IPS-课件-Induced-Pluripotent-Stem-Cells(iPSCs)

InducedPluripotentStemCells(iPSCs)—acandidateofEmbryonicstemcells武栋成武汉大学基础医学院生化教研室海外实验室BoneMarrowTransplant造血干细胞造血干细胞是指骨髓中的干细胞，具有自我更新能力，能发育生成各种类型的血细胞。造血干细胞可以救助很多患有血液病的人们，最常见的就是白血病。脐带血干细胞80年代，科学家发现脐带血液含有高浓度可用来供医疗用途的干细胞。脐带血干细胞取自脐带，不需要进行麻醉，对产妇、婴儿无痛和副作用。——废物利用，变废为宝资源丰富对供者无不良影响病毒感染风险低对人类白细胞抗原（HLA)配型要求低能快速获得移植供者移植后免疫排斥（GVHD）低优点间充质干细胞是属于中胚层的一类多能干细胞，主要存在于结缔组织和器官间质中。骨髓间充质干细胞不仅可分化为造血细胞，还具有分化为肌细胞、肝细胞、成骨细胞、软骨细胞、基质细胞等多种细胞的能力，也可分化为各种神经细胞。脐带间充质干细胞来源广泛，易获得，表达多种胚胎干细胞的特有分子标志，并具有间充质细胞的所有特性。100×间充质干细胞神经干细胞神经干细胞是来源于中枢神经系统的多能干细胞，终身具有自我更新能力，可以被诱导分化为各种类型的成熟神经细胞（神经元、星形胶质细胞和少突胶质细胞）。神经干细胞是神经系统形成和发育的源泉。我们培养的神经干细胞（P1）我们培养的神经干细胞（P4）成体干细胞存在于发育成熟机体器官组织中的具有高度自我更新和增殖潜能的未分化细胞，可以分化成为组成该组织的特定细胞类型，并可横向分化为至少2~3种以上其他的组织细胞。干细胞自我复制产生大量细胞增殖多向分化任何类型体细胞诱导、分化干细胞是一类人体内未充分分化、具有自我复制和多向分化能力的细胞。自我复制多向分化潜能干细胞胚胎干细胞造血干细胞脐带间充质干细胞脐带血干细胞成体干细胞骨髓间充质干细胞神经干细胞干细胞分类诱导多功能干细胞(iPS)胚胎干细胞胚胎干细胞是指当受精卵分裂发育成囊胚时内细胞团的细胞，它具有体外培养无限增殖、自我更新和多向分化的特性。胚胎干细胞是最理想的全能干细胞。Embryonicstemcells(ESCs)derivedfromtheinnercellmassofmammalianblastocyststheabilitytokeepself-renewalwhilemaintainingpluripotencyandtheabilitytodifferentiateintocellsofallthreegermlayersself-renewalplentyofcellsproliferationpluripotencyAnykindofsomaticcellsinductiondifferentiationEmbryonicstemcells(ESCs)HumanEScellsmightbeusedtotreatahostofdiseases,suchasParkinson’sdisease,spinalcordinjury,anddiabetes……InJanuary2009,theUSFoodandDrugAdministration(FDA)approvedthefirstclinicaltrialforusinghumanEScellstotreatpatientswithspinalcordinjuryEmbryonicStemCellsinnercellmassfeederlayerSetupESsystemStoreforever，numerouscopiesEmbryonicstemcells(ESCs)HoweverClinicallimitationethicaldifficultiespotentialtumorformationtissuerejectionOnewaytosolvetheseissuesisthegenerationofpluripotentcellsdirectlyfromthepatients’owncells.ReprogrammingStrategiesReprogramming：SomaticcellscanbereprogrammedtoastateofpluripotencyHypothesisOocytesandEScellscontainfactorsthatcanconfertotipotencyorpluripotencytosomaticcells.thefactorsthatplayimportantrolesinthemaintenanceofEScellidentityalsoplaykeyrolesintheinductionofpluripotencyinsomaticcells.TheThirdReprogrammingStrategiesInductionofPluripotentStemCellsfrommousefibroblatsbydefinedfactors24factorstranscriptionfactors:Oct3/4、Sox2、Nanog......genesupgreatedintumors:Stat3、E-Ras、c-myc、Klf4、β-catenin..............ThefactorsareexpressedinEScells，butnotinsomaticcells.Cell2006introduceeachofthe24candidategenesintomouseembryonicfibroblasts(MEFs)fromFbx15βgeo/βgeoembryos(retroviraltransduction)(abgeocassette(afusionoftheb-galactosidaseandneomycinresistancegenes)intothemouseFbx15genebyhomologousrecombinationFbx15βgeo/βgeoinactivated,noG418-resistantcoloniesStrategytotestcandidatefactorsSchematicdrawingrepresentingthestrategyforreprogrammingJamesThomson’sfactorsYamanaka’sfactorspSin-EF2-Oct4-PuromycinOct4pSin-EF2-Sox4-PuromycinSox2pSin-EF2-Nanog-PuromycinKlf4pSin-EF2-Lin28-Puromycinc-Myc-4day0day3day5dayRetrovirustransductionPlateonFeederESmedium+bFGFiPSgenerationSomaticcells5*104Day1:细胞准备Whenhumanadultfibroblast(HAF)cellshavereached80%confluence,aspiratemedium,washoncewithPBS,covercellswith0.05%trypsin,incubatefor5minat37°C.InactivatetrypsinwithHAFmedium,collectcellsina50-mlconicaltube.Centrifugethecellsat200gatroomtemperaturefor4minanddiscardthesupernatant.从体细胞到干细胞的重编程Day1:细胞准备Resuspendthecellsin1mlHAFmediumanddeterminecellnumberusinghemacytometer.DilutecellsuspensionwithHAFmediumto1104cellsml-1.Transfer1mlHAFsuspension(totally1104cells)perwellof12-wellplate(matrigelcoated).Incubateat37°C,5%CO2,for24h.Day2:病毒感染细胞Aspiratethemedium,replacewith1mlfreshHAFmedium,addpolybrene(final8ug/ml).AddOCT4,SOX2,Nanog,Lin28lentivirus100ul(oneT75flaskproducedvirushasbeenresuspendedin1mlmedium).Incubateat37°C,5%CO2,for24h.Day3:弃病毒Aspiratemedium,washcellsthreetimeswith3mlPBS,add1mliPSmedium.100×初始细胞形态/FibroblastDay4—day21:培养Changemediumeverydayforthreeweeks.Atthisstage,youcouldseedifferentcolonies,whichshowdifferentcellmorphologycomparedwithHAF.Day22:挑取ES样细胞克隆/传代MechanicalpickupsinglecloneandseedtothesinglewellswhichcontainingiMEFs.48hours,changemediumeverytwodays.Oneweekslater,theexactlyiPScolonieswillappear.livestainingbyTra1-60willbepositive.24factors22G418-resistantcolonieswhetherclonespossessedhaveEScell-likemorphologyandproliferationpropertiesdoublingtimeofiPSwasequivalenttothatofEScellsEScellmarkers(RT-PCR)RTminus:negativecontrol,addeverythingexceptreversetranscriptaseThepromotersofFbx15andNanog(Bisulfitegenomicsequencing)blackpointsindicatemethylatedCpGs;whitepointsindicateunmethylatedCpGdinucleotideswhichofthe24candidateswerecritical/importantmethod:theeffectofwithdrawal/deletionofindividualfactorsfrom24genesontheformationofG418-resistantcolonies.•result:identified10factors(3,4,5,11,14,15,18,20,21,and22)Combinationofthese10genesaloneproducedmoreEScell-likecoloniesthantransductionofall24genesdidEffectoftheremovalofindividualfactorsfromtheselected10factorsontheformationofG418-resistantcoloniesCombinationof4factorsidentify4factorsCombinationof3factorsCombinationof2factorsFourfactorsSomaticcellsc-MycOct3/4Klf4Sox2InducedInducedPlur