stem cell biology 2017 - washington university in st. louis · developmental potential of ipscs is...
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StemCellBiology
JimHuettner11/21/2017
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suggestedreadings• SolterD.(2006)Fromteratocarcinomastoembryonicstemcellsandbeyond:
ahistoryofembryonicstemcellresearch.NatRevGenet.7:319-27.• BuganimY,FaddahDA,JaenischR.Mechanismsandmodelsofsomaticcell
reprogramming.NatRevGenet.2013Jun;14(6):427-39.• BuganimY,MarkoulakiS,vanWietmarschenN,etal.(2014)The
developmentalpotentialofiPSCsisgreatlyinfluencedbyreprogrammingfactorselection.CellStemCell.15:295-309..
• DeLosAngelesetal.(DaleyGQ).(2015)Hallmarksofpluripotency.Nature525:469-478.
• FoxIJ,DaleyGQ,GoldmanSA,HuardJ,KampTJ,TruccoM.(2014)Stemcelltherapy.Useofdifferentiatedpluripotentstemcellsasreplacementtherapyfortreatingdisease.Science345(6199):1247391.
• SchwartzSD,RegilloCD,LamBLetal.,(2014)Humanembryonicstemcell-derivedretinalpigmentepitheliuminpatientswithage-relatedmaculardegenerationandStargardt’smaculardystrophy:follow-upoftwoopen-labelphase1/2studies.Lancet.e-pubOctober15.
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StemCells:definition
• SelfRenewal-undifferentiatedcellsthatcandividerepeatedlywhilemaintainingtheirundifferentiatedstate.
• Pluripotency–abilitytodifferentiateintoavarietyofdifferentcelltypes
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Donovan and Gearhart, 2001
Invitrodifferentiation:• Cell/tissuereplacementtherapies• Humanmodelsystemsofdiseaseanddevelopment
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TypesofStemCellsEmbryonic–fromtheinnercellmassofpre-implantationembryos,priortoformationofthe3germlayers(ectoderm,mesoderm,endoderm)
Somatic–undifferentiatedcellsfoundinspecificlocationsin“mature”tissues
iPScells–inducedpluripotentstemcellsgeneratedbyreprogrammingdifferentiatedcells(orcellnuclei,i.e.therapeuticcloning)
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Potency
• Totipotent–abletogenerateeverycelltypeincludingextraembryonictissues
• Pluripotent–abletogeneratecellsfromallthreeembryonicgermlayers
• Multipotent–abletogenerateavarietyofcellsfromaparticularsomaticstructure
• Unipotent–onlygenerateonecelltype
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TimeLine
fertilization gastrulation
totipotent pluripotent multipotent
somaticdifferentiation
zygote morula blastocyst
implantation
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http://stemcells.nih.gov/info/scireport/pages/chapter1.aspx
Innercellmass
Epiblast:embryoHypoblast:yolksac
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EarlyEmbryology
http://stemcells.nih.gov/info/scireport/pages/appendixa.aspx
Human Mouse
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makingaknockoutmouse
http://en.wikipedia.org/wiki/Knockout_mouse
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FirstIsolationofEScellsMouse:
EvansMJ,KaufmanMH.(1981)Establishmentincultureofpluripotentialcellsfrommouseembryos.Nature.292:154-6.MartinGR.(1981)Isolationofapluripotentcelllinefromearlymouseembryosculturedinmediumconditionedbyteratocarcinomastemcells.P.N.A.S.USA.78:7634-8.
Human:ThomsonJA,Itskovitz-EldorJ,ShapiroSS,WaknitzMA,SwiergielJJ,MarshallVS,JonesJM.(1998)Embryonicstemcelllinesderivedfromhumanblastocysts.Science.282:1145-7.
GeneticandDevelopmentalNormality(140cycles):SudaY,SuzukiM,IkawaY,AizawaS.(1987)Mouseembryonicstemcellsexhibitindefiniteproliferativepotential.JCellPhysiol.133:197-201.
