Id,enrificrutro-:n of .Adipo:g,enesls aD~ Osr""o-genE'sls Pru[!Ilways 'OifDiifiHlentillt",,~l BOine i!lital.·.I!1O-W StE'm.

Cells lfl 11liro in Rllhbit

A.I-liImmBm Hameed", ThFIIhim, iR_1, .Al-Jamfr3!llIl.)' Km:im.~, ZW::l, Ahz3~lld! .. A..zllirni, III ,R:a!ma~y, R4

p IkptIrt!wmto:fCJmir.ai~rudifi:;. '~llt oj';pmc.fil:1ial! :;~ F.1EufW oj'~I.;;.rint:lry jJk.did~Y UPM!:s-iimg , SilJ;it.M~j<itilf ~OD;[{.fi.r<i'1a ~

'Ikpmm;;mtq! J><i'l~<!!«f:YIFac,ufJ;y <IJ' jJk.mcm>2' mrd H~tIJM !ki'lllMw "'Co.m'~ptJ.I'ld'.;llt <i'JJ.riwr. li',am01lilt_<i'lmI:rnJ~\tilwo.aI.\IlI

T=s(!!jffi!-~nti~t:i.GlJJ.tS ~proce'SSJ;!,iherebya.oI!'J!lit!1l'2·ooll!!ll!!ittelilroaad!pro,~iimg,aJ.oJJg,a~pl!dfic d'er.'l!'loJ!l·m&ltaJ lliles:ge !mitdll!S into ,amo:the:ir ol!'ll t'!1l'2s. The ,diljl!di",~ Oliiful5. si1lld1'];\\1IS to' ,IISSl2SS 'whetbl!-rrnillifitmesenabiyIn!al stem Ol!'l!ls (iM:SCs) premlll!llliterl! to gilie me'I!-!1Ch~~ O;!ilI !iDl!<ilgelmrl5illiifefi!lJ~3!<"e in ~~poo!I'~ to' ind:iactfu.-e e1IDlmiUIlffilrClIl.es to ex-pamrl,a.d1J!ltM:SCs. BGne,l!le5emchjlm!al ~telll'[~UIS (BMSQ;:) 00ll!dlred! from lIi:UIll.lilf ,a.dldt mar~rnilli!·jt ,ro·ltlJ!lcrised! lJ.etero,ge!!leOll.S ig:rorlIJ!l6 (If oells ,aftl!oI' ~,g ami grcm-ing; m. ail!tme p1;'tl!!;~ }\fter ininaJ phllriJlg;, I!!e. >!iilltl!l<!nt ol!'ll!J ,l!1cll!iIbitedl!1ll!!3l~.rowtli1M, s!liDdle.-:!ihaped! 2Illd exhi:DLtedliJbrdblll!11:"Ui};e·mmpbdrog)' in ~rihl!l:g OOo&l!-llO~. l'lJi.iIiIR E:MSC:S dlliferel!lliiatell1. inte a.dlpocytes and oste.ocyte ,lIS a. ,amumt!lllifun of mtt,aoe,')1uW: ~lpjd imIJ!ll.ets and oaJ.cium ,Il1.epo.SLtio!ltJlrn,lJ!,~lthe ctiI~ lilil!oI 21..fu!Jys.

