StemCellBiology
JimHue0ner11/17/2015
suggestedreadings• SolterD.(2006)Fromteratocarcinomastoembryonicstemcellsandbeyond:
ahistoryofembryonicstemcellresearch.NatRevGenet.7:319-27.• BuganimY,FaddahDA,JaenischR.MechanismsandmodelsofsomaSccell
reprogramming.NatRevGenet.2013Jun;14(6):427-39.• BuganimY,MarkoulakiS,vanWietmarschenN,etal.(2014)The
developmentalpotenSalofiPSCsisgreatlyinfluencedbyreprogrammingfactorselecSon.CellStemCell.15:295-309..
• DeLosAngelesetal.(DaleyGQ).(2015)Hallmarksofpluripotency.Nature525:469-478.
• FoxIJ,DaleyGQ,GoldmanSA,HuardJ,KampTJ,TruccoM.(2014)Stemcelltherapy.UseofdifferenSatedpluripotentstemcellsasreplacementtherapyfortreaSngdisease.Science345(6199):1247391.
• SchwartzSD,RegilloCD,LamBLetal.,(2014)Humanembryonicstemcell-derivedreSnalpigmentepitheliuminpaSentswithage-relatedmaculardegeneraSonandStargardt’smaculardystrophy:follow-upoftwoopen-labelphase1/2studies.Lancet.e-pubOctober15.
StemCells:definiSon
• SelfRenewal-undifferenSatedcellsthatcandividerepeatedlywhilemaintainingtheirundifferenSatedstate.
• Pluripotency–abilitytodifferenSateintoavarietyofdifferentcelltypes
Donovan and Gearhart, 2001
InvitrodifferenSaSon:• Cell/Sssuereplacementtherapies• Humanmodelsystemsofdiseaseanddevelopment
TypesofStemCellsEmbryonic–fromtheinnercellmassofpre-implantaSonembryos,priortoformaSonofthe3germlayers(ectoderm,mesoderm,endoderm)
SomaSc–undifferenSatedcellsfoundinspecificlocaSonsin“mature”Sssues
iPScells–inducedpluripotentstemcellsgeneratedbyreprogrammingdifferenSatedcells(orcellnuclei,i.e.therapeuSccloning)
Potency
• ToSpotent–abletogenerateeverycelltypeincludingextraembryonicSssues
• Pluripotent–abletogeneratecellsfromallthreeembryonicgermlayers
• MulSpotent–abletogenerateavarietyofcellsfromaparScularsomaScstructure
• Unipotent–onlygenerateonecelltype
TimeLine
ferSlizaSon gastrulaSon
toSpotent pluripotent mulSpotent
somaScdifferenSaSon
zygote morula blastocyst
implantaSon
h0p://stemcells.nih.gov/info/scireport/pages/chapter1.aspx
Innercellmass
Epiblast:embryoHypoblast:yolksac
EarlyEmbryology
h0p://stemcells.nih.gov/info/scireport/pages/appendixa.aspx
Human Mouse
makingaknockoutmouse
h0p://en.wikipedia.org/wiki/Knockout_mouse
FirstIsolaSonofEScellsMouse:
EvansMJ,KaufmanMH.(1981)EstablishmentincultureofpluripotenSalcellsfrommouseembryos.Nature.292:154-6.MarSnGR.(1981)IsolaSonofapluripotentcelllinefromearlymouseembryosculturedinmediumcondiSonedbyteratocarcinomastemcells.P.N.A.S.USA.78:7634-8.
Human:ThomsonJA,Itskovitz-EldorJ,ShapiroSS,WaknitzMA,SwiergielJJ,MarshallVS,JonesJM.(1998)Embryonicstemcelllinesderivedfromhumanblastocysts.Science.282:1145-7.
GeneScandDevelopmentalNormality(140cycles):SudaY,SuzukiM,IkawaY,AizawaS.(1987)MouseembryonicstemcellsexhibitindefiniteproliferaSvepotenSal.JCellPhysiol.133:197-201.
Pluripotencymarkers• Stage-specificanSgens:AnS-SSEA3and4recognizeglobo-seriesgangliosides
• Tra1-60andTra1-81:keraSnsulfatesurfaceanSgens
• Oct3/4,Sox2,Nanog–transcripSonfactorsinvolvedwithmaintainingpluripotency
• Normalkaryotype,andpre-X-inacSvaSon?
TwotypesofEScells?
