Klf4在维持胚胎干细胞干性方面的研究课件

Klf4在维持胚胎干细胞干性方面的研究EpiblastMammalianembryosproduceextraembryoniccellspriortodefiningthefounderpopulationfortheembryoproper(Gardner,1983;SelwoodandJohnson,2006).Theprimaryroleoftheextraembryoniclineagesistomediateuterineimplantationandsubsequentmaternalsustenanceofthegrowingembryoandfetus.

Inrecentyears,ithasbeendiscoveredthatextraembry-onictissuesalsosupplypowerfulinductivesignalsthatspecifyandpatternearlydevelopment(BeddingtonandRobertson,1999).Toformtheembryo,apoolofuncommittedcellsmustbeestablishedandpoisedtorespondtothosesignals.Thispopulationistheepiblast.TheLimitationofTotipotency

Becauseitcangiverisetoanentireembryo,themammalianeggisoftendescribedastotipotent.However,thisdescriptiondoesnotmeanthattheeggitselfhastheabilitytodifferentiateintoallcelltypes.Thus,theeggandblastomeresproducedirectlyonlytwocelltypes,thetrophoblastandtheinnercellmass(ICM).Forsubsequentdevelopment,cellswithintheICMmustacquirethecapacitytogenerateothercelltypesandtodosoinaflexiblemanner(GardnerandBeddington,1988).TheICMproducesasecondextraembryoniclineage,thehypoblast,andaroundthesametime,theremainingcellsdevelopintopluripotentepiblast.Theepiblastisfunctionallyandmolecularlydistinctfromblastomeresandearlyinnercellmass(Gardner,1998;Kajietal.,2007;Kurimotoetal.,2006).

Thus,ratherthanrepresentingadiminutioninpotencyfromtheegg,wesuggestthattheepiblastconstitutesthegroundstate,meaningafullyunrestrictedpopulationthatharborstherequisitedevelopmentalpotencyandflexibilitytoproduceallembryoniclineages.

NicholsandSmithhavesuggestedthatthetwotypesofESCdifferfundamentallyinthegenenetworksthatmaintaintheirpluripotencyandnamedtheLIF-dependentESC“naive”andtheFGF2-dependenttype“primed.”TheGroundStateandTrueEmbryonicStemCells

Thenewlyformedepiblastisaclusterof10–20unspecializedcellssandwichedbetweenthetrophoblastandthehypoblast.Theepiblastgeneratestheentirefetusandsinglemouseepiblastcells,isolatedatthisstageandmicroinjectedintoanotherblastocyst,cancontributetoalllineages(Gardner,1998).Functionally,therefore,preimplantationepiblastisthedevelopmentalgroundstate.

ESCsalsoshareanepigeneticfeaturewithpreimplantationepiblast.ThistraitisthepresenceoftwoactiveXchromosomesinfemalecells.

Infemaleembryos,thepaternallyinheritedXchromosomeissilencedduringcleavageandremainssilentinextraembryoniclineages.Reactivationoccurstransientlyinthepluripotentlineagepriortoimplantation(Heard,2004).InXXembryos,oneoftheXchromosomesundergoesrandominactivationinearlyeggcylinderepiblastcells(Heard,2004).Theepiblastisthensubjecttoasystematictopologicalbombardmentwithinductivefactorsemanatingfromtheadjacentyolksacandtrophoblasttissue(BeddingtonandRobertson,1999).Eggcylinderepiblastcells,therefore,becomeinstructivelyspecifiedaccordingtotheirlocation.However,postimplantationepiblastcellscannotcontributetoblastocystchimeras(Rossant,2008),norcantheygiverisetoESCs.EpiSCscanalsobeproducedfromESCsinculture(Guoetal.,2009).Consistentwithatruedifferentiationevent,onecopyoftheXchromosomeinXXcellsisepigeneticallysilencedasESCsbecomeEpiSCs.However,EpiSCsstillexpressthecanonicalpluripotencyfactorsandcanbereprogrammedtonaivepluripotencybytransfectionwithjustasinglefactor,Klf4(Guoetal.,2009).TheresultingiPSCsshowreactivationoftheXchromosome,exhibittheESC-specifictranscriptionalprofile,producehighcontributionsomaticchimeras,andgivegermlinetransmission.GroundstatenaivepluripotencyisestablishedintheepiblastofthematureblastocystandmaybecapturedinvitrointheformofESCs.Shortlyafterimplantation,theepiblasttransformsintoacup-shapedepitheliumandbecomesprimedforlineagespecificationandcommitmentinresponsetostimulifromtheextraembryonictissues.EpiSCsaretheinvitrocounterpartofprimedepiblast.ESCscanbeinducedtodifferentiateintoEpiSCsbyexposuretoactivinandFgf,butthereversetransitionrequirestransfectionwithKlf4orotherreprogrammingfactors.