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Pluripotencymarkers• Stage-specificantigens:Anti-SSEA3and4recognizeglobo-seriesgangliosides
• Tra1-60andTra1-81:keratinsulfatesurfaceantigens
• Oct3/4,Sox2,Nanog–transcriptionfactorsinvolvedwithmaintainingpluripotency
• Normalkaryotype,andpre-X-inactivation?
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TwotypesofEScells?
• Blastocystchimera(+)• Highcloningefficiency• Shortdoublingtime• XaXa• DistalOct4enhancer• HighNanog,Klf2/4,Rex1
• Blastocystchimera(-)• Lowcloningefficiency• Longdoublingtime• XaXi• ProximalOct4enhancer• LowNanog,Klf2/4,Rex1
“Naïve”(ICM-like)
“Primed”(Epi-SC)
Bothtypescanselfrenewandgiverisetocellsfromall3germlayersinteratomasorfollowinginvitrodifferentiation
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maintenanceofpluripotency-1• Initialworkdoneonmouseembryonicfibroblast(MEF)
feedercellsinmediumsupplementedwithanimalserum
• OnefactorproducedbyfeedercellsthathelpsmaintainmouseEScellsintheirundifferentiatedstateisleukemiainhibitoryfactor(LIF)whichactivatestheStat3pathway.
• GoodManufacturingProcess(GMP)–guidelinesforisolationandpropagationofcellsthatwouldbeusedforreplacementtherapy.Ideallytheywouldbexeno-free.
• Thepushforxeno-freeconditions,combinedwithworktooptimizereprogramming,hasdrivenscreeningoffactorsthatcanenableserum-freemaintenanceofpluripotency
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maintenanceofpluripotency-2
• LIF-Stat3• BMP4-Smad1/5• Wnt(GSK-3inhibitors)• IGF
• TGFβ/activin-Smad2/3• FGF2• ERK1/2
• TGFβ/activin–Smad2/3• FGF2• ERK1/2• Wnt(GSK-3inhibitors)• IGF
• BMP4–Smad1/5
“naïve” “primed”PositiveRegulators
NegativeRegulators
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maintenanceofpluripotency-3
• LIF-Stat3• GSK-3inhibitors(Wnt)• ERK1/2inhibitors
(2i/LIF)
• LIF–Stat3• GSK-3inhibitors(Wnt)• ERK1/2inhibitors• PKCinhibitor• p38inhibitor• JNKinhibitor• ROCKinhibitor• FGF2• TGF-β1
Mouse(2008) Human(2013)
“CurrentStandard”Conditions
Forserumfreegrowthalsoneed:Insulin,transferrin,progesterone,putrescine,selenium
butsee:Takashimaetal.,Cell158:1254-1269(2014)
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Attemptstodefine“Stemness”• Earlymicroarrayprofilesshowedsurprisinglackofagreement
(limitationsinmicroarraytechnologyorplatform/lab/primarycellorcelllinedifferences)(Science302:393,2003)
• RelativelyweakoverlapbetweenmouseandhumanEScells(~25%)comparedto>90%typicalfordifferentiatedtissues.(StemCellReviews1:111-118,2005)butthismayreflectconfusionbetweennaïveandprimedEScells
Recentevidencefortotipotentmousestemcells:
Blockadeofpro-differentiationsignalingpathwayscanmaintaincellsatapproximatelythe8-cellmorulastageordrivenaïvemEScellstoa“pre-naïve”8-celllikestateSeeYangetal.(2017)Establishmentofmouseexpandedpotentialstemcells.Nature550:393-397.
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Invitrodifferentiation• DifferentcultureconditionsalterthefateofEScellsinvitro• Protocolsexistforallthreegermlayers
• Many,butnotall,protocolsinvolveaggregationofEScellsin“embryoidbodies”
• Mostprotocolsdonotyieldasingletypeofcell
• Selectionstepscanhelptoremoveundesiredcelltypes
• Needtoask:Howfar?&Howfaithful?
