Epigenetic Memory: Lessons From iPS Cells Derived From ...

Epigenetic Memory in iPSCs Derived From Human β Cells ... Directed in-vitro differentiation of iPSCs into β-like cells is a promising approach for ... ThisarticleispartoftheResearchTopic AdvancesinStemCellTechnologytoModelandTreatDiabetes Viewall 13 Articles Articles HolgerA.Russ TheBarbaraDavisCenterforDiabetes,UniversityofColoradoAnschutzMedicalCampus,UnitedStates SentaGeorgia Children'sHospitalofLosAngeles,UnitedStates RosaGasa InstitutdeRecercaBiomèdicaAugustPiiSunyer(IDIBAPS),Spain Theeditorandreviewers'affiliationsarethelatestprovidedontheirLoopresearchprofilesandmaynotreflecttheirsituationatthetimeofreview. AbstractIncompleteReprogrammingIntoiPSCsiPSCDifferencesRelatedtotheCellTypeofOriginiPSCDifferencesRelatedtoDonorsandStochasticVariabilityEpigeneticMemoryiniPSCsDerivedFromHumanβCellsConclusionAuthorContributionsConflictofInterestAcknowledgmentsReferences SuggestaResearchTopic> DownloadArticle DownloadPDF ReadCube EPUB XML(NLM) Supplementary Material Exportcitation EndNote ReferenceManager SimpleTEXTfile BibTex totalviews ViewArticleImpact SuggestaResearchTopic> SHAREON OpenSupplementalData MINIREVIEWarticle Front.Endocrinol.,19January2021 |https://doi.org/10.3389/fendo.2020.614234 EpigeneticMemory:LessonsFromiPSCellsDerivedFromHumanβCellsShimonEfrat*DepartmentofHumanMolecularGeneticsandBiochemistry,SacklerSchoolofMedicine,TelAvivUniversity,TelAviv,IsraelIncompletereprogrammingofsomaticcellsintoinducedpluripotentstemcells(iPSCs)mayberesponsiblefortheheterogeneityindifferentiationcapacityobservedamongiPSClines.Itremainsunclearwhetheritresultsfromstochasticreprogrammingevents,orreflectsconsistentgeneticorcell-of-origindifferences.SomeevidencesuggeststhatepigeneticmemorypredisposesiPSCstoenhanceddifferentiationintotheparentalcelltype.WeinvestigatediPSCsreprogrammedfromhumanpancreaticisletβcells(BiPSCs),asastepindevelopmentofarobustdifferentiationprotocolforgenerationofβ-likecells.BiPSCsderivedfrommultiplehumandonorsmanifestedenhancedandreproduciblespontaneousandinduceddifferentiationtowardsinsulin-producingcells,comparedwithiPSCsderivedfromisogenicnon-β-celltypesandfibroblast-derivediPSCs(FiPSCs).Genome-wideanalysesofopenchromatininBiPSCsandFiPSCsidentifiedthousandsofdifferentialopenchromatinsites(DOCs)betweenthetwoiPSCtypes.DOCsmoreopeninBiPSCs(Bi-DOCs)weresignificantlyenrichedforknownregulatorsofendodermaldevelopment,includingbivalentandweakenhancers,andFOXA2bindingsites.Bi-DOCswereassociatedwithgenesrelatedtopancreasdevelopmentandβ-cellfunction.ThesestudiesprovideevidenceforreproducibleepigeneticmemoryinBiPSCs.Bi-DOCsmayprovidecluestogenesandpathwaysinvolvedinthedifferentiationprocess,whichcouldbemanipulatedtoincreasetheefficiencyandreproducibilityofdifferentiationofpluripotentstemcellsfromnon-β-cellsources.IncompleteReprogrammingIntoiPSCsReprogrammingofsomaticcellsintoinducedpluripotentstemcells(iPSCs)usingYamanaka’scombinationoffourtranscriptionfactors(OCT4,SOX2,KLF4,andc-MYC,togethertermedOSKM)(1)hasopenednewavenuesforin-vitrogenerationofmultiplehumandifferentiatedcelltypesfordiseasemodeling,drugscreening,andregenerativemedicine.InitiallyiPSCsderivedfrommultiplecelltypeshavebeenexpectedtomanifestsimilardifferentiationcapacities,resemblingthoseofhumanembryonicstemcells(ESCs).However,accumulatedexperiencehasshownagreatheterogeneityindifferentiationcapacityamongiPSClines.Thisheterogeneityisthoughttoresultfromincompletereprogramming.ThemechanismsactivatedbyectopicexpressionofOSKMinsomaticcells,whichresultinreprogrammingtopluripotency,areonlypartlyunderstood.Thisprolongedprocess,lastingseveralweeks,involvesmultipleevents,includingsilencingofsomaticcellgenesandactivationofpluripotency-associatedgenes.ThelowefficiencyofOSKM-mediatedreprogrammingisthoughttoreflectthestochasticnatureofthesecomplexevents.Onlyasmallfractionofcellsacquirepluripotency,asjudgedbyacceptedassays(e.g.differentiationintocellsfromthethreeembryonicgermlayersinembryoidbodies(EB),andteratomaformationassay).OSKMfactorsfunctionbybindingtochromatinregionsandinducingtheirremodeling,therebyactivatingorrepressinggeneexpression.Evidencesuggeststhatbroadepigeneticchangesareamongearlykeyeventsofthereprogrammingprocess(2).Theepigeneticlandscape,shapedbyDNAmethylationandhistonemodifications,iscriticalformaintainingcellidentity.Erasingcell-specificpatternsofepigeneticmodifications,andreplacingthemwithpluripotencypatterns,arecentraltocellreprogrammingtopluripotency.AmpleevidencesupportstheincompleteandvaryingerasureoftheoriginalepigeneticmarksinbothmouseandhumaniPSClines.However,itremainsunclearwhetherthesevariationsrepresentconsistentpatterns,basedonthecelltypeoforigin,orgeneticdifferencesamongdonors,orreflectstochasticdifferencescausedbylowefficiencyofthereprogrammingmechanisms(Table1).WhileepigeneticmemorymaynotnecessarilyaffectgeneexpressionpatternsiniPSCs(3),likelyduetomissingtranscriptionfactors,itisexpectedtoaffectdifferentiationcapacitytowardsspecificcellfates.ThismaylimitsomeapplicationsofiPSCs,butatthesametimemaypredisposeiPSCstoenhanceddifferentiationintotheparentalcelltype,therebyfacilitatinggenerationofcellsforspecificuses.TABLE1Table1PossiblesourcesofiPSCvariability.iPSCDifferencesRelatedtotheCellTypeofOriginMouseiPSCSEarlycomparisonofmouseiPSCsderivedfrombonemarrowprogenitorcells,dermalfibroblasts,andneuralprogenitorcells,identifiedresidualDNAmethylationsignaturescharacteristicofthesomatictissueoforigin(4).ThesedifferencesfavorediPSCdifferentiationtowardsthedonorcelltype,whilerestrictingalternativecellfates.Theseresultswerecontrastedtothemethylationanddifferentiationpatternsofnuclear-transfer-derivedpluripotentstemcells,whichweremoresimilartothoseofESCs.Similarfindingswerereportedinearly-passageiPSCsobtainedfrommousefibroblasts,hematopoieticandmyogeniccells,whichexhibiteddistinctepigeneticpatterns(5).ThesepatternswerereflectedindifferenttranscriptionalprofilesoftheiPSCs,andintheirdifferentiationefficiencyintoembryoidbodies(EBs)andhematopoieticcelltypes.ThesedifferenceswereerodedwithiPSCpassaging,suggestingthatepigeneticmemorywasatransientphenomenon.iPSCsderivedfrommouseneonatalcardiomyocytes(CMs)werealsoshowntodifferentiatetowardCMsmoreefficientlythanfibroblast-derivediPSCs(FiPSCs)ormouseESCs(6).HumaniPSCsAnumberofreportsdocumentedsimilarepigeneticmemoryinhumaniPSCs.Kimet al.comparedDNAmethylationprofilesanddifferentiationpotentialofiPSCsderivedfromhumanumbilicalcordbloodandneonatalkeratinocytes(7).Theyidentifieddistinctgenome-wideDNAmethylationpatternsiniPSCsderivedfromeachcelltype,resultingfrombothincompleteerasureoftissue-specificmethylationandaberrantde-novomethylation.Thesedifferencesdidnotdisappearuponextendedpassaging.Ohiet