10.多能干細胞與重編程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，包皮纖維原細胞，非胚胎干細胞來源：纖維原細胞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:功能細胞，動物模型，安全問題，免疫排斥★Functionalcellsimmaturepancreaticendocrinecells未成熟胰腺內(nèi)分泌細胞，造血干細胞在卵黃囊中是相似的，;Hematopoieticstemcells(HSCs)resemblingthoseoftheyolksac;hepatocyteshavinganembryonicidentity.Longcultureinvitro(between45daysandupto3months).★Animalmodelsforspecificdiseasesandrobustinvitrofunctionaltests動物模型對于特定疾病★Safetyissues

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

Solutions:促進分化流程，保證多能干細胞不參與，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)性多能干細胞）成纖維細胞中轉(zhuǎn)入關(guān)鍵基因，得到iPS細胞Oct4Sox2Klf4c-Myc小鼠成纖維細胞TheNobelPrizeinPhysiologyorMedicine2012Cell126,1-14,August252006Cell,誘導(dǎo)性多能干細胞

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鐮狀細胞性貧血transductionstrategyandfactorselectiondonorcellresourceselectionandcharacterizationmethodsapplicationofiPScellsKeystepsofestablishingiPScelllines重編程來源細胞表皮成纖維細胞：細胞數(shù)量多，重編程技術(shù)成熟，難以采集。外周血單核細胞：便于采集（醫(yī)院），不易污染。腎小管上皮細胞：便于采集（新鮮中段尿液中收集，對提供者不造成損傷）。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胞附加體質(zhì)粒轉(zhuǎn)染：相對高效，易于操作，不整合外源基因，適用于多種體細胞（表皮成纖維細胞、外周血單核細胞、腎小管上皮細胞等）Sendai病毒附加體質(zhì)粒（episomalplasmid）重編程技術(shù)皮膚成纖維細胞來源的iPSC系A(chǔ)pplicationofiPScells★

Cellreplacementtherapy★

Studydiseases★

ScreenfordrugsEssentialstep:

DifferentiationiPS細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)生的個體特異性；治療的個體化。分化全能重編程建立患者特異的疾病模型研究疾病發(fā)生機制藥物篩選和個體性治療基因改造正常細胞病變細胞自我更新非遺傳性疾病遺傳性疾病細胞替代治療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