Mesenchymilil sl:em CeJ!~~, dBriMed hm. hol:l:e l!IiI.fill7O'IY'repriesenrl: is. seueee ofphlwirpmte:!IlJt rel]s (HOI'!.~dZ eJ: ,af .. , l002J, mamfe;;:t JmI~hl:iilllellge dtiifiliBrealIiillUOIil ,c;apil.cilty, indloo1i:ng mltoo~BIIe:ljjS clloodrngen.esis\ andadipogenesis (Znii: et (if., 20m). IJrns, MSQ; l'1l0']0IItecl. and ClIil:tured&o1ilJJ varieus iIi!ll:me;;'~FpeilF to be mOl]liroiogj.ciill'y sJimilaF and. fr:ni'e simillirur epl1opeproMes mid llie ~si.on ilfCOIm!lwillYlISM. eel surfaee marrJle!S (MlISiulIeJ; ,at, 2005, JIjI1 eJ: (it., 1m),1). Bene m3lrrm~ dlBN\iied me§eD.c:h:,)'ll!la]. :!l:telilJJ. ceb(M'SlCs) are 3Jttinlcnve CilUdti.cl.ate!l, tor Qe:lI~ba:s.ed ilieFapies. HO\'!j(ever, MSiC:;, 'eKp:ll!DdedJillJ. ,colJJve!lIliuol!l;l] mOllOhiI.yeF -DllilltUIefllamks peruits, pFlCIgJlessmg cell prol!i.f'e:m!UOIil (Ki1lm1Fuill IT aI., 2009'). The obj,eclive 'Of~ll ~:tl1'd~ 'IY'III11 to' assess w]J:e.ilie[mbhi!t meSBl!lChymml. skml oeJ]]S (FMSCs}pFecOmDllliIiteCUo gID;e mesenciItymili Qed!I.Ime3.i5eb.as~eJ:lrtii3Jre m. liespolllle fo indJumbi.re ecKJtrrllrel!lwarrrues to e:;;pilillilll.chJU Ms.cs.

f~'Q,~.1tiQiNl aiN'd ,[-rdlfu;fe"fJ1J'dS;e~ ... ~i ItJ .. d~ed Mediltl:

iFtabM,g, 'l'll'ereinaes&e&led u'ilmg- m~Ol1!l!a:Fmj:ecli(llJJ of 3i.llI1!ixtwieof 15mg~:gg;BtilIlilll!le iItymocMoride (jB:ioii:ela!n, Vetoqu.i:ool.Brcmr,e.t, SiP. 20_0, Frulloe}, .5.:gofXy.latzoine hydooclI!lmiide (nJiUM X"'iJLJ\ZIL~bl}, AustrrlllkOl) ,md! ftoejJTOlmIziillte lilJJ*afe 1 mglkg (C,t!lmive,t Veroqllmot ltd. Lure oedex, Frll!!lce)_ The 3il"lea from. eK!teE:iiLal!. ilI!I;gle af lli.eiliillm. to the hiiPjom!tw~s cliRed! alldJdi£imecledi, 3JUiliBll 1 j :m!lof bone mamm.r wilsoollleated hm i!litm:JJ. 11ImilIlg sJrini5.e (Sm[) \~i.1ih needle gauge 18:. The bene llilfl!IIDI'Ii"il:BpiIalted.'!i\i!'l:B, imme&illtely J:llixecl.'!i\iith 3d of titre Dub'lecoo"£ modlilied E~;gle'£mewll:m.()DMiEM).md!.lliig!ht ghl'l::ose-DMEM: wa:s, S!1Ipplemenlted. MIt!] 30~/o retal.bo • .me SBIlWl(jFBS), HliU/lIl:l!l!. pBlIlCillm. G, ]!I)UIm!l :strreptomycill, l]liIJJglruI. amp'iroteNcm B, l%IIl(llles~!biilil amill'!Ja.cid ~!i,}iiboo),mdJsodillm.p')'FL1Villte (lOOI!lP"j:m!l) alII. sup,plired by G[BCO~InI'!.t-iJtrroj5:elJJ. 'Co:rpomliiOIL ~ mJ of FB:S loWS placed!.m a 75mIl!.~ lII.aS!li::fo[ 3 n:rimdes and!.I:llBll ·~iIlillix:ed!.IDe&!'I. lovijlib. bOlremam)w was ad!dledro ·~fllil:Bk. The llIaEili::'!i\i.!'I:S, iilloullillted 3ilt 37~ in 5% 0)2 iill :D]fo[ 3 cl.a}fs"ii!nd!ilftBr Tlllmmtlle'lIloll"ildllleaient reillJll'!AicBrerem.ol!;red loVlliIile rt\Placill!g tile meW1IIm. After 12 cl.:lIys,ilie Cl!I~twie Jleac.arted.ro oo:uilIlue!lllce stage .<l!IIIid!.1!b.e IIlJ:O!lO~eJj