al.observedthathumaniPSCsgeneratedfromhepatocytes,skinfibroblasts,andmelanocytes,retainedsometranscriptionalcharacteristicsoftheoriginalcellsatlowpassages,whichcouldbepartiallyexplainedbyincompletepromoterDNAmethylation(8).Theynoticedthatincompletelysilencedgenestendedtobeisolatedfromothergenesthatwererepressedduringreprogramming,indicatingthatsilencingofisolatedgenesmaybelessefficient.GlobalDNAmethylationanalysesofiPSCsreprogrammedfromhumancornealimbalepithelialstemcells(LESC)showedgenemethylationpatternssimilartothoseoftheparentalcells,anddifferentfromthoseofFiPSCs(9).Upondifferentiation,LESC-derivediPSCsexpressedhigherlevelsofLESCmarkers,comparedwithFiPSCs.iPSCDifferencesRelatedtoDonorsandStochasticVariabilityIncontrasttothesefindings,whichassociatedepigeneticanddifferentiationdifferencesamongiPSCswiththeircelltypeoforigin,otherstudiessupportedadonor-relatedorstochasticbasisforthesedifferences.Inonesuchstudy(10),whole-genomeprofilingofDNAmethylationinfivehumaniPSClinesderivedfromadipocytesandfibroblasts(including3subclonesofasingleline)identifiedover1,000differentiallymethylatedsites,mostofthemassociatedwithCGislandsandgenes,indicatingstochasticinterclonereprogrammingvariability.Twootherstudies(11,12)surveyed16–18iPSClineseach,derivedfromfibroblastsandperipheralbloodcellsoffourhumandonorsineachstudy.BothstudiesconcludedthatthemajorityoftranscriptionalandDNAmethylomedifferencesamongiPSCs,aswellasdifferencesintheirdifferentiationcapacitytowardsthehematopoieticcelllineage,couldbeattributedtothedonor,ratherthanthecelltypeoforigin,indicatingthatgeneticdifferencesamongdonorscanresultinreproduciblereprogrammingdifferences.Finally,twostudies,whichanalyzeddifferencesingeneexpressionpatternsinhumaniPSCs,concludedthatgeneticdifferencesbetweenindividualdonorswerethemajorcauseoftranscriptionalvariationbetweenlines.Oneofthesestudies(13)compared25iPSClinesfromfourdonorsandthreetissues.ThesecondstudycomparedtwohumanESClineswithgeneticallymatchediPSCsderivedfromfibroblastsdifferentiatedfromeachESCline(14).TheirfindingsshowedreproduciblegeneexpressionpatternsamongeachESClineandthreeiPSCclonesderivedfromit,andvariationscomparedwiththeotherESClineanditsiPSCderivatives.ThestudyconcludedthatESCsandiPSCsexhibitedsimilargeneexpressionpatterns,andthatthedonorgeneticbackgroundwasresponsiblefortranscriptionalvariationsamongpluripotentstemcelllines.ThedonorgeneticbackgroundmayalsoinfluencethedifferentiationcapacityofESClines.However,incontrasttotherelativelylargenumbersofiPSClinesthathavebeengeneratedbymultiplelaboratories,dataondifferentiationofESClinesaccumulatedtodateisbasedonasmallnumberofestablishedESClinescommonlyusedbyallresearchgroups.Thislimitednumber,whichisaresultofethicalbarrierstogenerationofnewESClines,doesnotallowpropercomparisonsofvariationsamongESClinesindifferentiationpotentialintospecificcelllineages.EpigeneticMemoryiniPSCsDerivedFromHumanβCellsDirectedin-vitrodifferentiationofiPSCsintoβ-likecellsisapromisingapproachforgenerationofabundantinsulin-producingcellsforcelltherapyofdiabetes,diseasemodelinganddrugscreening.Despitesignificantprogress(15–21),currentdifferentiationprotocolsresultincellswithheterogeneousandimmaturephenotype,andsufferfromlowefficiencyandvariabilityamongiPSClines.Inasteptowardsdevelopingamorerobustdifferentiationprotocol,weinvestigatediPSCsreprogrammedfromhumanpancreaticisletβcells(BiPSCs).Thisapproachdependedonlineagetracingofhumanβ-cell-derived(BCD)cellswithintheheterogeneouscellpopulationinculturesofisolatedhumanislets(22).Thestablegeneticlabelallowedpositiveidentificationoftheβ-celloriginofindividualiPSCclones,thatotherwisewouldbedifficulttodistinguishfromiPSCsderivedfromnon-β-celltypespresentintheexpandedisletcellculture.InitialstudiesoffourBiPSClinesderivedfromβcellsofthreenondiabetichumandonorsestablishedtheirpluripotency,asjudgedbystandardassays(23).Nevertheless,chromatinimmunoprecipitation(ChIP)analysesshowedthatthelevelsofhistoneH3acetylation,ahallmarkofopenchromatinstructure,attheINSandPDX1lociinBiPSCsweresimilartothosefoundinhumanisletsandBCDcells,andsignificantlyhigherthanthoseinFiPSCs,aswellasintwoiPSClinesderivedfromisogenicisletnon-βcells(termedPiPSCs)fromtwoofthedonors.BiPSCsalsoexhibitedsignificantlylowerDNAmethylationlevels,characteristicoftranscribedgenes,ingenesexpressedinhumanisletcells,suchasPDX1,compareswithFiPSCs.Despitetheopenchromatinmarks,β-cellgeneswerenotexpressedinBCDandBiPScells.TheepigeneticmemoryofBiPSCswasassociatedwithhigherexpressionlevelsofPDX1,FOXA2,andINStranscriptsfollowingspontaneousdifferentiationintoEBsandteratomas,comparedwiththosederivedfromFiPSCsandPiPSCs,andanenhancedinduceddifferentiationcapacityintoinsulin-producingcellsinmicetransplantedwithBiPSC-derivedendocrineprogenitors,followingtheprotocolofKroonetal.(24).TheepigeneticphenotypeanddifferentiationcapacityofBiPSCswerereproducibleamongthefourlinesobtainedfromthreehumandonors,andappearedstablewithinthepassagerangeanalyzed(passages10–20).Toidentifygenesandpathways,whichmayberesponsiblefortheenhancedandreproducibledifferentiationcapacityofBiPSCs,weperformedaglobalanalysisofchromatinsitesdifferentiallyopeninBiPSCs,comparedwithFiPSCs,usinganAssayforTransposaseAccessibleChromatinwithhigh-throughputsequencing(ATAC-seq)(25).ForthisanalysiswegeneratedfivenewBiPSClinesfromthreenondiabeticdonors,whichwerecomparedtofiveFiPSClinesfromtwonondiabeticdonors.Alltheselinespassedpluripotencyassays,andEBsgeneratedfromBiPSCsatpassages9-12showedenhancedspontaneousexpressionofFOXA2,PDX1,andINS,comparedwiththosederivedfromFiPSCs(25),similarlytothefourBiPSClinesintheinitialstudy.DespitehighoverallsimilarityinopenchromatinbetweenthetwoiPSCtypes,theATAC-seqanalysisidentifiedthousandsofsignificantlydifferentialopenchromatin(DOC)sitesbetweenBiPSCsandFiPSCs,mostofwhichweremoreopeninBiPSCs(Bi-DOCs).Bi-DOCSoverlappedgeneregulatoryelementsknowntobeinvolvedindevelopment,suchasweakenhancers(markedbyH3K4me1)andbivalentenhancersandpromoters(markedbyH3K27me3),especiallyneargenesinvolvedinendodermaldevelopment,suchasFOXA2anditstargetgenes,andpancreasdevelopment,suchasPDX1,andNKX2-2,aswellasgenesexpressedinmatureβcells,suchasINS.TheATAC-seqdataforthesefourgenes(FOXA2,PDX1,NKX2-2,andINS)wasvalidatedbyH3K4me3ChIPanalysis,whichfoundhigherlevelsofthisopenchromatinmarkinthepromoterregionsofallfourgenesinBiPSCs,compareswithFiPSCs.ThesefindingscouldexplaintheenhancedexpressionofthesegenesinEBsgeneratedfromBiPSCs,comparedwiththosederivedfromFiPSCs.TherelevanceofBi-DOCstodifferentiationtowardsisletcellswasanalyzedbycomparingdirecteddifferentiationofBiPSCsandFiPSCsintodefinitiveendoderm(DE)andpancreaticprogenitor(PP)cellsaccordingtotheprotocolofRezaniaetal.