10.多能干细胞与重编程124

PluripotentStemCells

andReprogrammingYingJinInstituteofHealthSciencesShanghaiInstitutesofBiologicalSciences,CAS/ShanghaiJiaoTongUniversitySchoolofMedicineyjin@Contents★

Concept

★EmbryonicStemCells

★Reprogramming

Contents★Concept

★

EmbryonicStemCells

★Reprogramming

Whatarestemcells?◆

Reproduceitself(self-renewal);◆Generateoneormoredifferentiateddescendantcelltypes;◆Functionallyreconstituteanorganorawholeorganism.SymmetricdivisionAsymmetricdivisionSomaticCellBlastocystImplantationEmbryonicdevelopmentPostnataldevelopmentAdultThesecellswilldifferfromoneanotherinsignificantways:6●

Methylation●Regulatorygeneexpression●X-inactivation●Positionalinformation●Imprintingstatus●MarkerexpressiontotipotentpluripotentmultipotentorunipotentEmbryonicstemcells,ESCellsSomaticstemcellsFetaltissuestemcells

AdulttissuestemcellsInducedpluripotentstemcells(iPScells)Themoststudiedstemcellsinclude:Contents★

Concept

★EmbryonicStemCells

★Reprogramming

Embryonicstemcells:◆

Derivationandculture◆

Characterization◆

DifferentiationEmbryonicstemcells(ESCs)Derivedfrominnercellmass(ICM)ofblastocyst.ThefirsthumanESClinewasestablishedin1998byJamesA.Thomson.

Science

282:1145,1998ICMAhumanEScellcolonyonfeederlayerJamesThomson,Ph.D.StemCellandRegenerativeMedicineCenterUniversityofWisconsinSirMartinJohnEvansisaBritishscientistwho,withMatthewKaufman,wasthefirsttoculturemiceembryonicstemcellsandcultivatetheminalaboratoryin1981.Heisalsoknown,alongwithMarioCapecchiandOliverSmithies,forhisworkinthedevelopmentoftheknockoutmouseandtherelatedtechnologyofgenetargeting,amethodofusingembryonicstemcellstocreatespecificgenemodificationsinmice.In2007,thethreesharedtheNobelPrizeinPhysiologyorMedicineinrecognitionoftheirdiscoveryandcontributiontotheeffortstodevelopnewtreatmentsforillnessesinhumans.EmbryonicGermCells(EGCs)Derivedfromfetaltissue,culturedfromtheprimordialgermcells(PGC)ofthegonadalridgeoffetus.PGCsareembryonicprecursorsofthegametes.ThefirsthumanEGcelllinesweregeneratedbyDr.JohnD.Gearhartin1998.Proc.Natl.Acad.Sci.USA95:13726,1998AhumanEGCColonyFeederlayerJohnD.Gearhart,Ph.D.StemCellBiologyProgramJohnsHopkinesUniversitySchoolofMedicinePerelman

SchoolofMedicine

attheUniversityofPennsylvaniaMouseearlydevelopmentE3.5MouseESCderivationFlushembryos(day3.5)fromtheuterinehorns;PlacethemindividuallyontofeederlayerinEScellculturemedium.Theembryoshatchfromthezonapellucidaandattachtofeederlayerbymigrationofthetrophoblastcells.WhenICM–derivedclumphasenlargedenough,dislodgeitfromtheunderlyingsheetoftrophoblastcellsbydrawnpasteurpipette.UsethepipettetodisaggregateEScellclumpintosmalleraggregates.Transferthesmallclumpintoafreshdishcontainingfeederlayer.Generally,after2days,primarycoloniesofcellswillvisible.Discretecoloniesofastemcellmorphologyareselectivelyremovedafterupto7-8daysofcultureandthendissociatedinmicrodropsoftrypsin/EDTA,andpassagedintofreshfeederwell(keepthecelldensityhigh).SmallnestsofEScellsappearwithin2-3daysofsubcultureandcanbeexpanded3-5dayslaterbytrypsinizingthewholewellandtransferringitscontentsontoalargerfeederdish.AnestablishedEScelllinerequirecarefulsubcultureat2-3dayintervals.MouseEScellmediumissupplementedwith

serum

and

leukemiainhibitoryfactor(LIF).◆FeedercellsandserumFeedercellsprovideleukemiainhibitoryfactor(LIF)Serumprovidesbonemorphogeneticproteins(BMPs)◆Feeder-andserum-freeLIFandBMP◆

