiments. A star (*) indicates a significant difference from the control (P<0.05). (S-100: S-100 protein, NF: neurofi lament, MBP: myelin basic protein, NeuN: neural-specific nuclear protein, NGF: nerve growth factor, p75NGFR: p75 nerve growth factor receptor, trkA: tyrosine kinase receptor A, VEGF: vascular endothelial cell growth factor, VEGFR: vascular endothelial cell growth factor receptor)Jun-Ho Byun et al: Isolation of human mesenchymal stem cells from the skin and their neurogenic differ-entiation in vitro. J Korean Assoc Oral Maxillofac Surg 2012

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producestoxicandstressfulcultureconditions.Therefore,

theseconditionscontributedtotherapiddisruptionoftheactin

cytoskeletonandshapechangetoaformresemblingnerve

cells34,35.Thesenerve-likecellswerenottrulydifferentiated

nervecells,andtheydidnotexhibitelectricalconductions.

Moreover, thechemical induction led tocell apoptosis

anddeath33,36.Inthisstudy,weobservedrapidneuron-like

morphologicalchangesofhSMSCsafterchemicalinduction.

After6hrs of induction, thehighest neuron-like cells

morphologyandpeakexpressionofneuralandangiogenic-

relatedproteinsweredetected.Withthepassageofinduction

time,however, themorphologyofneural inductedcells

deteriorated,andthecellnumberandexpressionofnerve-

relatedproteinsdecreased.Interestingly,whentheinduction

mediumwas changed toDMEMwithFBS, theneural

differentiatedcellsreturnedtotheiroriginalshape,andthe

cellnumberalsoincreased.Theseresults indicatethat the

chemicallyinducedneuron-likecellsofthisstudywerenot

regardedastruenervecells.Whenstemcellsdifferentiate

intotargetcells,itisdifficultforthemtoreturntostemcells

especiallyinashorttimeperiod37.Therefore, thechemical

inductionprotocolwhereintheinductionmediacontained

BME,DMSO,andBHAwasnotasuitablechoiceforthe

neuraldifferentiationmethodofhSMSCs,even though

neuron-likemorphologyandvariousnerve-relatedproteins

wereobservedshortlyafterinduction.

Inpreviousreports,SKPs,consideredoneof theneural

crestoriginated-stemcells,weredifferentiated intonerve

cellsandmaintainedbysupplementationwithvariousgrowth

factorsandneurotrophinssuchasB27,NGF,brain-derived

neurotrophicfactor(BDNF),neuro-3(NT-3),andFBS8,18,19.

Thisissimilartopreviouslyreportedneuraldifferentiation

methodsofBMSCsinvolvingvariousgrowthfactorsand

neurotrophins25.Although further studiesareneeded to

determine themostoptimalmethod forinvitro neuraldifferentiationfromMSCs,theuseofthesegrowthfactors

andneurotrophinscanbeconsideredasubstitutemethodfor

theinvitroneuralinductionofSMSCs.

Severalresearchershavedemonstratedtheenhancednerve

regenerationaftertheinvivotransplantationofdifferentiatedSchwanncellsorneuronsfromBMSCs4,5,38.Nonetheless,

undifferentiatedMSCsarewell-knowntohavepotentialfor

differentiationintotargetcellsafterinvivotransplantation.Moreover, undifferentiatedBMSCs expressedvarious

neurotrophic factors afterin vivo transplantation intothenervedefect site4.Sinceprevious studieshavenot

suggestedanobviousinvitroneuraldifferentiationmethod

weisolatedandculturedadifferent typeofporcineskin-

derivedstemcellsundercultureconditiondifferentfromthat

ofSKPsaswellasdifferentserum-containingandadherent

cell culturingmethod and observed the distinctMSC

characteristicsintheseporcineskin-derivedcells15.Similarly,

inthisstudy,humanskin-derivedcellswereisolatedfrom

thesubmandibular skinsegments,andmultipotentMSC

characteristicsweredetected,i.e.,expressionoftranscription

factors(Oct4andNanog),detectionofMSCmarkers(CD29,

CD44,CD90,CD105,andvimentin),andpotentialofinvitroosteogenic,adipogenic,andchondrogenicdifferentiations.

