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Lineageconversionofmousefibroblaststopancreaticα-TianjinLiu*1,LiangliangSun2,BeigeJiang3,JinCen1,XiaotaoChen1,ZhaoyunZhang4,QinghuaWang5,XinCheng1,LijianHui*11.InstituteofBiochemistryandCellBiology,InstituteofHealthSciences,ShanghaiInstitutesforBiologicalSciences(SIBS),ChineseAcademyofSciences(CAS)（200031）2.DepartmentofEndocrinologyandMetabolism,ChangzhengHospital,SecondMilitaryMedicalUniversity,Shanghai,PRChina.（200003）3.DepartmentofHepaticSurgery,EasternHepatobiliarySurgeryHospital,SecondMilitaryMedicalUniversity,Shanghai,PRChina.（200438）4.DepartmentofEndocrinologyandMetabolism,HuashanHospital,Shanghai,5.DivisionofEndocrinologyandMetabolism,theKeenanResearchCentreintheLiKaShingKnowledgeInstitute,St.Michael’sHospital,Toronto,Canada.AbbreviatedTitle:InvitroreprogrammingoffibroblasttoendocrineKeyterms:αWordcount:Numberoffiguresandtables:TheseauthorscontributeequallytothisCorrespondingauthorand towhomreprintrequestsshouldbeaddressed:TianjinLiu，PhDInstituteofBiochemistryandCellBiology,InstituteofHealthSciences,ShanghaiforBiologicalSciences(SIBS),ChineseAcademyofSciencesNo.320,RoadYueyang,Shanghai,PRChina，200031ThelaboratoryofL.H.isfundedbyNationalKeyBasicResearchandDevelopmentProgramofChina（2013CB967103）,strategicPriorityResearchProgramoftheChineseAcademyofSciences（XDA ）,theHundredTalentsProgram（ NaturalScienceFoundationofChina（ ）andNaturalScienceFoundationofShanghaiMunicipality，PRChina（13ZR α-cellsareresponsibleforsynthesizingandsecretingthepeptidehormoneglucagontoelevatetheglucoselevelintheblood.Hence,thedysfunctionofα-cellswillresulttoseverehypoglycemiaandshock.Furthermore,α-cellsarerequiredforislettransplantationtherapyfordiabetes.Recentlystudiesshowα-cellssupportβ-cellssurvivalandareevenabletotrans-differentiatetoβ- caseofdiabetes.However, theunderstanding ofα-cells pathologicalandclinicalapplicationsremainselusiveduetounavailableofmatureα-cells.Herewepresentanewtechniquetogeneratefunctionalα-cells-likecells(iAlphacells).iAlphacellswereconvertedfrommousefibroblastsbytransductionoftranscriptionfactors(TFs)includingHhex,Foxa3,Gata4,Pdx1andPax4,whichcanspecificallyexpressα-cellspecificgenesandsecreteglucagoninresponsetoKclandArgstimulation.iAlphacellspresenttheα-cellsfunctionsinvitroandareabletodisturbthebloodglucoselevelinvivo. TransplantationofiAlphacellsledthenudemicetoinsulin-andincreaseβ-cellsproliferation.Ourstudydemonstratesanovelnewstrategytogeneratefunctionalα-likecellsforthepurposeofdiseasemodelingandregenerativemedicine.Currenttherapiesforthetreatmentoftype1diabetesincludedailyadministrationofexogenousinsulinand,lessfrequently,whole-pancreasorislettransplantation.However,insulininjectionsoftenresultininaccurateinsulindoses,andexposingthepatienttohypoand/orhyperglycemicepisodesthatleadtolong-termcomplications.Transplantationofisolatedisletsorentirepancreasestorecoverdysfunctionofpatient’sislethasyieldedsomepromisingresults(1,2).Withinislets,β-cellssecreteinsulin,whileα-cellssecreteglucagon,andbloodglucosebalancecontrolledbythemisessentialformaintainingenergyhomeostasis.However,limitedavailabilityofhigh-qualityisletdonorshasrestrictedclinicalapplicationsofisletstransplantation(3,4).Therefore,theformationofmature,singlehormone-expressingendocrinecellsincultureremainsamajorhurdle(5).Alternativesourcesofisletshaveattractedgreatattentions(6).Inducedendocrinecellsdifferentiationinvitrohasbeenachievedpreviouslyusingembryonicstemcellsandinducedmulti-potentialstemcells(7-10).However,thesecellsweremalfunctionedandco-expressedmixedpancreatichormonesinvitro.Inaddition,theclinicalusageofpartiallydifferentiatedcellsfromESandiPSmaypresentanunacceptableriskoftumorformation.Thus,theprotocolsofendocrinecellsre-differentiationarerequiredfurtherDynamicchromatinremodelingduringES/iPSdifferentiationwasprovedasarepressorimpairingendocrinecellmaturation(5).Ontheotherhand,reprogramminginducedbytranscriptionfactorsmayfacilitatethefullyconversionofendocrinecellsbyregulatechromatinremodeling.Ithasshownthatforcedexpressionoflineage-specifictranscriptionfactorsdirectlyconvertterminallydifferentiatedcellsintosomeothercell-typelineages(11).Recently,studieshaveshownthatcardiomyocytes,neurons,andhepatocytescanbeinducedfrommousefibroblastsbyover-expressionofdefinedtranscriptionfactors(12-14).Wepreviouslyhavereportedthatover-expressionofHnf1a,gata4andFoxa3canconvertmousefibroblastsintofunctionalhepatocyte-likecells(iHepcells)(12).Also,ithasbeenreportedthatreprogrammingpancreaticexocrinecellstoβ-likecellsbyexpressionofPdx1,Ngn3andMafainvivo(15,16).Therefore,wesupposethatreprogrammingterminaldifferentiatedcellsinvitrocangenerateendocrinecells.Thegenerationoffullydifferentiatedendocrinecells,especiallyα-cells,thatexhibitgenepatternandareabletophysiologicallyregulatehormonesecretioninvitrohasnotyetbeenachieved.Itlargelyimpairsyzingα-cells’roleinpathologicalandclinicalapplications.Inthisstudy,wesetupaprotocolofinvitroconversionofmousefibroblaststofunctional,terminallydifferentiatedendocrinecells.WefoundforcedHhex,Foxa3,Gata4,Pdx1andPax4over-expressioncaninducefibroblastsexpressα-cellspecificgenesandacquiredα-cellsfunctioninvitro,suchcellswerenamediAlphacells.Furthermore,iAlphacellsinthekidneyofnudemiceinduceinsulin-andpromoteβ-cellsproliferation.Basedonthisstudy,iAlphacellscanprovideatoolfordrugdiscovery,diabetesdiseasemodelingandregenerativemedicine.InductionofGcg–producingcellsfrommouseinducedhepatocytes(iHep)andtailtipfibroblasts(TTFs).Ithasbeenreportedthatcellreprogrammingcouldbemoresuccessfulifthestartingcelltypesharesacommondevelopmentalhistorywiththedesiredcelltype(17).Hepatocytesandpancreaticendocrinecellsshareacommonprogenitorcellduringembryonicdevelopment(18).Markedly,hepatocyteshavebeenreprogrammedtoinsulin-positivecellsbyforcedexpressionofPdx1invivo(19).However,itremainsunclearwhetherhepatocytescouldbeconvertedintospecificendocrinecellsinvitro.Fortheproliferationinhibitionofprimaryhepatocytes,weuseiHepcells,whichshowanexpressionprofileandhepaticfunctionclosetothoseofmaturehepatocytes,andiHepcellswereinducedfrommouseTTFbyover-expressionofGata4,Hnf1aandFoxa3andtheinactivationofp19Arf-/-(12).WefirstyzedthepossibilityofendocrinecellsreprogrammingfromiHepcellsinvitro.Wedesignedascreenfortranscriptionfactorscriticalforendocrineinductionusing11pancreatictranscriptionfactors(Fig.Table1).（原图未提供，请见下表）Table1TranccriptioniHepcellswereinfectedbylentescarryingoneofthecandidatetranscriptionfactors.TheexpressionofendocrinegenesIns2andGcgweremeasuredatday7tomonitortheconversiontopancreaticα-cellsorβ-cells.Notably,themRNAlevelsofGcg(α-cellsspecific)butnotIns2(β-cellswerelargelyinducedinPdx1,HhexandPax4transducedcells(Fig.1b),图，即原图中FigindicatingthatiHepcellscanbeconvertedtoglucagon-producingcells.Next,iHepcellsweretransducedwiththecombinationofPdx1,HhexandPax4toenhancesuchconversion.ExpressionsofGcgweresignificantlyincreasedupontransductionofPdx1,HhexandPax4.Moreover,anotherα-cell-markergene,Arxwasalsoincreased(Fig1c).