Induction of Pluripotent Stem Cells from Mouse Emb.docx

Induction of Pluripotent Stem Cells from Mouse Emb.docx

《Induction of Pluripotent Stem Cells from Mouse Emb.docx》由会员分享，可在线阅读，更多相关《Induction of Pluripotent Stem Cells from Mouse Emb.docx（14页珍藏版）》请在冰豆网上搜索。

InductionofPluripotentStemCellsfromMouseEmb

InductionofPluripotentStemCellsfromMouseEmbryonicandAdultFibroblastCulturesbyDefinedFactors

SUMMARY

Differentiatedcellscanbereprogrammedtoanembryonic-likestatebytransferofnuclearcontentsintooocytesorbyfusionwithembryonicstem（ES）cells.Littleisknownaboutfactorsthatinducethisreprogramming.Here,wedemonstrateinductionofpluripotentstemcellsfrommouseembryonicoradultfibroblastsbyintroducingfourfactors,Oct3/4,Sox2,c-Myc,andKlf4,underEScellcultureconditions.Unexpectedly,Nanogwasdispensable.Thesecells,whichwedesignatediPS（inducedpluripotentstem）cells,exhibitthemorphologyandgrowthpropertiesofEScellsandexpressEScellmarkergenes.SubcutaneoustransplantationofiPScellsintonudemiceresultedintumorscontainingavarietyoftissuesfromallthreegermlayers.Followinginjectionintoblastocysts,iPScellscontributedtomouseembryonicdevelopment.Thesedatademonstratethatpluripotentstemcellscanbedirectlygeneratedfromfibroblastculturesbytheadditionofonlyafewdefinedfactors.

INTRODUCTION

Embryonicstem（ES）cells,whicharederivedfromtheinnercellmassofmammalianblastocysts,havetheabilitytogrowindefinitelywhilemaintainingpluripotencyandtheabilitytodifferentiateintocellsofallthreegermlayers（EvansandKaufman,1981;Martin,1981）.HumanEScellsmightbeusedtotreatahostofdiseases,suchasParkinson’sdisease,spinalcordinjury,anddiabetes（Thomsonetal.,1998）.However,thereareethicaldifficultiesregardingtheuseofhumanembryos,aswellastheproblemoftissuerejectionfollowingtransplantationinpatients.Onewaytocircumventtheseissuesisthegenerationofpluripotentcellsdirectlyfromthepatients’owncells.

Somaticcellscanbereprogrammedbytransferringtheirnuclearcontentsintooocytes（Wilmutetal.,1997）orbyfusionwithEScells（Cowanetal.,2005;Tadaetal.,2001）,indicatingthatunfertilizedeggsandEScellscontainfactorsthatcanconfertotipotencyorpluripotencytosomaticcells.WehypothesizedthatthefactorsthatplayimportantrolesinthemaintenanceofEScellidentityalsoplaypivotalrolesintheinductionofpluripotencyinsomaticcells.

Severaltranscriptionfactors,includingOct3/4（Nicholsetal.,1998;Niwaetal.,2000）,Sox2（Avilionetal.,2003）,andNanog（Chambersetal.,2003;Mitsuietal.,2003）,functioninthemaintenanceofpluripotencyinbothearlyembryosandEScells.Severalgenesthatarefrequentlyupregulatedintumors,suchasStat3（Matsudaetal.,1999;Niwaetal.,1998）,E-Ras（Takahashietal.,2003）,c-myc（Cartwrightetal.,2005）,Klf4（Lietal.,2005）,andb-catenin（Kielmanetal.,2002;Satoetal.,2004）,havebeenshowntocontributetothelong-termmaintenanceoftheEScellphenotypeandtherapidproliferationofEScellsinculture.Inaddition,wehaveidentifiedseveralothergenesthatarespecificallyexpressedinEScells（Maruyamaetal.,2005;Mitsuietal.,2003）.

Inthisstudy,weexaminedwhetherthesefactorscouldinducepluripotencyinsomaticcells.Bycombiningfourselectedfactors,wewereabletogeneratepluripotentcells,whichwecallinducedpluripotentstem（iPS）cells,directlyfrommouseembryonicoradultfibroblastcultures.

RESULTS

Weselected24genesascandidatesforfactorsthatinducepluripotencyinsomaticcells,basedonourhypothesisthatsuchfactorsalsoplaypivotalrolesinthemaintenanceofEScellidentity（seeTableS1intheSupplementalDataavailablewiththisarticleonline）.Forb-catenin,c-Myc,andStat3,weusedactiveforms,S33Y-b-catenin（Sadotetal.,2002）,T58A-c-Myc（Changetal.,2000）,andStat3-C（Brombergetal.,1999）,respectively.BecauseofthereportednegativeeffectofGrb2onpluripotency（Burdonetal.,1999;Chengetal.,1998）,weincludeditsdominant-negativemutantGrb2DSH2（Miyamotoetal.,2004）as1ofthe24candidates.

Figure1.GenerationofiPSCellsfromMEFCulturesvia24Factors

（A）Strategytotestcandidatefactors.

（B）G418-resistantcolonieswereobserved16daysaftertransductionwithacombinationof24factors.Cellswerestainedwithcrystalviolet.

（C）MorphologyofEScells,iPScells（iPS-MEF24,clone1-9）,andMEFs.Scalebars=200mm.（D）GrowthcurvesofEScells,iPScells（iPS-MEF24,clones2-1–4）,andMEFs.33105cellswerepassagedevery3daysintoeachwellofsix-wellplates.

（E）RT-PCRanalysisofEScellmarkergenesiniPScells（iPS-MEF24,clones1-5,1-9,and1-18）,EScells,andMEFs.Nat1wasusedasaloadingcontrol.

（F）BisulfitegenomicsequencingofthepromoterregionsofOct3/4,Nanog,andFbx15iniPScells（iPS-MEF24,clones1-5,1-9,and1-18）,EScells,andMEFs.OpencirclesindicateunmethylatedCpGdinucleotides,whileclosedcirclesi