Pancreaticβcells:• PagliucaFW,etal.(2014)GenerationofFunctionalHumanPancreaticβCellsInVitro.Cell.Oct,159:428-39.
• RezaniaA,etal.(2014)Reversalofdiabeteswithinsulin-producingcellsderivedinvitrofromhumanpluripotentstemcells.NatBiotechnol.Nov,32:1121-33.
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EScells → neurons• pluripotent• functionallyimmortal• genetically&developmentallynormal
• postmitotic• polarized• excitable• heterogeneous
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4 d 4 d 4 d 6 d
Serum Free Medium
+ RA
ESC
SFD
SF+RA
Kimetal.,DevelopmentalBiology328:456-471,2009
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β-tubulin nestin Hoechst
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GABA β-tubulin Hoechst GAP43 MAP2 Hoechst
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voltage-gatedNa+andK+currents
DevelopmentalBiology168:342-357,1995
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JournalofNeuroscience16:1056-65,1996
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DevelopmentalBiology328:456-471,2009
HierarchicalclusteringbyfrequencyofGeneOntologyterms
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Reprogramming
• SCNT–somaticcellnucleartransfer(reproductiveandtherapeuticcloning)–deterministicandfairlyrapid
• iPS–inducedpluripotentstemcells–slowandstochastic(untilrecently)
• Transdifferentiation–conversionofoneterminallydifferentiatedcelltypeintoanotherwithoutde-differentiationtoanimmaturephenotype.Mustruleoutcellfusionorotherexplanations.
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Reprogramming:somaticcellnucleartransfer
http://www.biotechnologyonline.gov.au/images/contentpages/scnt.gif
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ReprogrammingFirsts:SCNTFrog:
GurdonJB.(1962)Adultfrogsderivedfromthenucleiofsinglesomaticcells.DevBiol.4:256-73.
Sheep:CampbellKH,McWhirJ,RitchieWA,WilmutI.(1996)Sheepclonedbynucleartransferfromaculturedcellline.Nature.380:64-6.
Human:(2004)–ClaimofhumanSCNTthatprovedtobeunfounded!TachibanaM,etal.(2013)Humanembryonicstemcellsderivedbysomaticcellnucleartransfer.Cell.153:1228-38.
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ReprogrammingFirsts:iPScellsMouse:
TakahashiK,YamanakaS.(2006)Inductionofpluripotentstemcellsfrommouseembryonicandadultfibroblastculturesbydefinedfactors.Cell.126:663-76
Human:TakahashiK,TanabeK,OhnukiM,NaritaM,IchisakaT,TomodaK,YamanakaS.(2007)Inductionofpluripotentstemcellsfromadulthumanfibroblastsbydefinedfactors.Cell.131:861-72.YuJ,VodyanikMA,Smuga-OttoK,etal.,(2007)Inducedpluripotentstemcelllinesderivedfromhumansomaticcells.Science.318:1917-20.
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GeneratingiPScells
• Expresstranscriptionfactors:Oct3/4,Sox2,Klf4andc-MycOROct3/4,Sox2,NanogandLin28
• Initialde-differentiationandproliferation(day1-3,enhancedbyMyc);histonemodificationandchromatinreorganization
• 2ndwaveofgeneexpression-stemcellanddevelopmentrelatedgenes(day9-12);DNAdemethylationandXreactivation
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GrafT.CellStemCell9:504-516,2011
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NatureReviewsGenetics14:427-439,2013
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Removingthebottleneck?
• Raisetal.,Nature502:65-70,2013implicateMbd3,acomponentintheNuRDcomplexthatmediatesgenerepressionviahistonedeacetylationandchromatinremodeling.
