cell differentiation in vitro model systems

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CellDifferentiationInVitro:SecondaryarticleModelSystemsArticleContents.IntroductionGeorgePStudzinski,UMD–NewJerseyMedicalSchool,Newark,NewJersey,USA.OutlineofMethods.ApplicationsBecauseconditionsofcultureinvitrocannotbeidenticaltothoseinthelivingsystem,cell.FutureDevelopmentslinesculturedinvitroarereferredtoas‘modelsystems’.Notallcelltypesarecapableofdifferentiationinvitro.Proceduresforstudyingdifferentiationinmodelsystemsdependonthetypeofculturethatcanbeemployed.Techniquesemployedincludeliquidsuspensioncultures,monolayerculturesandcomplexculturesystemsinvolvingsemisolidmatricesorliquidoverlaymethods.Suchinvitroculturetechniqueshaveapplicationstothestudyofdifferentiationinbasicresearchandclinicalapplications.Introductionapproximatingthoseofthemorematurecells.TheseconditionsareprovidedbyhormonesorchemicalagentsWhenmammaliantissuesaredispersedintosinglecellsor(differentiationinducers)orbyalterationsincultureaggregatesofcellsandplacedinasterileenvironmentthatmediumorconditions,suchasgrowthinmonolayersorresemblestheenvironmentofthecellsinthebody,theyareinsuspension,thatinitiatethepathwaysnecessaryfortheoftenabletoproliferateforaconsiderabletime.Thesubsequentchangesingeneexpression.conditionsforgrowthcanbemanipulatedtoallowtheproliferationofasinglecelltype.Thus,studiescanbemadeonhomogeneous,ornearlyhomogeneous,cellpopulationsunderconditionsthatlimitthenumberofvariablesOutlineofMethodsencounteredbythecellswhentheyarepartoftissues,andthussimplifytheanalysisofthecomplexprocessesthatProceduresforstudyingdifferentiationinmodelsystemsconstitutethelifeofthecell.Originally,tissueculturedependonthetypeofculturethatcanbeemployed.Someexperimentswereperformedinglassbottlesorplatesandcellscanbeculturedinsuspensionandthisisaconvenientweresaidtobeconducted‘invitro’fromtheLatinwordforandcost-effectiveaswellaslabour-efficientwayofglass,vitrum.However,sincetheseconditionsareneverproducinglargemassesofcellsforbiochemicalstudies.exactlythesameasthoseinthelivinganimal,‘invivo’–SuspensionculturesareroutinelyusedforcultivationoffromtheLatinvivere,tolive–theyarereferredtoas‘modelleukaemiacells,butothercelltypes,e.g.HeLacellsderivedsystems’.Inmostcases,however,particularlyifthecellsfromcancerousepitheliumofuterinecervix,havebeenarenottransformedintoneoplastic(cancerous)variants,adaptedtogrowinsuspension.Mostmammaliancells,thecellsceasetoproliferate.Thiscaneitherbebecauseofhowever,growasmonolayersonsolidsupportsurfaces,theinherentlimitationoftheirproliferativeability(i.e.whichnowadaysareusuallyplastics.Inaddition,cellsenescence)orbecauseofinductionofaprogrammeforcellaggregatescanbecultivatedinmorecomplexculturedeath–apoptosis.Morerarely,whenthecellsareinearlysystems.developmentalstages,theyacquirecharacteristicsoffunctioning,maturecellsthatmimicsomestageinamoreadvancednormaldevelopment.ThislastprocessisLiquidsuspensionculturesdesignatedascell‘differentiation’.HaematopoieticcellsdonotnormallygrowattachedtoNotallcelltypesarecapableofdifferentiationinvitro.solidsurfaces,sotheycanbegrowninsuspension.First,thecellshavetobeeitherinthecelldivisioncycleDifferentiationofleukaemiccellscanbestudiedusing(cyclingcells)orcapableofenteringthecellcycle(G0cells)primaryculturesorestablishedcelllines.Theprincipalgivenanappropriatemitogenicstimulus.Second,thecellsadvantageofprimaryculturesisthattheeventsaremorehavetobecapableofsurvivalandproliferationinvitro,likelytoreflectthenormalprocessesthattakeplaceinvivo.whichinsuccessfulattemptsisachievedbysupplyingtheThedisadvantagesarethatthesuccessrateofmaintainingenvironment,nutrientsandgrowthfactors/cytokinessufficientnumberofcellsincultureforextensiveexperi-necessaryforspecificcelltypes.InthecaseofG0cellsthismentsislow,sothatparametersofdifferentiationthatcanisachievedbyremovinggrowthrestraintspresentinvivo.bestudiedarelimited,anditmaybedifficulttorepeattheThird,conditionshavetobeprovidedconducivetothefindings.Conversely,establishedcelllinesmayhavedriftedacquisitionofphenotypicandfunctionalcharacteristicsgeneticallyfromtheirinvivoprogenitors,whichmayskewENCYCLOPEDIAOFLIFESCIENCES/&2001NaturePublishingGroup/www.els.net1 CellDifferentiationInVitro:ModelSystemstheresultsofdifferentiationinduction,butthereisnolimitester12-O-tetradecanoylphorbol-13-acetate(TPA)pro-oncellnumbersthatcanbeobtained.Ideally,theducesamacrophage-likephenotype,andthehormonalconclusionsderivedfromexperimentsinwhichestablishedformofvitaminD(1,25-dihydroxyvitaminD3;1,25D3)orcelllinesareusedshouldbeconfirmedwithprimarythenucleosideanaloguearabinocytosine(AraC)producescultures.amonocytic-likephenotype.Theinducersarenotpheno-Primaryculturesareinmostcasesobtainedfromhumantype-specific;intheK562cellsystemTPAinducessomebloodorbonemarrowsamplestakenwiththesubject’sfeaturesofmonocyticdifferentiation,whileAraCinducesconsent.Inessence,theprocedurestartswiththeremovalerythroiddifferentiation,and1,25D3inhibitsthisactionofofpatient’scellsfromtheserumandfromacidicheparin,AraC.UnlikeHL60cells,U937cellsexposedtoretinoicwhichmayinhibitcellgrowth,andseparationofthecellsaciddifferentiateintomacrophage-resemblingcells.intotheprincipalbloodcomponentsusingadensityForanoptimaldifferentiationsystem,theconditionsgradientsuchasFicoll-Hypaque.Thebuoyantcellsatmustbecarefullystandardized.Ifestablishedcelllinesarethegradient–specimeninterfaceincludelymphocytes,used,itispossibletoobtainwelldifferentiatingsublinesmonocytes,neutrophilprecursorsfromblasttomyelocytethatprovidemorehomogeneouscellpopulationsandstage,proerythroblastsanderythroblasts,andleukaemicfacilitateperformanceofkineticexperiments.Thiscanbecellsincasesofleukaemia.Thecellsinthepelletconsistachievedbysubcloningcellpopulationsandselectingpredominantlyoferythrocytes,butgranulocytesarealsoappropriatecolonies.Differentiation-resistantsubclonespresent.Tlymphocytescanbeseparatedfromtheotherarealsousefulascontrolsindiversedifferentiationstudies,cellsbyformingrosetteswithneuraminidase-treatedsheepandmayalsobeidentifiedinsubcloningexperiments,buterythrocytes.Toseparatemonocytes/macrophagesfromcultivationinincreasingconcentrationsofadifferentiationtheothercellsintheinterface,theinterfacecellsareagentisamoreeffectivemethodfordevelopmentofwashed,resuspendedin10%fetalcalfserum(FCS)andresistance.Forexample,HL60-Gcells,subclonedfromanplacedhorizontallyinatissuecultureflaskplacedina378CearlypassageofHL60cells,showsignsofdifferentiationincubatorfor1–3hours.Removalofthesupernatantfluidwithin6hoursofadditionof1,25D3andallcellsintheandunattachedcellsleavestheadherentmonocytesandcultureexpressCD14,anearlymarkerofdifferentiation,macrophagesontheflasksurface.Theseparatedcellscanwithin48hours(Zhangetal.,1994).ThisisincontrasttobegrownintocoloniesandattemptscanbemadetoreportsfromvariouslaboratoriesthatHL60cellsrequireestablishlong-termcultivationiftheyarederivedfrom4–10daysfordifferentiation.Conversely,HL60-40AFimmortalizedcellssuchasleukaemicstemorprogenitorcellsshowcompleteresistancetothedifferentiatingactionscells.Alternatively,thecellscanbeusedasdifferentiationof1,25D3.modelsinprimarysuspensionculture.ItisusualtoaddOptimalconditionsforcelldifferentiationalsorequireantibacterialandantimycoplasmalagentsduringestab-attentiontocultureconditions.Thereisusuallyarangeoflishmentofcellcultures.However,thisdoesnotguaranteecellconcentrationsatwhichthecellscanbeseededandmycoplasma-freeculturesandtestingisimportanttoallowedtogrow,withoutthenecessityofsubculturing,excludeaninapparentinfection,asinfectedcultureswhichdisturbscultureequilibriumandcomplicatesproducemisleadingresultsinstudiesofdifferentiation.quantificationinkineticexperiments.Concentrationsless421Thechoiceofcelltypeandthematurityofthecellsthan210mlmaynotallowHL60cellstoproliferate,421dependsontheobjectiveofthedifferentiationstudy.andbelow510mldifferentiationispoor.WhenModelsareavailablethatmimicdifferentlineagesandconcentrationsaretoohigh,manycellsdie.Thus,inthisstagesofhaematopoieticcelldifferentiation.Forinstance,differentiationsystemtheoptimalstartingcellnumberis5521thereisagreatvarietyofhumanmyeloidcellsubtypes,310+110ml.ThenatureofthemediumshouldsuchasHL60cells,whicharepromyelocytictypecellsthatbethesameasthemediumrecommendedforoptimalcandifferentiatetothegranulocytic,monocyticormacro-growth,andsupplementationwithserum/growthfactorsisphagephenotype;U937cells,whichdifferentiatetowardsgenerallynotacriticalfactor.Intheauthor’slaboratorythemonocyticphenotype;andK562cells,whichhavethethelessexpensivebovinecalfserumsupplementationwascapacitytodifferentiatetoerythroidormegakaryocyticfoundtobesuperiortofetalcalfserumfordifferentiationphenotypes,dependingonthenatureofthedifferentiationofHL60cells,eventhoughitislessrichingrowthfactors.inducer.OtheravailablemyeloidcellsincludeHELcells,Also,asmentionedabove,inapparentinfectionwithKU812,MEG01andML-1.mycoplasmahasledtoerroneousconclusionsinvariousInductionofdifferentiationofhaematopoieticcellsisdifferentiationsystems.Suchinfectionsaremorecommonprimarilyaccomplishedbytheadditionofselectedwhenantibioticsareroutinelyusedforculturemainte-chemicalsorbiologicalsignallingmoleculestotheculturenance,sofrequenttestingformycoplasmaisrecom-system.Forexample,HL60cellscanbeinducedtowardsmended.Asimpletestthatcanbeperformedinmostthegranulocyticphenotypebyexposuretoappropriatelaboratorieshasbeenpublished(Studzinskietal.,1973),concentrationsofthesolventdimethylsulfoxideorthebutiffacilitiesareavailablethiscanbedonebypolymerasemorphogenretinoicacid,whilethecarcinogenicphorbolchainreaction(PCR)ormicrobiologicaltechniques.2ENCYCLOPEDIAOFLIFESCIENCES/&2001NaturePublishingGroup/www.els.net