见下图，即原图中FigMorphologically,iHepcellsalsotransferedfromtypicalepithelial-likemesenchymal-likewithPdx1,HhexandPax4over-expression(Fig.1d).图，即原图中FigSinceiHepcellswerederiveddirectlyfromTTFs,wedeterminedwhetherGcg-producingcellscouldbeacquireddirectlyfromTTFs.Wetransducedp19Arf-/-TTFswithiHepinductionfactors,Gata4,Hnf1aandFoxa3,andPdx1,Hhex,Pax4(collectivelyreferredas6TF),whichcouldformclones14later(Fig1e).见下图，即原图中FigGcgmRNAexpressionweredetectedafter6TFsinfection(Fig1f).Fig1g.Wenextremovedindividualfactorsfromthecombination6TF.GcgexpressionincreaseduponremovingofHnf1afrom6TF(Fig.1f).下图，即原图中FigHence,wetransducedTTFsbythecombinationtoGata4,Pdx1,Hhex,andFoxa3(collectivelyreferredtoas5TF).Removinganyfactorfrom5TFreducetheexpressionofGcg(Fig.1g,Supplementaryfigure1a).原图中FigMarkedly,GcgexpressionwasnotinduceduponwithdrawalofHhexFoxA3,Incontrast,over-expressionofHhexandFoxa3weresufficienttoinduceGcgexpression(SupplementaryfigureHowever,mRNAlevelsofGcgandArxin,HhexandFoxa3inducedcellssignificantlylowerthanthatin5TFtransducedcells(Supplementaryfigure见下图，即原图中supplementaryFigProteinlevelsofglucagonwerealsoexpressedatalowlevelinHhexFoxa3inducedcellscomparewithiAlphacells(SupplementaryfigurePreviousstudieshaveshownthatcultureconditioniscriticalfordifferentiationofendocrinecellsinvitro(20).WenextimprovedGcginductionbyoptimizingtheculturemedium.Wefoundoneconditionalmedium,whichcontainedneuralbasalmedium,N-2supplement,B-27supplement,nicotianamineandbFGF,wasmorepotentininducingGcgexpressionthanDMEM,orDMEM-F12media(Fig.1h).见下图，即原图FigTheexpressionofGcgincreasedgraduallyduringtheinduction,thattheconversionisaprogressiveprocess(Fig.1i见下图，即原图FigTakentogether,weconcludedthatTTFareconvertedintoGcg-producingcellswith5TFoverexpressionandneural-basalmedium-basedmedium(Fig.1jTodeterminetheindividualfunctionof5TFininduction,wemonitoredtheexpressionsoflineagespecificmarkersafterremovingindividualfactorsfrom5TF,includingendodermmarker(Emoes,Wnt3a,Gata6),pancreaticprogenitormarker(Sox9),liverspecificmarker(Alb),βcellspecificmarker(Mafa),δcellspecificmarker(sst)andexocrinecellspecificmarkerwithdrawalanyfactorfrom5TFswillinducetheup-regulationof的(Supplementary的(Supplementaryfigure2).Fig2blineagemarkers做修改.Overall,5TFinfectioninwildtypeTTFandpancreatictriggeredGcgandArxexpression(SupplementaryFigure3a,b)supplementaryFig2b,5TFs组Gcg和Arx表达明显升高,indicatingthepossibilitytoinduceα-cellfromfibroblastinvivo.p19Arf-/-TTFswereconvertedintoαcell-likeWecomparedtheglobalexpressionprofilesamongiAlphacells,TTFs,andislet.MicroarraydatarevealedthatnumerousisletfunctionalgenesupregulatediniAlphacellscomparedtoTTFs(Fig.2a,supplementary4a,b).原图未提供，看能不能查阅些文献，文献中的结果。1079and3942outof43400annotatedgeneswerefoundtobe5-doubllingup-regulatediniAlphacellsandislet,separay,andabout694genesintheseup-regulatedgenewereoverlap.TocharacterizetheseGcg-producingcells,weyzedexpressionofα-cellspecifictranscriptionfactors,includingArx,Dpp4,Pcsk2andIrx2.ThesemarkergeneswereallinducedinGcg-producingcells.Notably,markergenesforotherpancreaticcells,suchasIns2,Nkx6.1andGlp1R(forβ-cells),Sst(forδ-cells),andAmy2a(forexocrinecells)undetectableorexpressedatalowlevel(Fig.2b).