(16).GlobaltranscriptomeanalysesbyRNA-seqidentified567protein-codinggenesexpressedathigherlevelsinBiPSC-derivedDE,comparedwithFiPSC-derivedDE,and181genesexpressedathigherlevelsinBiPSC-derivedPP,comparedwithFiPSC-derivedPP(25).ThesegenesweresignificantlyenrichedforgenesmappingnearBi-DOCs.AmonggenesexpressedathigherlevelsinBiPSCatbothstages,themostprominentwasestrogenreceptor1(ESR1;3.5-foldand53.4-foldhigherinDEandPP,respectively)andseveralofitstargetgenes.17β-estradiol(E2)hasbeenshowntoprotectmouseβcellsfromapoptosisbysignalingthroughestrogenreceptor(ER)αencodedbyESR1(26).ERαactivityhasbeenreportedtoincreaseNeurog3expressionandβ-cellproliferationinamousemodelofpancreaspartialductligation,andduringmouseisletdevelopment(27).IthasbeensuggestedtoregulateendocrinelineagespecificationthroughdownregulationofNOTCHsignaling.Thus,inclusionofE2intheculturemediumatkeystagesofthein-vitrodifferentiationprotocolmayincreaseitsefficiencyandreproducibility.ConclusionOverall,theanalysesofBi-DOCssupporttheexistenceofreproducibleepigeneticmemoryinBiPSCs.TheassociationbetweenBi-DOCsandgeneexpressionlevelsatearlystagesofthein-vitrodifferentiationprotocolprovidesaplausibleexplanationfortheenhanceddifferentiationcapacityofBiPSCsintotheβ-celllineage,comparedwithpluripotentstemcellsfromanon-β-cellsource.BothchromatinstructureanddifferentiationcapacitywerereproducibleinacombinednumberofnineBiPSClinesfromsixhumanisletdonorsintwoseparatestudies.Bi-DOCsmayprovidecluestogenesandpathwaysinvolvedinthedifferentiationprocess,whichcouldbemanipulatedtoincreasetheefficiencyandreproducibilityofdifferentiationofpluripotentstemcellsfromothersources.SuchmanipulationscouldincludeactivationofcandidategenesusingCRISPR-onapproachesorsmall-moleculecompounds.AuthorContributionsTheauthorconfirmsbeingthesolecontributorofthisworkandhasapproveditforpublication.ConflictofInterestTheauthordeclaresthattheresearchwasconductedintheabsenceofanycommercialorfinancialrelationshipsthatcouldbeconstruedasapotentialconflictofinterest.AcknowledgmentsThisresearchinmylaboratorywassupportedbytheJuvenileDiabetesResearchFoundation(JDRF),theIsraelScienceFoundation,andtheInnovativeMedicinesInitiativeoftheEuropeanUnionSeventhFrameworkProgram.HumanisletsforthesestudieswereprovidedthroughtheJDRFECITIsletsforBasicResearchProgramandtheIntegratedIsletDistributionProgram.References1.TakahashiK,YamanakaS.Inductionofpluripotentstemcellsfrommouseembryonicandadultfibroblastculturesbydefinedfactors.Cell(2006)126:663–76.doi: 10.1016/j.cell.2006.07.024PubMedAbstract|CrossRefFullText|GoogleScholar2.WatanabeA,YamadaY,YamanakaS.Epigeneticregulationinpluripotentstemcells:akeytobreakingtheepigeneticbarrier.PhilosTransRSocLondBBiolSci(2013)368:20120292.doi: 10.1098/rstb.2012.0292PubMedAbstract|CrossRefFullText|GoogleScholar3.CahanP,DaleyGQ.Originsandimplicationsofpluripotentstemcellvariabilityandheterogeneity.NatRevMolCellBiol(2013)14:357–68.doi: 10.1038/nrm3584PubMedAb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