Interestingly,unliketheporcineSMSCsofapreviousstudy15,

wecouldnotobserveSox2expressioninthehSMSCsofthis

study.Thisisconsistentwiththeresultofotherresearch,i.e.,

highvisibilityofOct4andNanogbutnegativeexpression

ofSox2 inhumanBMSCs24. Inaddition, theyobserved

stronglyexpressednestin,aneuralprecursormaker, inall

kindsofMSCsoriginatinginthebonemarrow,dermis,and

adiposetissues,similar tothisstudy.Actually, thisneural

precursormarkerwasusuallyseen inSKPs, regardedas

neuralcrest-originatedstemcell inskin19.Takentogether,

theisolatedandcultivatedhumanskin-derivedcellsinthis

studyareconsideredmultipotent,hSMSCsdemonstratingthe

characteristicsofaneuralprecursor.

TheinvitroneuraldifferentiationmethodsofBMSCshave

beeninvestigatedbyseveralresearchers.First,variousmolecules

involved inneuraldevelopment, suchasgrowthfactors,

neurotrophins,cytokines,andretinoicacid,wereused25,26.

Second,neuraldifferentiationwasobtainedbyincreasingthe

intracellularcyclicadenosinemonophosphate27.Third,specific

chemicalcompoundssuchasβ-mercaptoethanol(BME),

DMSO,andBHAwereused in theserum-freeculturing

mediumforneural induction16,17.Mostrecently, theneural

differentiationofBMSCswasalsoobservedwhencultured

in inflammatoryastrocyte-containingmedium28. In these

variousinvitroneuralinductionprotocols,chemicalprotocolshavebeenwidelyusedandstudiedbecauseitwasasimple

approachthatrapidlyyieldedneuron-likecelldifferentiation

fromBMSCs29-31.Morphologicalchanges intonerve-like

cellsandexpressionofneuron-specificmakershavebeen

observedwithinafewhoursofchemicalneuralinduction.In

addition,morethan70%ofdifferentiatedneuron-likecells

fromBMSCswereobservedafterchemicalneuralinduction,

whichwasextremelyhigherconcentrationcomparedwiththe

otherinductionmethod32,33.

Inrecentstudies,however,theuseofchemicalcompounds

inserum-freeconditionsfortheneuralinductionofBMSCs

J Korean Assoc Oral Maxillofac Surg 2012;38:343-53

352

masisonCA,HoppingSB,etal.Characterizationandisolationofstemcell-enrichedhumanhairfolliclebulgecells.JClinInvest2006;116:249-60.

14. RiekstinaU,MucenieceR,CakstinaI,MuiznieksI,AncansJ.Characterizationofhumanskin-derivedmesenchymalstemcellproliferationrateindifferentgrowthconditions.Cytotechnology2008;58:153-62.

15. KangEJ,ByunJH,ChoiYJ,MaengGH,LeeSL,KangDH,etal. Invitroandinvivoosteogenesisofporcineskin-derivedmesenchymalstemcell-likecellswithademineralizedboneandfibringluescaffold.TissueEngPartA2010;16:815-27.

16. WoodburyD,SchwarzEJ,ProckopDJ,BlackIB.Adultratandhumanbonemarrowstromalcellsdifferentiate intoneurons.JNeurosciRes2000;61:364-70.

17. WoodburyD,ReynoldsK,Black IB.Adult bonemarrowstromalstemcellsexpressgermline,ectodermal,endodermal,andmesodermalgenesprior toneurogenesis. JNeurosciRes2002;69:908-17.

18. FernandesKJ,Mckenzie IA,MillP,SmithKM,AkhavanM,Barnabé-heiderF,etal.Adermalnicheformultipotentadultskin-derivedprecursorcells.NatCellBiol2004;6:1082-93.

19. FernandesKJ,KobayashiNR,GallagherCJ,Barnabé-heiderF,AumontA,KaplanDR,etal.Analysisoftheneurogenicpotentialofmultipotentskin-derivedprecursors.ExpNeurol2006;201:32-48.