LIF-andBMP-free(2i)Thefibroblastgrowthfactor/extracellularsignal-relatedkinase(Fgf/Erk1/2)inhibitorGlycogensynthasekinase3(GSK3)inhibitorCultureofmouseESCsSun,etal.Sun,etal.HumanearlydevelopmentInvitrodevelopmentofIVF体外受精frozenembryos

HumanearlyembryosThephotoisprovidedbyprofessorFengofRplementAnti-humanserumantibodyImmunosurgeryABCEScellsBlastocystReubinoffetal.,2000MichaelAmitetal,JAnat.200:225,2002+Ab+CDerivationofthreenewChinesehumanEScelllines.(A)MorphologyofasurplushumanblastocystfromtheIVFclinics;(B)PrimaryICMoutgrowth(Passage0);(C)Typicalundifferentiatedcoloniespassagedbymechanicalsplitting(Passage5);(D)HighermagnificationofahumanESCcolonypassagedbymechanicalsplitting(Passage9).MechanicalremovaloftrophectodermP0P5P9Lietal.(2010)InvitroCell&Dev.Biol.

–Animal46:186-191.CultureofhumanESCsFeedercells(producingActivin激活素A):basicfibroblastgrowthfactor(bFGF)Passcells,notsinglecells

(enzymes,ormechanicalsplitorcombinationofenzymeandmanualsplitting)InthepresenceofRockinhibitor丝氨酸苏氨酸蛋白激酶,singlecellOKTrypsin胰蛋白酶-singlecellsAccurase-singlecellsDispase分散酶-clumpsCollagenase胶原酶:feedercellsMechanicalpassageprocedureofhumanEScells.Typicalundifferentiatedcolonieswereseparatedfromfeederlayersusingglassneedles.Thencolonieswerecuthorizontally,andthenverticallytoformuniformclumpsandtransferredontonewlypreparedfeederlayers.Feeder-freeculturehEScellsFeedercellsderivedfromhumantissue:Fetalmuscle,fetalskinAdultfallopiantubuleepithelialcellsForeskinfibroblastAdultmarrowcellsAdultlungUterineendometriumhEScellderivedfibroblast胎儿：肌肉皮肤成人：上皮，子宫内膜，包皮纤维原细胞，非胚胎干细胞来源：纤维原细胞NatureBiotech2006DerivationofhESCsindefinedconditionsTeSR1mediumcontaining:bFGF,LiCl氯化锂,GABAγ氨基丁酸,pipecolicacid哌丁酸,andTGFb转化生长因子，成纤维细胞生长因子

-sufficienttosupportfeeder-independenthESCculture.Thehumanmatrix-coatedplateswerecomposedof:

hCollagenIV胶原蛋白4,hVitronectin玻璃黏连蛋白,hFibronectin纤维粘连蛋白,andhLaminin层粘连蛋白

JamesAThomson,NatureBiotechnology20062006EstablishmentofhumanfeedercelllineswithoutanimalcomponentsBasalCultureMedium/HumanSerum/HumanGelatin/HumanTrypsinSHhES6SHhES7SHhES8DerivationofXeno-freeHumanESCellLinesLietal.,unpublished3Linesfrom32EmbryosBasicCharacteristicsofESCells1.Karyotypicallynormal;2.Proliferateinvitroindefinitelyunderwell-definedcultureconditions;3.Mostofcellsrecoverafterfreezingandthawing;4.Differentiateintoavarietyofcelltypesinvitroandinvivo.Science300:913,2003Reproduceitself(self-renewal)＊