oeillBlv,ere wamedtl.vice lril!h 2 mi. ofpllor.plla,te linlfi'eFed S!lil:iE.e (mSlGpH 1.2)_ 'Ihe eultures.were dtige;;ted w.~tJ]. [,vo ml.oE{jl_10%.I:E)'iP'min.-0'Jl~% eHIyl1BJre mi"iilllliiillle temllaoeic acoid!. (EDIA, S,iigm<l, USA) aLI!Il1. msmtmtoo.ol!lilie swfuce .oEthelaye[ :f!lF1IIll!imites\,i!lilllec~kmgilie oe:llJr. ti!de:rilie microscope til!l. 'I!he. !egai!i!ecl! their !oll!!ld.ed!. shape .wmiliBlilWSClIJrdecl! thell)']lmine. DIIb[ecoo"s m.odifi.ed E~glle's med!ium. OOll!tamillg 10% ffiS was aJded tlllllie medilliI!I:l1. andtlien g<:!I!lit1J tl1PiPed to d_etadJ. reb fm nexl four slib C1!llitW;es (]l4i)-

H'O~ie "MaaTow ,mwr '[~ftAdipo.gel,"es,~:!'

'The celli;. \,VeFie han<eillted 1:1), clecau1iiwgl!hemoewum fi"rulII. the lII.<11~,\~~:sihUng\~ith. PBS, two mil .oE ~120%ilIypf>m.-0'.m:% e,tJ]y]ene d.iamim~l:e1irn:!Icelic ilIcid.~-][)[A, Siglwl,lUSA)b:a1!hirng .md JI~~£ing\~i.th Hi mil. ofDMiEM_ Themednua .mJ. ceilJjll\~eFecoHected. iinsferne testtubes, oellilrit'ng>ed at 2000 ~. roF 10 :minui:e;;l" ~t\Pi"iilltedJ the pFecipltOlltedpelil.el;s. audJhclofDi.li.ffiM WO'lS then.lIclded to the pellets. 1IDduKed!_ 'COlllllJt 'HIe II!lllli!btl .of Oe:]]ll1llf>i!I!I.g 11. henmcyrometer.

Gel] S!!IspellSlolli. WOlS p]!'I!ted iDto· me!l>E'lI.dhymcl. srem oel!l. expaIll\llonmedil!lW. (MCs.aQ at d!emi.ty of cells. (Hi~) :m.il 24-lV,e:m eWlnl!Jie dlish with llli:l!l. voiume peF lV,eJ]]_ eel!ls\~eFe mcMoolted <'lit 37'>C ina 5% COl~ed.illl::l!Ibato[ Oi\temi;gl1ti"mdJ completed. l:lolill1llen~!lft5r 24 ll.mmlof mmlhilluolIL '\¥hen the ·celli;. meHl!ll% ·colIlJIhrent, GlIeiillJr 3~-p:Katedilie mMlWll[rolIIJJ. elIc_]]W,eJ]] and <lIdd llllIil <lIWpoogBJILef>]llillUUauolIJ. med:il!U!lIl(DMElli.!"I.ow glh!l~e, EetOl]boi!i.me se:mm. HII%, WmMI Dexi'!!I!l.l:etJ]~solIl.e, (lj M 3-i~obniy[-I-metihyllxmth:i!I!l.e, 1(1 mg/miL !ieoomhmmt llumau iD8utin. and 10 mMJh!Jd.omeitllacin, pBmcdlirn .imd sl:rrepromycin},tmll mMl!U!IIlWM. cha~d corre~pcmdll to dtiEilieFeI!l!tiiilltiioil <'lit firet dlilY. AdipogeJILef>]r. inducuwm:edii1lll!lIl. WalllllfiBd at hit, 31.:1 au-d! )1110 .d<ly, wll!ille at 1()> .d1ly w~sreplaced!. hyawpogeller.is maill!rema!I!l.eemMl.!lm(Dlli.llEM-~o\..l.gmoo!!!e.re.tmllJcr\i.me semm 10%" m mu.,jmI. Jie;[)OllII!moan~ llmm1lD. iD8uliu. and. penic~tin. and. ~;mep,IDmyc:iIl)_ Ou 91", 1]111, .md ]]111, day, cells \'ve:Iie eWltnred n!ling~rllirEmgenesi:s. inmmti.on mMDIl, lilt lSlII'li!lillg~di.pogenoes.is mamreID1ll0e :medium.~IlJ. <'lit 17~', 19(}> alldJ 21" day;cW!ooed 1iImg!lrllipogenesi:s. irulucu01!l. medium_ Lipid dooplletllcan. be deteatedby microsoopic examID3Inon as el1l"l~r~ 5 di3lYS mtoilie mfferell!li1Inon perioo.

('jli,rl.li1:et1' 0 Slfm:r.!i~ig Pl:oiooo'

.~er 2Id<1y:s. ofdiffeF,entiiatiol:l!, 'HIe mediIwl Er0lIi:lJ ,oo.cJh wel!l. l'!fas!l£llir.l<I,ted care:ful!l.y_ The <lIdipocy[es Wefiernxed. b}' mruootiing in4i%pa:rrilroImllIdshyd!efOI 40 nIllrmIrell <lit mOIIIJJ. tempeF<IItwe, fiX<llIiV(e carefhl!J.Yilspmalted. au-d!. nnsedtareetisues (Hilll!i.!mJtes ,oocJh) \,vitllit IX J2'BS_ En01ilg!h OJ]. R,ed SQluu.o1IJ. to OOVeF the\~el!Is. Cl WipeF wil!l.)" mDllblliitedilJt !oomtfmpe.fil.tmie ror 5g)'lIll!imites, llien.rell!lOl!;rediilie oii~ J,Bdi.oomtii.onimd lll'lImedHIe\~eillB tbJiBemraellwilili. ] .DIillvllfrer aLI!Id. Srnirn::d oeillB llm::l.e1iIr.with B"ematol{yllm So11i1,uon (05 mil vo1\!Jme) fur 15 m:i!mJites. Fim!l.lY!1di.pocytes oontllmmg l!i¢d mopl.etsweFe stained red byilie Oil. iIted.OOhiJtWIIl will.l.e the ·ce]~ IDIcleiwiere stained b]arkilblli1e fi"Cll!IJ. ili.elIten:ulltmq'l!in. (Flrttenger et al,. ]999)

H'o.~ie· "MaaTow St~wr .[er~ .Osi';eogru,!e.:!',~:!'

Mer the eefls lv,ereilIIaro.esi:ed m. UmI.ofDMiEM l'!far. m:entii.onedi. aheve; .~. ceilJJ. SlIS]lieJ:!illO!l. wllr.p'Lalted lin DMEM at 3. clit:m~t3' OEce]~!l. (Hi~)peFwel!I.m.the\'1i.troneatimloQI]ilgen oOillteda 24- we]!l. CluillRliI"e dtif>b.wlth.1 mL'IwiUmepeFWiel!l.. Cel!l.~.l!,ferreill.ci.!lbatedillt :n~c lin ill. 5% 'COl ht!m!i.dmed mmlhillIDF ffiiieTIIIIiight 1l!Ildcomljll.ere oOOliB!1ilellted aclMe,redi. 1II1'iter 24 heurs ·ofi.ncJ!abaumJi.Wlle:nilie oeillB a!Jie 1(I(]I% omill.lIIelIlt,ilie mMmtlJ.'!.vilS ClIreR]l!l.y !l:spmil,ted. mom eilch.wel!I.ll!I!Id 1.m]. ositeogenoes.is iDrluclioIl. mMmtlJ. (TIMill-Eow glJliOO!!!e, fetal. iElol!;Jffie S·emm I'(]I%, Hi mMJ DB>:=rae1!ihta!lolle solli1hon, J\.:oooroic Acid!. 2-PihLoopJlerute soiuliolli, L-lnt!illl!i!I!l.e and Penicilli!I!l.illl-d!. SITeptomycin},ildded thi.,;. medium. W.!IiS clrnll!geci!. wFFe8poodiIJJg to diJill.eFiB!IlJtiiatWllJ. at W;llt di1l1fimdilien It\}lllil,QedW~th. fresh ooteogeElelli~. mililc1iiolli. medtilliI!I:l1. ,every 1-3 d1JJyll fo[ 2] d1JJysof CluillRlilli!ng.