20. HuntDP,MorrisPN,SterlingJ,AndersonJA,JoannidesA,JahodaC,etal.Ahighlyenrichednicheofprecursorcellswithneuronalandglialpotentialwithinthehairfollicledermalpapillaofadultskin.StemCells2008;26:163-72.

21. ZhaoM,IsomSC,LinH,HaoY,ZhangY,ZhaoJ,etal.Tracingthestemnessofporcineskin-derivedprogenitors(pSKP)backtospecificmarkergeneexpression.CloningStemCells2009;11:111-22.

22. ShihDT,LeeDC,ChenSC,TsaiRY,HuangCT,TsaiCC,etal.Isolationandcharacterizationofneurogenicmesenchymalstemcellsinhumanscalptissue.StemCells2005;23:1012-20.

23. MarchesiC,PluderiM,ColleoniF,BelicchiM,MeregalliM,FariniA,etal.Skin-derivedstemcellstransplantedintoresorbableguidesprovidefunctionalnerveregenerationaftersciaticnerveresection.Glia2007;55:425-38.

24. RiekstinaU,CakstinaI,ParfejevsV,HoogduijnM,JankovskisG,MuiznieksI,etal.Embryonicstemcellmarkerexpressionpatterninhumanmesenchymalstemcellsderivedfrombonemarrow,adiposetissue,heartanddermis.StemCellRev2009;5:378-86.

25. Sanchez-ramosJ,SongS,Cardozo-pelaezF,HazziC,StedefordT,WillingA,etal.Adultbonemarrowstromalcellsdifferentiateintoneuralcellsinvitro.ExpNeurol2000;164:247-56.

26. KimBJ,SeoJH,BubienJK,OhYS.Differentiationofadultbonemarrowstemcellsintoneuroprogenitorcellsinvitro.Neuroreport2002;13:1185-8.

27. DengW,ObrockaM,FischerI,ProckopDJ.Invitrodifferentiationofhumanmarrowstromalcells intoearlyprogenitorsofneuralcellsbyconditionsthatincreaseintracellularcyclicAMP.BiochemBiophysResCommun2001;282:148-52.

28. WangFW,JiaDY,DuZH,FuJ,ZhaoSD,LiuSM,etal.Rolesofactivatedastrocytesinbonemarrowstromalcellproliferationanddifferentiation.Neuroscience2009;160:319-29.

29. BossolascoP,CovaL,CalzarossaC,RimoldiSG,BorsottiC,DeliliersGL,etal.Neuro-glialdifferentiationofhumanbonemarrowstemcellsinvitro.ExpNeurol2005;193:312-25.

30. LeiZ,YongdaL,JunM,YingyuS,ShaojuZ,XinwenZ,etal.Cultureandneuraldifferentiationofratbonemarrowmesenchymalstemcellsinvitro.CellBiolInt2007;31:916-23.

31. YamaguchiS,KurodaS,KobayashiH,ShichinoheH,YanoS,HidaK,etal.Theeffectsofneuronalinductionongeneexpressionprofileinbonemarrowstromalcells(BMSC)--apreliminarystudyusingmicroarrayanalysis.BrainRes2006;1087:15-27.

fromhSMSCs,undifferentiatedautologousSMSCscould

be suggested as reasonable transplantmaterial for the

regenerationofnervedefects.

V. Conclusion

IsolatedhSMSCsfromadultskinshowedMSCcharacte-

risticspossessingmultipotencyandneuralprecursormarker,

suggestingthathumanskinmaybeusedasavailableadult

stemcellsource.Nonetheless,whethertheinvitroneurogenicdifferentiatedcellsfromhSMSCsunderthechemicalneural

inductionprotocolproduceactualnervecellswasdoubtful,

even thoughneuron-like cellmorphology andvarious

nerve-relatedproteinsweredetected in thesechemically

inducedcells.Therefore,wesuggestthattheundifferentiated

autologoushSMSCs-insteadofchemicallyinducedneuron-

likecells-beconsideredforuseastransplantmaterialforinvivonerveregenerationinthefuture.

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