• Blastocystchimera(+)• Highcloningefficiency• ShortdoublingSme• XaXa• DistalOct4enhancer• HighNanog,Klf2/4,Rex1
• Blastocystchimera(-)• Lowcloningefficiency• LongdoublingSme• XaXi• ProximalOct4enhancer• LowNanog,Klf2/4,Rex1
“Naïve”(ICM-like)
“Primed”(Epi-SC)
Bothtypescanselfrenewandgiverisetocellsfromall3germlayersinteratomasorfollowinginvitrodifferenSaSon
maintenanceofpluripotency-1• IniSalworkdoneonmouseembryonicfibroblast(MEF)
feedercellsinmediumsupplementedwithanimalserum
• OnefactorproducedbyfeedercellsthathelpsmaintainmouseEScellsintheirundifferenSatedstateisleukemiainhibitoryfactor(LIF)whichacSvatestheStat3pathway.
• GoodManufacturingProcess(GMP)–guidelinesforisolaSonandpropagaSonofcellsthatwouldbeusedforreplacementtherapy.Ideallytheywouldbexeno-free.
• Thepushforxeno-freecondiSons,combinedwithworktoopSmizereprogramming,hasdrivenscreeningoffactorsthatcanenableserum-freemaintenanceofpluripotency
maintenanceofpluripotency-2
• LIF-Stat3• BMP4-Smad1/5• Wnt(GSK-3inhibitors)• IGF
• TGFβ/acSvin-Smad2/3• FGF2• ERK1/2
• TGFβ/acSvin–Smad2/3• FGF2• ERK1/2• Wnt(GSK-3inhibitors)• IGF
• BMP4–Smad1/5
“naïve” “primed”Posi%veRegulators
Nega%veRegulators
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”Condi%ons
Forserumfreegrowthalsoneed:Insulin,transferrin,progesterone,putrescine,selenium
butsee:Takashimaetal.,Cell158:1254-1269(2014)
A0emptstodefine“Stemness”
• Earlymicroarrayprofilesshowedsurprisinglackofagreement(limitaSonsinmicroarraytechnologyorplatorm/lab/primarycellorcelllinedifferences)(Science302:393,2003)
• RelaSvelyweakoverlapbetweenmouseandhumanEScells(~25%)comparedto>90%typicalfordifferenSatedSssues.(StemCellReviews1:111-118,2005)butthismayreflectconfusionbetweennaïveandprimedEScells
InvitrodifferenSaSon• DifferentculturecondiSonsalterthefateofEScellsinvitro• Protocolsexistforallthreegermlayers
• Many,butnotall,protocolsinvolveaggregaSonofEScellsin“embryoidbodies”
• Mostprotocolsdonotyieldasingletypeofcell
• SelecSonstepscanhelptoremoveundesiredcelltypes
• Needtoask:Howfar?&Howfaithful?
PancreaScβcells:• PagliucaFW,etal.(2014)GeneraSonofFuncSonalHumanPancreaScβCellsInVitro.Cell.Oct,159:428-39.
• RezaniaA,etal.(2014)Reversalofdiabeteswithinsulin-producingcellsderivedinvitrofromhumanpluripotentstemcells.NatBiotechnol.Nov,32:1121-33.
EScells → neurons• pluripotent• funcSonallyimmortal• geneScally&developmentallynormal
• postmitoSc• polarized• excitable• heterogeneous
4 d 4 d 4 d 6 d
Serum Free Medium
+ RA
ESC
SFD
SF+RA
Kimetal.,DevelopmentalBiology328:456-471,2009
β-tubulin nestin Hoechst
GABA β-tubulin Hoechst GAP43 MAP2 Hoechst
voltage-gatedNa+andK+currents
DevelopmentalBiology168:342-357,1995
JournalofNeuroscience16:1056-65,1996
DevelopmentalBiology328:456-471,2009
HierarchicalclusteringbyfrequencyofGeneOntologyterms
Reprogramming
• SCNT–somaSccellnucleartransfer(reproducSveandtherapeuSccloning)–determinisScandfairlyrapid
• iPS–inducedpluripotentstemcells–slowandstochasSc(unSlrecently)
• TransdifferenSaSon–conversionofoneterminallydifferenSatedcelltypeintoanotherwithoutde-differenSaSontoanimmaturephenotype.MustruleoutcellfusionorotherexplanaSons.
Reprogramming:somaSccellnucleartransfer
h0p://www.biotechnologyonline.gov.au/images/contentpages/scnt.gif
ReprogrammingFirsts:SCNTFrog:
GurdonJB.(1962)AdultfrogsderivedfromthenucleiofsinglesomaSccells.DevBiol.4:256-73.
Sheep:CampbellKH,McWhirJ,RitchieWA,WilmutI.(1996)Sheepclonedbynucleartransferfromaculturedcellline.Nature.380:64-6.
Human:(2004)–ClaimofhumanSCNTthatprovedtobeunfounded!TachibanaM,etal.(2013)HumanembryonicstemcellsderivedbysomaSccellnucleartransfer.Cell.153:1228-38.
