StemCellBiology

JimHuettner11/21/2017

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.

StemCells:definition

•  SelfRenewal-undifferentiatedcellsthatcandividerepeatedlywhilemaintainingtheirundifferentiatedstate.

•  Pluripotency–abilitytodifferentiateintoavarietyofdifferentcelltypes

Donovan and Gearhart, 2001

Invitrodifferentiation:• Cell/tissuereplacementtherapies• Humanmodelsystemsofdiseaseanddevelopment

TypesofStemCellsEmbryonic–fromtheinnercellmassofpre-implantationembryos,priortoformationofthe3germlayers(ectoderm,mesoderm,endoderm)

Somatic–undifferentiatedcellsfoundinspecificlocationsin“mature”tissues

iPScells–inducedpluripotentstemcellsgeneratedbyreprogrammingdifferentiatedcells(orcellnuclei,i.e.therapeuticcloning)

Potency

•  Totipotent–abletogenerateeverycelltypeincludingextraembryonictissues

•  Pluripotent–abletogeneratecellsfromallthreeembryonicgermlayers

• Multipotent–abletogenerateavarietyofcellsfromaparticularsomaticstructure

•  Unipotent–onlygenerateonecelltype

TimeLine

fertilization gastrulation

totipotent pluripotent multipotent

somaticdifferentiation

zygote morula blastocyst

implantation

http://stemcells.nih.gov/info/scireport/pages/chapter1.aspx

Innercellmass

Epiblast:embryoHypoblast:yolksac

EarlyEmbryology

http://stemcells.nih.gov/info/scireport/pages/appendixa.aspx

Human Mouse

makingaknockoutmouse

http://en.wikipedia.org/wiki/Knockout_mouse

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.

Pluripotencymarkers•  Stage-specificantigens:Anti-SSEA3and4recognizeglobo-seriesgangliosides

•  Tra1-60andTra1-81:keratinsulfatesurfaceantigens

•  Oct3/4,Sox2,Nanog–transcriptionfactorsinvolvedwithmaintainingpluripotency

•  Normalkaryotype,andpre-X-inactivation?

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

maintenanceofpluripotency-1•  Initialworkdoneonmouseembryonicfibroblast(MEF)

feedercellsinmediumsupplementedwithanimalserum

•  OnefactorproducedbyfeedercellsthathelpsmaintainmouseEScellsintheirundifferentiatedstateisleukemiainhibitoryfactor(LIF)whichactivatestheStat3pathway.

•  GoodManufacturingProcess(GMP)–guidelinesforisolationandpropagationofcellsthatwouldbeusedforreplacementtherapy.Ideallytheywouldbexeno-free.

•  Thepushforxeno-freeconditions,combinedwithworktooptimizereprogramming,hasdrivenscreeningoffactorsthatcanenableserum-freemaintenanceofpluripotency

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

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)

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.

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.

EScells → neurons•  pluripotent•  functionallyimmortal•  genetically&developmentallynormal

•  postmitotic•  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–somaticcellnucleartransfer(reproductiveandtherapeuticcloning)–deterministicandfairlyrapid

•  iPS–inducedpluripotentstemcells–slowandstochastic(untilrecently)

•  Transdifferentiation–conversionofoneterminallydifferentiatedcelltypeintoanotherwithoutde-differentiationtoanimmaturephenotype.Mustruleoutcellfusionorotherexplanations.

Reprogramming:somaticcellnucleartransfer

http://www.biotechnologyonline.gov.au/images/contentpages/scnt.gif

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.

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.

GeneratingiPScells

•  Expresstranscriptionfactors:Oct3/4,Sox2,Klf4andc-MycOROct3/4,Sox2,NanogandLin28

•  Initialde-differentiationandproliferation(day1-3,enhancedbyMyc);histonemodificationandchromatinreorganization

•  2ndwaveofgeneexpression-stemcellanddevelopmentrelatedgenes(day9-12);DNAdemethylationandXreactivation

GrafT.CellStemCell9:504-516,2011

NatureReviewsGenetics14:427-439,2013

Removingthebottleneck?

•  Raisetal.,Nature502:65-70,2013implicateMbd3,acomponentintheNuRDcomplexthatmediatesgenerepressionviahistonedeacetylationandchromatinremodeling.

•  Arguethatthereprogrammingfactorsrecruitbothrepressive(Mbd3/NuRD)andde-repressive(Wdr5andUtx)complexes,andreprogrammingonlyoccurswhentheMbd3/NuRdrepressionloses.

•  Achievenearly100%reprogrammingwithin7daysincellswithMbd3reducedoreliminated.

Skippingthebottleneck?

•  Jaenischlab(CellStemCell15:295-309,2014)usedSNELfactorsfromthedeterministicphase(Sall4,Nanog,EsrrbandLin28).

•  Obtainedfewerbut“higherquality”mouseiPSCcoloniesasjudgedbyproductionofall-iPSCmicefrom4nblastocystinjections,andlackoftrisomy8.

•  Hasnotworkedyetinhumans

•  Isthisde-differentiationortransdifferentiation?

Transdifferentiation

•  Conversionfromonedifferentiatedcelltypetoanotherwithoutevidentde-differentiationandre-differentiation

• Mustnotbeconfusedbycellfusionorselectionforrarepluripotentcellsinthesourcematerial.

•  InducedbyexpressionoftranscriptionfactorsandmicroRNAs

GrafT.CellStemCell9:504-516,2011

Fibroblaststoneurons

• Wernigandcolleaguesscreened19transcriptionfactorsvialentiviralexpression

•  Found5weremostcriticalAsc1,Brn2,Olig2,Zic1andMyt1l,and3weresufficient

•  20%conversionwithin2weeks•  ForhumanfibroblastconversionalsorequireNeuroD1anditislessefficient(2-4%)andslower(5-6weeksforfunctionalsynapses)

Yangetal.,CellStemCell9:517-525,2011

Conversionprocess•  Asc1bHLHtranscriptionfactorbindstomanyofthesamegenomiclociwhenexpressedinfibroblasts,myoblastsorneuralprogenitors.

•  Thesesitesaremarkedbyspecifichistonemodifications(H3K4me1,H3K27acetyl,H3K9me3)

•  thesesitesarenotaccessibleinkeratinocytesorosteoblasts,whichresisttransdifferentiationintoneurons.

•  Brn2Pou-HomeodomaintranscriptionfactorisrecruitedbyAsc1toasubsetoflocations

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.

Aldhous,2001

GoalsofReprogramming:• Modelsofhumandisease

•  Isogeniccellsforreplacementtherapy

Muffatetal.(2016)CNSdiseasemodelswithhumanpluripotentstemcellsintheCRISPRage.CurrOpinCellBiol.43:96-103.

Wuetal.(2016)Stemcellsandinterspecieschimaeras.Nature540:51-59.

ProofofConcept

Hannaetal.,Science318:1920-1923,2007