Indefinitelyproliferation(>70passages);＊Karyotypicanalysis;(GlobalSNPScan)＊MarkersforundifferentiatedEScells;＊Telomeraseactivity;＊Colonyformingassay.Nature.2007Nov22;450(7169):497-502.Science300:913,2003MolecularmarkersofundifferentiatedhEScellsSSEA-4TRA-1-60

SSEA-1Oct-4AKPTRA-1-81Sun,etal.(2006)HumanMolecularGeneticsTra-1-81Tra-1-60SSEA-4SSEA-1Oct4APKbufferfeederPositivecontrolheatHES（P20）heatTelomeraseactivityinhEScellsSun,etal.UndifferentiatedEScolony

semi-differentiatedEScolony

differentiatedEScolony(AKPpositive)(AKPnegative)(AKPmixture)OverexpressionofStk40couldreduceEScellself-renewalabilityColonyformingassayonstk40overexpressedEScellsStk40overexpressedandcontrolcellswereplatedon100mmdishataverylowdensityandculturedforabout1weeks.TheESCcolonieswerefixed,stainedwithAKP(alkalinephosphatase)andcountedaccordingthreedifferenttypes.Ivanova,Netal.Nature442:533,2006AcompetitionstrategyRegulationofembryonicstemcellself-renewalandpluripotencybyleukaemiainhibitoryfactorHiroyukiHirai,PeterKarian,andNobuakiKikyo1Pluripotency

＊EmbryoidBody(EB)formation,invitro;＊Teratomaformation,invivo;＊Developmentalpotentialinchimericoffspring.EmbryoidBody拟胚体(EB)Todate,thebest-studiedmodeofEScelldifferentiationistheformationinsuspensioncultureofmulti-cellularaggregates聚集calledEBs.Withintheseaggregates,complexinteractionsbetweenheterologouscelltypesresultintheinductionofdifferentiationofstemcellstoderivativesofallthreeembryonicgermlayers.PlatingoftheEBscausesfurtherdifferentiationandoutgrowth.

A.G.Smith,2003SimpleEBCysticEBAEBbulgesoutOutgrowthfromanEBSun,etal.hESEBd3EBd13EBd18EBofhESCells

InvitrodifferentiationassayPluripotency

＊EmbryoidBody(EB)formation,invitro;＊Teratomaformation,invivo;＊Developmentalpotentialinchimaericoffspring.Pluripotency

＊EmbryoidBody(EB)formation,invitro;＊Teratomaformation,invivo;＊Developmentalpotentialinchimeric嵌合体offspring.

DeterminationofdevelopmentalpotentialofEScellsEScellscontributetodifferentcelltypes,includinggermlinecells,inchimericoffspring.Embryonicstemcelltrialsformaculardegeneration:apreliminaryreportTheLancet(2012)StevenDSchwartz,Jean-PierreHubschman,GadHeilwell,ValentinaFranco-Cardenas,CarolynKPan,RosaleenMOstrick,EdmundMickunas,RogerGay,IrinaKlimanskaya,RobertLanzaInterpretationThehESC-derivedRPEcellsshowednosignsofhyperproliferation,tumorigenicity,ectopictissueformation,orapparentrejectionafter4months.Thefuturetherapeuticgoalwillbetotreatpatientsearlierinthediseaseprocesses,potentiallyincreasingthelikelihoodofphotoreceptorandcentralvisualrescue.LANCET,201418patients22monthfollowupVisualacuityImprovedin10Remainedsamein7Decreasedin1StemCellReportsMay12,20154Asianpatients1yearfollowupVisualacuityImprovedin3Remainedstablein1ChallengesofapplicationofhESC-derivedcells:功能细胞，动物模型，安全问题，免疫排斥★Functionalcellsimmaturepancreaticendocrinecells未成熟胰腺内分泌细胞，造血干细胞在卵黄囊中是相似的，;Hematopoieticstemcells(HSCs)resemblingthoseoftheyolksac;hepatocyteshavinganembryonicidentity.Longcultureinvitro(between45daysandupto3months).★Animalmodelsforspecificdiseasesandrobustinvitrofunctionaltests动物模型对于特定疾病★Safetyissues

animalcomponents:mousefeedercells,fetalbovineSerumtumorformation:undifferentiatedorpartiallydifferentiatedESCs,heterogeneouspopulations,karyotypicinstability.★Immuno-rejection