见下图，即原图Fig2d，并未检测Amy2aTheculturedisletsandiAlphacellsshowsimilarpatternofglucagon(Fig2c).见下图，即原图Fig2cThedatasuggestedthatTTFswereconvertedintoα-cellslikecells(iAlphacells),butnototherpancreaticlineagecells.Toassaythestabilityofconversion,wedetecteddynamicchangesofTF (SupplementaryFigure5a-e见下图，即原图Fig3a-e.ThemarkersGata6andWnt3awereup-regulatedatday5(Supplementary5f,g)见下图，即原图Fig3f,g,whileα-cellspecificmarkerIrx2maintainexpressionduringthecourse(SupplementaryFigure5h)见下图，即原图Figh.Hence,α-cellconversionmayundertakeaprocessofreversiontoCharacterizationofiAlphacellsThereceptorsofglucose,insulinandglucagoninα-cellarecriticalforglucagonsenseandsecreting(21).Therefore,wedetectedandfoundthesereceptorswereup-regulatediniAlphacells(Fig.3a),见下图，即原图Fig4aGlucagoncontentiniAlphacellswere50percentofthatinislets(Fig.andinsulinproteincouldnotbedetectediniAlphacells(Fig3cBiologicallyactiveglucagonsecretionwasdetectediniAlphacells,iAlphacellsexhibitedsimilarglucagonsecretionpropertyinresponsetoKclandArgstimulationasislets,suggestingthatglucagonbutnotinsulinsecretioncanbephysiologicallyregulatedinthesecells(Fig3d,e)见下图，即原图Fig4b-Notably,iAlphacellsestablishedα-cellfunctionsinToverifythephysiologicfunctionofiAlphacells,sensitivitytostimuluswastested.Nudemiceweretransplantedwith1*106iAlphacellsorTTFsinkidneycapsule.Bodyweights,insulintolerancetest(ITT)andglucosetolerancetest(GTT)weremeasuredat2,4and8weeksaftertransplantation.At1-2weeks,therewasnosignificantdifferencebetweenbodyweightsofnudetransplantedwithTTFsandiAlphacells(Fig.4a,SupplementaryFigure6a).原图Fig5a.iAlphacellrecipientsexhibitedsimilarresponseforITTandatfirstweek(SupplementaryFigure6b,c)即原图Fig5b-c.While,at2wefoundadelayedbloodglucoselevelsreduction(Fig.4b)即原图Fig5b recipients(supplementarymovie1),Movie1未提供indicatingthatiAlphacellsmayaffectinsulinrelatedmetabolicfunctioninvivo.GTTresultsshowthattherewasnosignificantdifferencebetweennudemicetransplantedwithTTFsoriAlphacellsat2weeks(Fig.4c)即原图Fig5c.At4weeks,thebodyweightsofiAlphacellstransplantednudemicesignificantlydecreasedcomparedTTFstransplantedones(Fig.4d)Fig5d.Therewassignificantdifferenceofbloodglucoseinfastandnormalstatecomparedwiththatoftransplantedanimals(Fig.4e,f)即原图Fig5e-f.ITTresultsshowthathavesignificantdifferenceiniAlphaandTTFtransplantedmice(Fig.4e)图Fig5e.At8weeksaftertransplantation,bodyweightsofiAlphatransplantedmicerecovered(Fig.4g)即原图Fig5g,however,therewassignificantdifferenceofITTandGTTcomparedwithcontrolanimals即原图Fig5h-i.Therefore,iAlphacellspresenttheα-cellsfunctionsinareabletodisturbthebloodglucoseregulationinvivo.这一段就是在讲FigiAlphacellsinducetheregenerationofisletcellsinThefunctionofiAlphacellsweredecreasedat8weeksaftertransplantation,sowedetectthesurvivalofiAlphacellsintherecipients,andfoundgraftcanbeseeninthecapsuleofrecipient’skidney(Fig.6a),andthesewereglucagon-positivebutnotinsulin-positive(Fig.6b,Supplementary7c).