INTRODUCTIONESandEpiSCsSimilarities:BothEScellsandEpiSCsarecapableofmultilineagedifferentiationinvitroandcanformteratomaswhengraftedintoadultmice(Bronsetal.,2007;Tesaretal.,2007).Bothcelltypesexpressthethreetranscriptionalregulators,Oct4(Pou5f1–MouseGenomeInformatics),Sox2andNanog,thataregenerallyconsideredtoconstitutethecorepluripotencynetwork(Boyeretal.,2005;Lohetal.,2006;Wangetal.,2006).Differences:However,therearesignificantdifferencesingeneexpressionbetweenEScellsandEpiSCs(Tesaretal.,2007).Furthermore,thecultureconditionsformaintainingthetwocelltypesarequitedistinct.EScellsself-renewinresponsetothecytokineleukaemiainhibitoryfactor(Lif)(Smithetal.,1988;Williamsetal.,1988)andeitherserum,bonemorphogeneticprotein,ortheinhibitionofMek/Erksignalling(Burdonetal.,1999;Yingetal.,2003;Yingetal.,2008).TheyaredrivenintodifferentiationbyFGF/Erksignalling(Kunathetal.,2007;Stavridisetal.,2007).EpiSCs,bycontrast,aremaintainedbyFGFandactivin(Bronsetal.,2007).RESULTSANDDISCUSSION(A)PhasecontrastandfluorescenceimagesofestablishedEpiSClineWederivedEpiSCsfromE5.75mouseembryoscarryingtheOct4GiPtransgene(Yingetal.,2002).Celllineswereestablishedandmaintainedwithoutfeedersinserum-freeN2B27medium(YingandSmith,2003)supplementedwithactivinAandFgf2(bFGF)(Bronsetal.,2007).Fig.1.EpiSCsaredistinctfrom,anddonotspontaneouslyconvertto,EScells.(B)qRT-PCRanalysisofmarkergeneexpressioninEScellsandEpiSCs.ES,EScellsin2i/Lif.Epi6andEpi7aretwoindependentEpiSClines.y-axis,relativeexpressionnormalisedtoGapdh.(D)EpiSCsloseOct4expressionanddifferentiateordiein2i/Lif.AF,EpiSCculturedinactivinAplusFgf2WeconcludethattheEpiSCrepresentsastablecellstatethatdoesnotnaturallyreverttonaïvepluripotentstatus.(E)qRT-PCRanalysisofEScelldifferentiationintoEpiSCsuponcultureinFgf2andactivin.Epi3andEpi10indicatecellsculturedinFgf2andactivinforthreeandtenpassages,respectively.y-axis,relativeexpressionnormalisedtoGapdh.(F)Oct4andme3H3K27immunostainingoffemaleEScell-derivedEpiSCs.EpiSCsbothexpressOct4andexhibitanuclearbodyindicativeoftheinactiveX(whitearrow).Bluearrowindicatesadividingcell.(C)ConstitutiveKlf4expressionpermitscontinuedrecoveryofEScellcoloniesaftercultureinactivinandFgf2.Onethousandcellswereplatedforeachsampleintriplicateattheindicatedpassage(P)number.MT,emptyvectortransfectants;K4,Klf4transfectants.Therefore,constitutiveKlf4eitherallowslong-termpersistenceofasmallfractionofundifferentiatedEScells,orenablesafractionofEpiSCstodedifferentiateandregainthegroundstate.(D)PiggyBacvectorforexpressionofKlf4(pGG137Klf4),andcontrolPiggyBacvector(pGG131).Arrows(P)indicatePCRprimersusedtoamplifythePBLTRfragmentafterCre-mediatedrecombination.(E)Hygromycin-selectedKlf4andcontrolvector-transfectedEpiSCs.(F)qRT-PCRanalysisshowingthatforcedKlf4expressiondoesnotinduceEScellmarkergeneexpressioninEpiSCculture.ES,EScells;Epi,EpiSCs;Vec,EpiSCtransfectedwithcontrolvectorpGG131;Klf4,EpiSCstransfectedwithpGG137Klf4.y-axis,relativeexpressionnormalisedtoGapdh.WeconcludethattheexpressionofKlf4atasimilarRNAleveltothatpresentinEScellsisnotalonesufficienttoresetEpiSCsandinstatefullpluripotencyincellsmaintainedinactivinandFgf2.