Solutions:促进分化流程，保证多能干细胞不参与，流式纯化特定标志，包含选择性基因★Improvedifferentiationprotocolsforgenerationofhomogenouspopulationoffullydifferentiatedcells;★

Guaranteetheabsenceofpluripotentcellsintransplantedcells:

◆purificationbyFACSusingspecificcell-surfacemakers;

◆

hESCscanbeengineeredtocontainaselectiongeneContents★

Concept

★

EmbryonicStemCells

★Reprogramming(重编程）

TheNobelPrizeinPhysiologyorMedicine2012SirJohnB.GurdonSirJohnB.GurdonBorn:1933,Dippenhall,UnitedKingdomAffiliationatthetimeoftheaward:GurdonInstitute,Cambridge,UnitedKingdomPrizemotivation:"forthediscoverythatmaturecellscanbereprogrammedtobecomepluripotent"AclassicexperimentItwaswhilehewasatOxford'sDepartmentofZoologythathecarriedoutaclassicexperimentpublishedin1962.Hehypothesizedthatthegenomeofamaturecellmightstillcontainalltheinformationneededtodriveitsdevelopmentintoallthedifferentcelltypesofanorganism.Hereplacedtheimmaturecellnucleusinaneggcellofafrogwiththenucleusfromamatureintestinalcell.Thismodifiedeggcelldevelopedintoanormaltadpole.TheDNAofthematurecellstillhadalltheinformationneededtodevelopallcellsinthefrog.Cloning'becameareality’ProfessorChrisGrahamofOxfordUniversity'sDepartmentofZoology,oneofSirJohn'sfirststudentswhoworkedwithhimatOxfordinthe1960s,says:'Heshowedthatyoucouldtakeseveralnucleifromoneindividualandproducegenetically-identicalanimals–thatwashisgreatachievement.PeoplehadtalkedaboutcloningagooddealbutwithJohnGurdon’sworkitbecameareality.1997NatureDollyisderivedfromamammaryglandcellTherapeuticCloningReproductiveCloningCibelli,2003Science33:1669,2004Science2004核移植

孤雌发育2007ByLifelineCellTechnology,USAandScientificCenterforObstetrics,Gynecology,andPerinatology,RussiaHumanEmbryonicStemCellsDerivedbySomaticCellNuclearTransfer1DivisionofReproductive&DevelopmentalSciences,OregonNationalPrimateResearchCenter,OregonHealth&ScienceUniversityCELL,2013Received:April30,2013Revised:May3,2013Accepted:May3,2013Published:May15,2013InvitroactivationoocyteoocyteDifferentiatedsomaticcellsPluripotentEmbryoniccells?ProgramReprogramInducedPluripotentStemCellsiPSCells(诱导性多能干细胞）成纤维细胞中转入关键基因，得到iPS细胞Oct4Sox2Klf4c-Myc小鼠成纤维细胞TheNobelPrizeinPhysiologyorMedicine2012Cell126,1-14,August252006Cell,诱导性多能干细胞