• Arguethatthereprogrammingfactorsrecruitbothrepressive(Mbd3/NuRD)andde-repressive(Wdr5andUtx)complexes,andreprogrammingonlyoccurswhentheMbd3/NuRdrepressionloses.
• Achievenearly100%reprogrammingwithin7daysincellswithMbd3reducedoreliminated.
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Skippingthebottleneck?
• Jaenischlab(CellStemCell15:295-309,2014)usedSNELfactorsfromthedeterministicphase(Sall4,Nanog,EsrrbandLin28).
• Obtainedfewerbut“higherquality”mouseiPSCcoloniesasjudgedbyproductionofall-iPSCmicefrom4nblastocystinjections,andlackoftrisomy8.
• Hasnotworkedyetinhumans
• Isthisde-differentiationortransdifferentiation?
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Transdifferentiation
• Conversionfromonedifferentiatedcelltypetoanotherwithoutevidentde-differentiationandre-differentiation
• Mustnotbeconfusedbycellfusionorselectionforrarepluripotentcellsinthesourcematerial.
• InducedbyexpressionoftranscriptionfactorsandmicroRNAs
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GrafT.CellStemCell9:504-516,2011
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Fibroblaststoneurons
• Wernigandcolleaguesscreened19transcriptionfactorsvialentiviralexpression
• Found5weremostcriticalAsc1,Brn2,Olig2,Zic1andMyt1l,and3weresufficient
• 20%conversionwithin2weeks• ForhumanfibroblastconversionalsorequireNeuroD1anditislessefficient(2-4%)andslower(5-6weeksforfunctionalsynapses)
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Yangetal.,CellStemCell9:517-525,2011
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Conversionprocess• Asc1bHLHtranscriptionfactorbindstomanyofthesamegenomiclociwhenexpressedinfibroblasts,myoblastsorneuralprogenitors.
• Thesesitesaremarkedbyspecifichistonemodifications(H3K4me1,H3K27acetyl,H3K9me3)
• thesesitesarenotaccessibleinkeratinocytesorosteoblasts,whichresisttransdifferentiationintoneurons.
• Brn2Pou-HomeodomaintranscriptionfactorisrecruitedbyAsc1toasubsetoflocations
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Evaluation• SCNTvsiPSCsfromisogeniccells:
MaH,MoreyR,O'NeilRC,etal.,(2014)Abnormalitiesinhumanpluripotentcellsduetoreprogrammingmechanisms.Nature511:177-83.JohannessonB,SagiI,GoreA,etal.,(2014)Comparablefrequenciesofcodingmutationsandlossofimprintinginhumanpluripotentcellsderivedbynucleartransferanddefinedfactors.CellStemCell15:634-642.
• Origin-dependenceafteriPSCdifferentiation:HargusG,EhrlichM,Araúzo-BravoMJ,etal.,(2014)Origin-dependentneuralcellidentitiesindifferentiatedhumaniPSCsinvitroandaftertransplantationintothemousebrain.CellReports8:1697-703.
• Anoptimizationstrategy?MorrisSA,CahanP,LiH,etal.,(2014)DissectingengineeredcelltypesandenhancingcellfateconversionviaCellNet.Cell158:889-902.
• Retentionofcellularage?HuhCJ,ZhangB,VictorMB,DahiyaS,BatistaLF,HorvathS,YooAS.MaintenanceofageinhumanneuronsgeneratedbymicroRNA-basedneuronalconversionoffibroblasts.Elife.2016Sep20;5.
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Aldhous,2001
GoalsofReprogramming:• Modelsofhumandisease
• Isogeniccellsforreplacementtherapy
Muffatetal.(2016)CNSdiseasemodelswithhumanpluripotentstemcellsintheCRISPRage.CurrOpinCellBiol.43:96-103.
Wuetal.(2016)Stemcellsandinterspecieschimaeras.Nature540:51-59.
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ProofofConcept
Hannaetal.,Science318:1920-1923,2007