10.多能干細(xì)胞與重編程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體外受精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絲氨酸蘇氨酸蛋白激酶,singlecellOKTrypsin胰蛋白酶-singlecellsAccurase-singlecellsDispase分散酶-clumpsCollagenase膠原酶:feedercellsMechanicalpassageprocedureofhumanEScells.Typicalundifferentiatedcolonieswereseparatedfromfeederlayersusingglassneedles.Thencolonieswerecuthorizontally,andthenverticallytoformuniformclumpsandtransferredontonewlypreparedfeederlayers.Feeder-freeculturehEScellsFeedercellsderivedfromhumantissue:Fetalmuscle,fetalskinAdultfallopiantubuleepithelialcellsForeskinfibroblastAdultmarrowcellsAdultlungUterineendometriumhEScellderivedfibroblast胎兒：肌肉皮膚成人：上皮，子宮內(nèi)膜，包皮纖維原細(xì)胞，非胚胎干細(xì)胞來源：纖維原細(xì)胞NatureBiotech2006DerivationofhESCsindefinedconditionsTeSR1mediumcontaining:bFGF,LiCl氯化鋰,GABAγ氨基丁酸,pipecolicacid哌丁酸,andTGFb轉(zhuǎn)化生長因子，成纖維細(xì)胞生長因子

-sufficienttosupportfeeder-independenthESCculture.Thehumanmatrix-coatedplateswerecomposedof:

hCollagenIV膠原蛋白4,hVitronectin玻璃黏連蛋白,hFibronectin纖維粘連蛋白,andhLaminin層粘連蛋白

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擬胚體(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嵌合體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:功能細(xì)胞，動(dòng)物模型，安全問題，免疫排斥★Functionalcellsimmaturepancreaticendocrinecells未成熟胰腺內(nèi)分泌細(xì)胞，造血干細(xì)胞在卵黃囊中是相似的，;Hematopoieticstemcells(HSCs)resemblingthoseoftheyolksac;hepatocyteshavinganembryonicidentity.Longcultureinvitro(between45daysandupto3months).★Animalmodelsforspecificdiseasesandrobustinvitrofunctionaltests動(dòng)物模型對(duì)于特定疾病★Safetyissues

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

Solutions:促進(jìn)分化流程，保證多能干細(xì)胞不參與，流式純化特定標(biāo)志，包含選擇性基因★Improvedifferentiationprotocolsforgenerationofhomogenouspopulationoffullydifferentiatedcells;★

Guaranteetheabsenceofpluripotentcellsintransplantedcells:

◆purificationbyFACSusingspecificcell-surfacemakers;

◆

hESCscanbeengineeredtocontainaselectiongeneContents★

Concept

★

EmbryonicStemCells

★Reprogramming(重編程）

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

孤雌發(fā)育2007ByLifelineCellTechnology,USAandScientificCenterforObstetrics,Gynecology,andPerinatology,RussiaHumanEmbryonicStemCellsDerivedbySomaticCellNuclearTransfer1DivisionofReproductive&DevelopmentalSciences,OregonNationalPrimateResearchCenter,OregonHealth&ScienceUniversityCELL,2013Received:April30,2013Revised:May3,2013Accepted:May3,2013Published:May15,2013InvitroactivationoocyteoocyteDifferentiatedsomaticcellsPluripotentEmbryoniccells?ProgramReprogramInducedPluripotentStemCellsiPSCells(誘導(dǎo)性多能干細(xì)胞）成纖維細(xì)胞中轉(zhuǎn)入關(guān)鍵基因，得到iPS細(xì)胞Oct4Sox2Klf4c-Myc小鼠成纖維細(xì)胞TheNobelPrizeinPhysiologyorMedicine2012Cell126,1-14,August252006Cell,誘導(dǎo)性多能干細(xì)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鐮狀細(xì)胞性貧血transductionstrategyandfactorselectiondonorcellresourceselectionandcharacterizationmethodsapplicationofiPScellsKeystepsofestablishingiPScelllines重編程來源細(xì)胞表皮成纖維細(xì)胞：細(xì)胞數(shù)量多，重編程技術(shù)成熟，難以采集。外周血單核細(xì)胞：便于采集（醫(yī)院），不易污染。腎小管上皮細(xì)胞：便于采集（新鮮中段尿液中收集，對(duì)提供者不造成損傷）。附加體質(zhì)粒（episomalplasmid）

重編程技術(shù)病毒轉(zhuǎn)染：造成插入突變蛋白質(zhì)誘導(dǎo)：低效，操作復(fù)雜小分子誘導(dǎo)：目前尚不適用于人體細(xì)胞附加體質(zhì)粒轉(zhuǎn)染：相對(duì)高效，易于操作，不整合外源基因，適用于多種體細(xì)胞（表皮成纖維細(xì)胞、外周血單核細(xì)胞、腎小管上皮細(xì)胞等）Sendai病毒附加體質(zhì)粒（episomalplasmid）重編程技術(shù)皮膚成纖維細(xì)胞來源的iPSC系A(chǔ)pplicationofiPScells★

Cellreplacementtherapy★

Studydiseases★

ScreenfordrugsEssentialstep:

DifferentiationiPS細(xì)胞誕生的重要意義解決免疫排斥問題；避開ESC建系的倫理問題；疾病發(fā)生的個(gè)體特異性；治療的個(gè)體化。分化全能重編程建立患者特異的疾病模型研究疾病發(fā)生機(jī)制藥物篩選和個(gè)體性治療基因改造正常細(xì)胞病變細(xì)胞自我更新非遺傳性疾病遺傳性疾病細(xì)胞替代治療DiseaseiPScelllinesSpinalmuscularatrophy(Nature,2008)Amyotrophiclateralsclerosis(ALS)(Science2008)Parkison’sdiseasepatient(Cell,2009)Type1diabetes(PNAS,2009)Fanconianemia(Nature,2009)Dysautonomia(Nature,2009)家族性自主神經(jīng)異常Amyotrophiclateralsclerosis(ALS,Science,2009)Klinefeltersyndrome,47,XXYanditsvariants,isthemostcommonchromosomalaberrationamongmen,withestimatedfrequencyof1:500amongnewborns.MenwithKlinefeltersyndromepresentwithsequelsofhor