A.~;Zdt~·~," .;4' S Stdi:I,!i~i8 Pl-oiocol

.~er 14-17 days ofdifiiBr,ellia!tlOllJ, th.eruewum was CO'lrefiallly lIIspirnrted from ,each lV,e:IlJ,

25

~VNALS OF It!ICROSCOPT

Vol 11, _-lpnllOl1

osteocytes by incubating in iced cold 70% ethanol for I hour at room temperature alcohol was aspirated and rinsed twice (10 minutes each) with water. Enough Alizarin Red solution was added to co rer the wells (1 mL per well), incubated at room temperature for 30 mIDU es. the alizarin red solution removed, and the wells washed four times with 1 mL water and aspira ed after each washing. Later on, 1 mL wa er was added 0 each well 0 prevent the cells from drying and examined using inverted microscope (Leica, Auterian). The Osteocytes containing calcium deposits were stained orange red by the Alizarin Red solution.

RE ULTS

BMSCs obtained from ilium of adult male rabbit comprised he erogeneous groups of cells after seeding and growing in culture pia es. After initial plating, the adheren cells exhibited small rounded, spindle-shaped, or large flattened morphology. Most cells grew and exhibited fibroblast-like morphology in reaching confluence. Interestingly the small rounded cells adhered to the surface of the e cell layers, These small rounded cells disappeared after repeated passage whereas the fibroblast-like cells became enriched. At passage 4, the fibroblast-like cells became morphologically homogeneous with more than 95% purity (Fig. 1).

Rabbit ~SCs were differentia ed and adipogenic after _1 days 0 mature adipocytes. The intrncelJular accumulation of lipid droplets as a lipid vacuoles (red) in the adipocytes were stained with Oil Red 0 solution (Fig. 2). Rabbit MSCs were differentiate after 14 days and demonstrated the mineral deposition throughout the culture as a mature osteocytes lineage as indicated by Alizarin Red S staining (Fig. 3).

DISCU SIO~

Stem cells differ from other precursor or progenitor cells in two essential ways. First, stem cells are self renewing, theoretically possessing the ability to proliferate and reproduce them limitlessly. Second, stem cells are multi or pluripotent and also have the potential to differentiate into multiple cell types. The plasticity and differentiation of stem cells are regulated by the microenvironment in which they reside. Bone marrow-derived cells can be aspirated directly from

1

Fig.I: Culrured BMSCs exhibited large, fibrobl:as -like mmp!lology in re3chiDl; cODfilleIKe. lmerest:Wgly!he small rounded calls adhered 0 W! surflce of these cell13yeIS (arrows). Sc.ale bar=lOO um

26

Fig. z: Cultmed BMSCs e.,wibiRd the accum1llarioll of iDmIcenular lipid droplets deRcted by Oil Red 0 staiDiDg (IIlTOWS). Scale b:u=lOO JUll

FIg. 3: CtJlrmed BMSCs e.:iliibited the. de~ilioD of calcum (arrows) was detemililed by AIizarm Red S smining. Scale bar- 00 IIlD-