Oct-4Sox2C-MycKlf4EBsanddifferentiationTeratomasectionImmunostaininginteratomaE13.5E7.5ContributionofiPScellstomouseembryonicdevelopment2/183/22Retroinfection;Nochimaericmicewereborn;Lowefficiency;GeneexpressionandepigeneticallydifferentfromEScellsThefirstgenerationofmouseiPScellsNature2007JulyYearof2007GenerationofOct4-andNanog-selectediPScells.Oct4locus6weeksChimericmouseb,c:twolivepupsafter2Nblastocystinjection;d:iPSembryosbyinjectioninto4Nblastocyst;E12.5e:E14.5embryofrom4NTetraploidBlastocystComplementationFromShaorongGaoinTongjiUniversityOct4-andNanog-selection;Viablechimaeras;Contributetogermline;Generatelivelate-termembryoswheninjectedintotetraploidblastocysts;ThebiologicalpotencyandepigeneticstateareindistinguishablefromthoseofEScellsThesecondgenerationofmouseiPScellsNature2007JulyChimericmaleXC57BL/6femaleTumorformationbyc-Mycreactivation121F1mice(8-41weeks)fromNanog-iPS20D17cellline;24diedorwerekilledduetoillness;13miceidentifiednecktumors,5micewithothertumors;20%Inthesetumors,retroviralexpressionofc-Mycisreactivated.NatureBiotechnology,2007OctoberNat.Biotech,2008129SvJae/C57B6/LE14.5ChimerasTheefficiencyforderivingiPScellsfromthenumberofpickedcolonieswasabout45%.Theoverallefficiencyofreprogrammingwasabout0.5%5-10timeshigherthanwhathasbeenachievedwiththedrug-selectionapproaches.ThethirdgenerationofmouseiPScellsYear2009Bygroupsof1.JamesThomsonpublishedinScience(Oct4,Sox2,NanogandLin28)withlentivirus

2.ShinyaYamanakapublishedinNatureBiotec.(Oct4,Sox2,Klf4andc-Myc)withretrovirus

3.GeorgeDaleypublishedinNature(Oct4,Sox2,Klf4,c-Myc,SV40largeT,hTERT)Yearof2007TreatmentofSicklecellanemiamousemodelwithiPScellsGeneratedfromautologousskinbyRudolfJaenischgroupScience

2007December镰状细胞性贫血transductionstrategyandfactorselectiondonorcellresourceselectionandcharacterizationmethodsapplicationofiPScellsKeystepsofestablishingiPScelllines重编程来源细胞表皮成纤维细胞：细胞数量多，重编程技术成熟，难以采集。外周血单核细胞：便于采集（医院），不易污染。肾小管上皮细胞：便于采集（新鲜中段尿液中收集，对提供者不造成损伤）。附加体质粒（episomalplasmid）

重编程技术病毒转染：造成插入突变蛋白质诱导：低效，操作复杂小分子诱导：目前尚不适用于人体细胞附加体质粒转染：相对高效，易于操作，不整合外源基因，适用于多种体细胞（表皮成纤维细胞、外周血单核细胞、肾小管上皮细胞等）Sendai病毒附加体质粒（episomalplasmid）重编程技术皮肤成纤维细胞来源的iPSC系ApplicationofiPScells★

Cellreplacementtherapy★

Studydiseases★

ScreenfordrugsEssentialstep:

DifferentiationiPS细胞诞生的重要意义解决免疫排斥问题；避开ESC建系的伦理问题；疾病发生的个体特异性；治疗的个体化。分化全能重编程建立患者特异的疾病模型研究疾病发生机制药物筛选和个体性治疗基因改造正常细胞病变细胞自我更新非遗传性疾病遗传性疾病细胞替代治疗DiseaseiPScelllinesSpinalmuscularatrophy(Nature,2008)Amyotrophiclateralsclerosis(ALS)(Science2008)Parkison’sdiseasepatient(Cell,2009)Type1diabetes(PNAS,2009)Fanconianemia(Nature,2009)Dysautonomia(Nature,2009)家族性自主神经异常Amyotrophiclateralsclerosis(ALS,Science,2009)Klinefeltersyndrome,47,XXYanditsvariants,isthemostcommonchromosomalaberrationamongmen,withestimatedfrequencyof1:500amongnewborns.MenwithKlinefeltersyndromepresentwithsequelsofhor