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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