27

deners.and cOlIl1frise hetBrogalet:lll'1l, ~op1IIbhOOlS of' oelIlswith ms1iirn::t j1l!a;;;,ti!.cily. These ce[l]js ,~eS8 IIlmlltifo;ienlti!ail a!Dd. dliE¥eFeolil~lte mto osreolbb1l1s, c~ge rel]s, sJleJleta~IIl111:gde ce[l]js, e:odotlleli!!l!J. rel]s, C.-diiilC nmscle cel!l!S!,Irep~Jt(ll(';ytes, .rurtd!. adipocyres bootiht j~\\,joo and tf~ liilliti'_ MSC:!;,illudmJed. \~ith. adlipogenic lm:lUaTIOIil m.em1!llll wer,e ilIbie~o dl:ilfereIltiatemtolifid-{lO!IJtilI:iilltmg 1J:dipocytes (Egliti, ilI!IId ~', 1997; l8Jrngll'li'il.ti 1I!Dd j{jll!, 2)004!). EvideJ!lce S11Igge;;hllThLait ~eC'feliOl:lJ.'of'varioml gro'\ll'iili.-fl7QlIl1'1J!bng~lIIlbfffilnce3' l!ikely [lliayg, ilIll irmporlan~ rel'e m Rmcllom1] of MSCs, in aJ:lJ.~lttemp,t toiden.i!i:fy .01. p>OOiSi:b]e merJiamsm. undledy.ing the Fegenemtive Elo;tel!lJti!Ol].of HM9Cs (Cihmet ,at, 10(7). :Studies mdliOlbte iliat ooatmg1:Jjs~uecuJjrure j1l!~£,TI~.va!Jie wilili "iilrnnecu andJ oobgen. I promore08teogelli.cdifieFiBliIJtiiatim:lJ. over DOIl-ocmted pbteS. AllizotriJ:JJ. Fed S :s,toillJilllIlg of' cl.emolIl~,iF<IItes uilileFa~ d.epwitl'lllli.tllFooghollJttlle cWltUlJie ilIIU osteo<genicpotenticl_ Viil:!"onec1ti.l!l.md 'COilagell I are ]lI17Q\.rnted illtlle li.t ad.IieOOIlIlll!lBOO. oOil,rnllgnSlglle C!II~tm:epl~sh(';-o[gla~lwaFei'l to .ilill!. irn. the d[ffu:renrb.3ltlonof ~>BDch)'lDcl.stem.ce1!l£, to ru,teobh"mts(SaiL!lrfiZlIlyk ill ,al_, 1(04). These FeS!IIHll are ooll!m!8tenli '\!.ilili. declines ill Oi~,teOgeJl!ic and pffllil~ adi~ocgeaic pOt>BDltiiil!l~ge,a!lth.ough. ~s~ge IIllW!lOeF:1Ill!1l)I' he mOFe :irmporlan~ than. ag;efm the iL!IIIite:[. Tile Fe5wli!S,OlFIf: ilIlIso ,con~is,tel!lJt,~ith. [llFe\'1iooll ~ortsilia,tmltoog;B=~smd ilIdlpo@Bl!lif:8l!ll deeliae .vitibt ilI@:e i3!ruil ~r.:Sill.~e ofmm]!le MSGscPFe\'1iou8 reports slro~~eclJ~t the :liesh!ly isobteclJbol!lif: JlIl1I![OO'W OODmilrts sigui~caJ:l!I!lYgJe<JIl:eI mI!lIllJOBI:S, of cel]llcolIilmilited to tile adlipogenic lirDBaj5e. There is ilIlIso e\<idenceffiitat mesencJlllymil!lEtem. ce[Ull oom:miJtted to an adi~ocgeaic [llF'Og:nlID canbe m.d!!I.ced tolmm-ulif[eFeoJ]!'Ilte t~} tire oiSreogell!ic patilitwO'ly_ MSC bm 1rum:.m.00&Em~e ,\!,BIe ilIlIso mporteclJ to lbeoo:me more m,teogeni.c <lit bre ~s~ge (9cWil!l:iIlg etal., 200®).

WewUI.tW .. lme to tilkehlriill 'OfpommiJtyw'~ a:m :stOlff mV-.rniogy an.dMeill.e;al Immlillology lLlIbornronesllWM fertlieir kind help.

HlItag\lvatl S, Jill, W. (2004!)_ lisobllon .all1i. ~e!l]jt (If siJl:lE:l.etalJ muscle [llFo@:emtm oeUs from. motme bolleWilml' .... ]3~ocIlBlIl:l1. iEl:iopll}'8. .Res .. Commin~ 3.U.; H9--.124_

Ohen CJ, (It] YC, [jjilO st, CltBl!l \flY, (;hel!l SY, WI.!. CW, \l,hmg CC, \\limgW'1{, Huang YS, H~I.!. SB. (2007_ TmlIlS]JiL3!II.t3JiIiilll.l(lfhClIlelIll!3!mHl,lE1iFo1Ill!3J~ eells fm ~plleru. merve Fepai![ .. up Noollti'L ],()4(1).:443-53..