ReprogrammingFirsts:iPScellsMouse:
TakahashiK,YamanakaS.(2006)InducSonofpluripotentstemcellsfrommouseembryonicandadultfibroblastculturesbydefinedfactors.Cell.126:663-76
Human:TakahashiK,TanabeK,OhnukiM,NaritaM,IchisakaT,TomodaK,YamanakaS.(2007)InducSonofpluripotentstemcellsfromadulthumanfibroblastsbydefinedfactors.Cell.131:861-72.YuJ,VodyanikMA,Smuga-O0oK,etal.,(2007)InducedpluripotentstemcelllinesderivedfromhumansomaSccells.Science.318:1917-20.
GeneraSngiPScells
• ExpresstranscripSonfactors:Oct3/4,Sox2,Klf4andc-MycOROct3/4,Sox2,NanogandLin28
• IniSalde-differenSaSonandproliferaSon(day1-3,enhancedbyMyc);histonemodificaSonandchromaSnreorganizaSon
• 2ndwaveofgeneexpression-stemcellanddevelopmentrelatedgenes(day9-12);DNAdemethylaSonandXreacSvaSon
GrafT.CellStemCell9:504-516,2011
NatureReviewsGeneScs14:427-439,2013
Removingthebo0leneck?
• Raisetal.,Nature502:65-70,2013implicateMbd3,acomponentintheNuRDcomplexthatmediatesgenerepressionviahistonedeacetylaSonandchromaSnremodeling.
• Arguethatthereprogrammingfactorsrecruitbothrepressive(Mbd3/NuRD)andde-repressive(Wdr5andUtx)complexes,andreprogrammingonlyoccurswhentheMbd3/NuRdrepressionloses.
• Achievenearly100%reprogrammingwithin7daysincellswithMbd3reducedoreliminated.
Skippingthebo0leneck?
• Jaenischlab(CellStemCell15:295-309,2014)usedSNELfactorsfromthedeterminisScphase(Sall4,Nanog,EsrrbandLin28).
• Obtainedfewerbut“higherquality”mouseiPSCcoloniesasjudgedbyproducSonofall-iPSCmicefrom4nblastocystinjecSons,andlackoftrisomy8.
• Hasnotworkedyetinhumans
• Isthisde-differenSaSonortransdifferenSaSon?
TransdifferenSaSon
• ConversionfromonedifferenSatedcelltypetoanotherwithoutevidentde-differenSaSonandre-differenSaSon
• MustnotbeconfusedbycellfusionorselecSonforrarepluripotentcellsinthesourcematerial.
• InducedbyexpressionoftranscripSonfactorsandmicroRNAs
GrafT.CellStemCell9:504-516,2011
Fibroblaststoneurons
• Wernigandcolleaguesscreened19transcripSonfactorsvialenSviralexpression
• Found5weremostcriScalAsc1,Brn2,Olig2,Zic1andMyt1l,and3weresufficient
• 20%conversionwithin2weeks• ForhumanfibroblastconversionalsorequireNeuroD1anditislessefficient(2-4%)andslower(5-6weeksforfuncSonalsynapses)
Yangetal.,CellStemCell9:517-525,2011
Conversionprocess• Asc1bHLHtranscripSonfactorbindstomanyofthesamegenomiclociwhenexpressedinfibroblasts,myoblastsorneuralprogenitors.
• ThesesitesaremarkedbyspecifichistonemodificaSons(H3K4me1,H3K27acetyl,H3K9me3)
• thesesitesarenotaccessibleinkeraSnocytesorosteoblasts,whichresisttransdifferenSaSonintoneurons.
• Brn2Pou-HomeodomaintranscripSonfactorisrecruitedbyAsc1toasubsetoflocaSons
EvaluaSon• SCNTvsiPSCsfromisogeniccells:
MaH,MoreyR,O'NeilRC,etal.,(2014)AbnormaliSesinhumanpluripotentcellsduetoreprogrammingmechanisms.Nature511:177-83.JohannessonB,SagiI,GoreA,etal.,(2014)ComparablefrequenciesofcodingmutaSonsandlossofimprinSnginhumanpluripotentcellsderivedbynucleartransferanddefinedfactors.CellStemCell15:634-642.
• Origin-dependencea�eriPSCdifferenSaSon:HargusG,EhrlichM,Araúzo-BravoMJ,etal.,(2014)Origin-dependentneuralcellidenSSesindifferenSatedhumaniPSCsinvitroanda�ertransplantaSonintothemousebrain.CellReports8:1697-703.
• AnopSmizaSonstrategy?MorrisSA,CahanP,LiH,etal.,(2014)DissecSngengineeredcelltypesandenhancingcellfateconversionviaCellNet.Cell158:889-902.
Aldhous,2001
GoalsofReprogramming:• Modelsofhumandisease
• Isogeniccellsforreplacementtherapy
ProofofConcept
Hannaetal.,Science318:1920-1923,2007