(B)qRT-PCRanalysisofmarkergeneexpressioninEScells,EpiSCsandderivativeEpi-iPScellsisolatedin2i/Lif.y-axis,relativeexpressionnormalisedtoGapdh.qRT-PCRanalysisshowedthemarkerproEScells,withupregulationofStellaandKlf2.Conversely,Fgf5andbrachyurymRNAswerelost.(C)me3H3K27stainingoffemaleEpiSCsandderivativeEpi-iPScells.Weexaminedme3H3K27immunostainingandfoundthatthenuclearbodycorrespondingtotheinactiveXchromosomewaslostinOct4-GFP-positivecellsaftertransferto2i/Lif(Fig.3C).However,ineachoftheGFP-positiveclonesweobservedpartialorcompletelossofvisibleDsRedexpression(Fig.3D,E),althoughqRT-PCRanalysisrevealedthatthetransgeneswerenotcompletelysilenced(Fig.3F).(G)ChimericmouseproducedfromtheK4C12Epi-iPScloneandagoutigermlineoffspring.Thisconfirmsthatthedevelopmentalcapacityhasbeenfullyderestrictedandtheauthenticpluripotentstateestablished.ThesecellsshouldthereforebeconsideredasEpiSC-derivediPScells,orEpi-iPScells.Fig.4.Retentionofgroundstatepluripotencyaftertransgeneexcision.(A)Splinkerette-PCRreveals1-3PBinsertionsineachiPSclone.WechosetwoDsRed-positiveclonesandtransfectedeachwithaCreexpressionplasmid.After5days,cellsthatnolongerexpressedDsRedwereisolatedusingflowcytometrywithsingle-celldepositioninto96-wellplates(Fig.4B).(B)FlowcytometryshowingtheDsRed-negativepopulationintheK4C3linebeforeandafterCretransfection.(C)GenomicPCRshowinglossoftheKlf4transgeneandgainofthePB-LTRfragmentintworevertantclones.(D)RT-PCRanalysisshowingthelackofKlf4transgeneandDsRedexpressioninexpandedCre-revertedcells.TwothirdsoftheexpandedclonesretainedonlythePBterminalrepeats.RT-PCRanalysisfailedtodetectexpressionoftheKlf4transgeneorDsRedfromtheserevertants(Fig.4D).TheyretainedEScellmorphology,Oct4-GFPexpressionandEScellmarkerpro.4E,F).(F)MaintainedmorphologyandOct4-GFPexpressioninaCre-revertedEpi-iPScellline.(G)me3H3K27stainingofKlf4transgene-deletediPScellsascomparedwithparentalEpiSCs.(H)ChimericmousemadewithrevertantK4C3-A3cells,andagoutioffspringdenotinggermlinetransmission.Femalechimaerasfromtwooutofthreeclonesproducedagoutioffspringintheirfirstlitter(Fig.4H),indicativeoftransmissionofiPScell-derivedoocytes.Therefore,completeremovaloftheKlf4transgenedoesnotdestabilisetheinducedgroundstate.Thisestablishesthatreprogramminghasbeenfinalisedanddoesnotdependuponongoingtransgeneexpressionorinsertionalmutagenesis.Humanandmouseembryonicstemcells(ESCs)arederivedfromblastocyst-stageembryosbuthaveverydifferentbiologicalproperties,andmolecularanalysessuggestthatthepluripotentstateofhumanESCsisolatedsofarcorrespondstothatofmouse-derivedepiblaststemcells(EpiSCs).mEpiSCsandhESCsshareaflattenedmorphology,intolerancetopassagingassinglecells,dependenceonTGFβ/Activinsignaling(15),inactivationoftheXchromosomeinmostfemalecelllinesisolated(16),andahighpropensitytodifferentiateintoPGCsinresponsetoBMP4invitro(17).HerewerewiretheidentityofconventionalhumanESCsintoamoreimmaturestatethatextensivelysharesdefiningfeatureswithpluripotentmouseESCs.

ThiswasachievedbyectopicinductionofOct4,Klf4,andKlf2factorscombinedwithLIFand