Eglim ili.!A, Mezey IE_ (]997). H,em;]!i:ojJore,tiic oe:[l]js d]jJIfeFeJIlti!Ollteillto lbotl]. mi.cJ"ogij!'l and macrogl!ia in tile lbrnmsof aJullt woe. Prec. Nati. Ac.aJ. SCi:. as'A.94, 4(J8'{}-4(M5.

Horwitz [EM, Gordon PL,K;.oo '\VK, §effi:ey C. MaE':, ~eJhae:l n. NeeJ!, Re!le y_ MeNdl, Linda M:u,u!l!, Ted. Hmfirl:l!!I!DiIl (2002)_ lisoiLml:eclJil!llogeDeicbolle w3!HOw-derivedJDILelllllch:')rma:! cel]ll eogmft aad sti!mll!l!~lte grrO!'i!llli irn cb:iW!riBl!l wiiliht 08tOOge=~1l imrpBrfeata:[mpl!ic~Iti!.Ollil f'm cell thBr<l]ry' of' bone, .F'wc .Natl Acad Sri ~ .'99:8932-8937_

Killl!llIDL1!lJ T, Uy Bool, L Sellv.!'IFIll1!a:a!m2" f::bellwChill!.la:i. (2009)_ Th:rriee-Dim.enmol!lal. CU!l!ibLlLFif: m Rabbit: iEl:one MaFFQW MesaJIcll)'mcl. StBlIl:l1. Ce:llJll Usill!g MicroOllime[ Beads irn. Spiro (;liI~rure. EW1J~i1~ Cells tlIrti Maim1att 18: 41

till TIll'll:, M~FIiiffiI.a. M, HU1im:3JcJlIltlDW, Hiai nI, hie EH, LiilluJ.B.. OOO'1} LdellittiIEiOilllloo of (;OIIllIDlOllJ.

Fatilitw!'l.}':S, Med!i~.tmg nLffe:reIlrbatioll!. (If BODe Manow- il!!Dd A&po~e TLS8111e-,DeEived!. Hmmm Mee.ch:yl1!lcl. Stem Cel!l!! irnto· ThJiee MeSBIOChymml LiIIle!'lge~. Stem. >CelIIs lS:75G1---761l

MliIsirui. RA, J8e&cllmov,'loES, SuklrUlll. GT (200(5). Ci:lmparisQlIl of'mesBEhymal stem cel]ll dhlairnedi. frrom dliWBre:o:l:lllU!I1!lall.mS!lle;:L B.ull EIp Brol Med 13.9: 5;()4 -50.9_

P.iltten§BI MF, M:ilIckayAM:, Beall:: 9(;, Jai!8\'!fIlll. JRK, noo~sR., MIllcdD, M.o(lmIall!.~, SiimooetlIi D'W, C~rug 3, Mmllotk DR.. (1999) Muil!WW:eage ~QI:en!i!l~ OE!'Idu]t h1iIm:m. mes>elldliLymcl. ~teiliLll. oe:[l]js_ Scirmce 2'84.: 1.43-.147.

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111

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\\hll!l .• Mffi"ltle:Jml.ch SH,ILClIbClla 00. (007)- Effects (IIf serial [ll!'ls~gillg Oll. the adlipogeni.c i3!Dd ClIfiI:etlg;emc dJifi'ereJ1!iirutioopotelJJtia]. of iIlI.dipo@B"deril'!.i'eclJImID!IIlmeselilrhymmi. s:telil:lJ.ceJ!h .. 1Iss:u,c' Eitlg 13(6): 'J2'9.1--B_

Zu!.:!PA, ZIm Mi, Amji.:.m F', DWeJl ft. De Ugarte, Jerry L ffu;mg, HiIroshi l¥.li:l!LIDO, Zem. C. IIJfGm!Cll, Job. K. fFilSBI, PrniSpe.rltl~, MOl["c H_ Hedrick O:OG(2)_Himn<1ll. adipoiSeili!E<llUe is a seuree o:fmuLtipo;talit ::;:re!I!I:lJ. ceJ!!Is. Mol Bio.l C~ll,; 13:42'79---4295_