CellDifferentiationInVitro:ModelSystemsAlterationofcultureconditionscanalsobeusedtoadherencetothesurfaceoftheculturevesseliscriticalforinducedifferentiation.Aninterestingexampleiscultiva-cellproliferation,whichislimitedbytheavailabilityofthistionoffreshlyisolatedbloodmononuclear(gradientsurfaceifcelldensityistoohighandthecellsremaininterface)cellswithmonolayersofhumanumbilicalveinconfluentforasignificantperiodoftime.Forceddetach-endothelialcellsgrownonanendotoxin-freecollagenousmentoftenoccursandresultsincelldeathbyaprocessmatrix(Randolphetal.,1998).Themonocytespenetrateknownas‘anoikis’,butitmayalsoresultindifferentiation.intothesubendothelialcollagenwithinonehour,butintheAdvantageistakenofthisinamodelforkeratinocytefollowingtwodaysapproximatelyhalftheCD14-positivedifferentiation.Here,mousekeratinocytesaredetachedmonocytesreversethemigrationbackthoughtheen-fromthesubstratumandareplacedinacomplexdothelium.Twoformsofdifferentiationhavebeenspecializedmediumcontaining1.45%methylcellulose,detectedinthissystem;thecellsremaininginthewhichprovidesasemisolidsupportforthecellsforsubendotheliummaturetomacrophages,whilethere-initiationofthedifferentiationprocess.verse-transmigratedcellsshowmorphologicalappear-Anotherdifferentiationsystemregulatedbycellcon-ancesandmarkersofmaturedendriticcells(Randolphfluenceisthemodelforterminaladipogenesis.Severaletal.,1998).establishedmouseproadipocytecelllines,e.g.3T3-L1Assessmentofdifferentiationdependsoncomparingthecells,areavailableandarebelievedtoprovidefaithfulappearanceandthefunctionalcapabilitiesofthemoremodelsofadipocytedifferentiationinvivo.Theevidencematurecellsagainsttheundifferentiatedorpartiallyforthisincludesthedevelopmentofnormalfatpadsindifferentiatedcellsusedtoinitiatethedifferentiationanimalsinjectedsubcutaneouslywithinvitrodifferentiatedsystem.Reductionorcessationofproliferativeactivity,adipocytes.Inthismodelsystemspontaneousconversionthoughsometimesincludedinthecriteriafordifferentia-totheadipocytephenotypecanoccurinasignificanttion,isaseparate(albeitrelated)process,andshouldonlyproportionofthecellpopulation2–4weeksaftertheybeconsideredasconfirmatoryofotherwisedemonstratedreachconfluence.Thus,eitherdetachmentfromsubstra-differentiation.Ontheotherhand,thenumerouscelltumorforcedconfluencecanresultindifferentiation,surfacemarkersavailableandflowcytometricanalysisdependingonthecelltype.havelargelysupplantedthemorelaboriouscytochemicalExamplesofothermodeldifferentiationsystemsinandimmunologicalmethodsforassessingdifferentiation-monolayercultureincludehumancoloncancerandrelatedchangesinthephenotype.Cellmorphologycanbeprostatecancercells,breastcancercellsthatcanbesuggestiveofthelineageofdifferentiation,butisdifficulttoinducedtomakemilkproteinsandfatdroplets,muscleinterpretandoflittlehelptothenonexpert.Thisisbecausecellsthatcanformcontractingmyotubeswhenchangedinvitrodifferentiationisacaricatureratherthananfromgrowthfactor-richFCS-supplementedmediumtoaccuratereplicaofinvivodifferentiation,sotherecognitionmediumsupplementedwithalowerconcentrationofhorseofmorphologyismoreanartthanascience.Functionalserum,osteoblastcellculturesthatcanformmineralizedcharacteristics,suchasphagocytosisbymaturingmyeloidbonematrix,andavarietyofneuronaldifferentiationcells,areeasytorecognizebuttheprocessisexcessivelysystems.Alsoveryfrequentlyusedareratpheochromocy-laborious.DemonstrationofenzymeactivitiesorproteinstomaPC12cells,whichcanbedifferentiatedintomatureassociatedwithdifferentiatedcellsisthereforeareasonableneuronswhenculturedwithnervegrowthfactor(NGF)compromise.Thisisshown,forinstance,bymeasuringtheandadenosine3’,5’cyclicmonophosphate(cAMP).Mouseactivityofanesterase(‘NSEreaction’)orbydetectingembryonicstem(ES)cellsaremultipotentcellsandcanbereactiveoxygenspeciesgeneratedduringphagocytosisinducedtodifferentiatetoneuroectodermalcells(neurons)(‘NBTreaction’)indifferentiatedmonocyticandmyeloidwithretinoicacid,andtomesodermalcellswithdimethylcells.Similarly,theappearanceofhaemoglobinsignifiessulfoxide.Othermultipotentialcelllineshavebeenerythroidlineagedifferentiation.Assessmentoflargeestablishedfromtheneuralcrestandtheneuroepithelium.numbersofcellsisfacilitatedbytheuseofflowcytometry,andchangesinthelevelsofexpressionofnumerousproteinsortheiraccessibilitytoantibodiesprovideComplexculturesystemsmarkersofdifferentiationandlineageidentification.CellsgrowninsemisolidculturematricessuchassoftagarormethylcelluloseformmulticellularcoloniesthatoftenMonolayerculturesdifferentiateaccordingtotheirinherentpotential.Col-lagencanalsoprovideamorephysiologicalmatrix.AlargenumberofmammaliancelldifferentiationsystemsInduceddifferentiationofbreastcancercellsinsemisolidhavebeenestablishedinmonolayerculture.Mostofthegelmatrixcanresultinformationofduct-likestructures.considerationsandcaveatsdiscussedwithreferencetoThree-dimensionalgrowthcanalsobeobtainedbysuspensionculturesapplyalsotothissystem.Itisespeciallygrowingcellsin‘liquidoverlay’,wherecellsareplacedimportanttopayattentiontocelldensityinculture,sinceintomediumaboveanonadherentlayer.WhencellclustersENCYCLOPEDIAOFLIFESCIENCES/&2001NaturePublishingGroup/www.els.net3 CellDifferentiationInVitro:ModelSystemshavegrowntosufficientsizetheyareplacedintoMAPKpathwayoperatingindifferentiatingPC12andmagneticallystirredsuspensioncultures,orintomultiwellHL60cells,aswellasinkeratinocytecultures.plates,wheretheycangrowinto‘multicellspheroids’Thereappearstobenolimittothewaysinwhichinvitro(Durand,1999).Insomecasesthesespheroidshavedifferentiationsystemscanbeutilizedformolecularandstructuresthatresembleorganizingtissues,suchascellbiologystudies.formationoftubularandtrabecularstructureswithinthespheroidsofrenalcarcinomacells(Lieubeau-Teilletetal.,Clinicalapplications1998).StructuresresemblingendometriumhavealsobeendescribedwhenendometrialcelllinesareculturedintheThemodalitiesthatcanbeusedforcancertherapyincludepresenceofovariansteroidsonartificialbasementdifferentiationtherapy.InvitrosystemsprovideauniquemembranescomposedofMatrigel(Mukherjeeetal.,testinggroundforthisformoftreatmentofhumancancer1993).Complexstructuressuchaspancreaticacinicansincehumancellscanbeusedtoevaluatetheeffectivenessalsobemaintainedincultureusingthemethodsdescribedofagentsproposedforclinicaluse.inthissection,butalthoughphenotypicmodulationofModeldifferentiationsystemsarealsousefulinmanysuchstructurescansometimesbeobserved,sofaronlyotheraspectsofcancerresearch.Incancerdiagnosis,limitedinformationondifferentiationprocesseshasbeenrecognitionofthelineageofcoloniesdifferentiatedinobtainedusingthesecomplexsystems.semisolidmediamayoccasionallybehelpfultodeterminethenatureofpoorlydifferentiatedleukaemiccellsandthusprovideinformationregardingtreatmentmodalitiesthatarelikelytobethemosteffective.DifferentiationsystemsalsoprovideguidanceforoptimizingproceduresaimedatApplicationsregeneratingdamagedhumantissuessuchasskin,muscleornerves.BasicresearchCurrently,oneofthemajorinterestsisinelucidatingtherelationshipbetweencellcyclecontrolanddifferentiation.FutureDevelopmentsSeveralsystemshaveprovedtobeparticularlyusefulinthisregard.AdipocytedifferentiationillustratesanunexpectedAmongtheemergingadvancesinthefield,twoappeartoaspectofthisrelationship.Monolayersof3T3-L1adipo-havespecialpotentialforamajorimpact.Fromapureblastscanbemaintainedinactivecellcycle,butwhenthetechnologystandpoint,microarrayhybridizationandcellsreachconfluencetheyaretemporarilyarrestedintherelatedsophisticatedanalysistoolsprovideavastcomple-G1phaseofthecellcycleandbegintoexpressearlymentofgeneswhoseexpressioncanbedeterminedbymarkersofadipocytedifferentiation.These‘preadipo-measuringtheabundanceofmRNA.Invitrodifferentia-cytes’re-enterthecellcycleandundergomitoticclonaltionmodelsystemsshouldprovetobeexceptionallyexpansionwhenexposedtoamediumcontaining3-amenabletothisformofanalysis,sincethesesystemsisobutyl-1-methylxanthine,dexamethasoneandinsulin.generallyconsistofhomogeneouscellpopulationsthatcanThisshortensthetimenecessarytoreachpermanentG1besampledincrementallyduringtheprogressionofthearrestandaccumulationoffatdropletsfromtwoweekstodifferentiationprogramme.Ofcourse,identificationofthreedays(Rubinetal.,1978),andshowsthatcellcycledifferentiationgeneswillrequireaconceptualsynthesisoftraverseacceleratesandmayevenberequiredfornetworksthroughwhichthesegenesrelatetooneanother,differentiationasshownbytheabilityofinhibitorsofaformidableundertaking.ThismaybecomefacilitatedbyDNAreplicationtoblockdifferentiationinboneandtheallianceoffortyUSlaboratoriesbeingestablishedtoerythroidcells.Anothersysteminwhichdifferentiationmapcellsignallingpathways.precedescellcyclearrestisexemplifiedbythevariousAnothernewdevelopmentindifferentiationresearchleukaemiacellsinducedtodifferentiate,inwhicharelatestotherecentfindingthatsomestemcellshavetrans-proliferativebursthasbeendescribedintheveryearlydifferentiationpotential.Surprisingly,neuralstemcellsstagesofinductionofdifferentiation.Further,theseeming(NSC)frommouseforebrain,embryonicoradult,irreversibilityof‘terminal’differentiationappearsinmanysystemicallyinjectedintoirradiatedmiceofadifferentcasestobeillusory–re-entryintothecellcyclehasbeengeneticstrainwerefoundtoproduceavarietyofbloodcelldemonstratedforsuchterminallydifferentiatedcellsasthetypesincludingmyeloid,lymphoidandearlyhaemato-myotubules(Endo,1992)and1,25D3-differentiatedHL60poieticcells(Bjornsonetal.,1999).SinceNSCscanbecells(StudzinskiandBrelvi,1987).continuouslyexpandedinculture,thissystemcanprovideInvitrodifferentiationsystemsalsoprovideexcellentasourceforrepopulationofbonemarrowwithunlimitedopportunitiesforstudiesofintracellularsignallingpath-numbersofhaematopoieticcellprecursors.Similarly,ways.PublishedstudiesincludeinvestigationsoftheRas/injectionofimmaturenervecellsderivedfrommouse4ENCYCLOPEDIAOFLIFESCIENCES/&2001NaturePublishingGroup/www.els.net 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