这里是指Fig6a-b，需 Confusingly,at8weeks,survivediAlphacellsdon’ttakeimportantrolesregulatingglucose.Wedetectedthesecretionofinsulinandglucagonplasmaofrecipients,andfoundthatbothglucagonandinsulininrecipientswerehigherthanthoseinTTFsrecipientsat8weeks(Fig6c,d).ThesedatadeterminethatiAlphacellstransplantationdisturbthefunctionofislet.ThenwedetecttheisletchangesagainsttokidneytransplantediAlphacells.Interestingly,ProportionofKi67positivecellsintheisletofiAlphacellsrecipients,aswellasβ-cells,werehigherthanthatinTTFrecipients,proportionofα-cellsinisletweredecreased(Fig6d见下图，即原图FigTheseresultsdemonstrateiAlphacellsdecreasetheratioofα/βcellsinrecipients’islet.Thesemaybecausedbytheαtoβcellstrans-differentiationinislet.Remarkably,iAlphacellsdonotformtumors8weeksafterxeno-innudemice,while,α-TC,aαtumorcellline,formtumors(supplement7a)见下图，应该为supplementfigureMoreover,iAlphacellsingraftisPCNAnegative(supplementfigure6b),datasuggestediAlphacellsarenottumor-prone.此段内容见下图，即原图supplementfigureTherehasbeenreportthatα-cellscanserveasprogenitorsofβ-cells,Basedontheabovedata,wefoundtheenhancedproliferationofislets’cellsandincreasedβ-cellsiniAlphacellsrecipients’islet,whichenhancedratioofβ/αwewhichmaybecausedbydedifferentiationofαcells,ifso,thisnewroleofα-cellstogeneratenewβ-cellsmightbetheirmostimportantone.Thedefectinα-cellsthatoccursintypeIIdiabetescausesimpairedglucosesensing.Abnormalglucagonsecretionisinvolvedinbothhypoglycemiaandhyperglycemiaindiabetes.Glucagonsecretionisregulatedbyavarietyofnutrient,neuralandhormonalfactors(22).Followinganovernightfast,plasmaglucagonrisesonceglucosefallsbelowathreshold(23)anddecreasesprogressivelyuntilplasmaglucoserisesabovethenormalrange(24).Duetothelimitedaccessibilityandtyofα-cellspopulations,thefunctionsofα-cellsinthepancreaticisletsremainasanenigma.Exceptmaintainingplasmaglucoselevels,α-cellshavealsobeensupposedtoprotectandtogeneratenewβ-cells.However,thepossibilityandmechanismhasremainedcontroversialfortheabsenceofinvitroevidence,asitisnoteasytopurifyα-cellsinvitro.Inthisstudy,weexperimentallydemonstratetheprotocoltogeneratedα-cellslikecells.Byoverexpression5transcriptionfactors(TFs),mousetail-tipfibroblasts(TTFs)canbeconverteddirectlytomaturepancreaticcells(iAlpha)inconditionalculture.Inducedcellsexpresstheα-cellsmarkersGcgandArx.TransplantediAlphacellsinthekidneycapsuledisturbthemetabolismofrecipient,i.e.inducinginsulin promotingβ-cellproliferation.iAlphacellsshowthecharacteristicofprimaryα-Formanyyears,thedogmawasupheldthatterminallydifferentiatedcellswerecommittedtoaspecificfunctionandcouldnolongerchangetheiridentity.Incontrast,progenitor/stemcellsareexpectedtoretainsomemultipotencycapacities,andthereforecanbemoresuitableforreplacementstrategies.However,theES/iPSderivedendocrinecellsusuallyshowbi-function(coexpressinsulinandglucagon)andimpaireglucosesensing.LimitedavailabilityoftheisletsandES/iPSderivedendocrinecellsrestrictthedevelopmentofcellreplacementtherapyfordiabetes.Thus,findinganewresourceofisletcellswhichincludingα-andβ-cellsisdemanded.Reprogramingofisletcellsfromsomaticcellsmaybeanalternativeway.Nowadays,moreandmoreexamplesofefficient“transdifferentiation”havebeenreportedintheabsenceofapluripotentstate.However,reprogrammedendocrinecellscannotbegeneratedinvitropreviously.Here,wesuccessfullyconvertfibroblastsintoendocrinecellsbyoverexpressionofendocrinelineage-specifictranscriptionfactors.Suchconversionavoidsanintermediatepluripotentstateandessentiallyremovestheriskofteratomaformation.α-cellsspecificmarkerGcgexpressionwasdrasticallyinducedinTFstransducedcells.Thefunctionofα-cellsinthepancreaticisletsiswellknowntoproduceglucagoninthepost-absorptivestatetomaintainplasmaglucoselevelsbystimulatinghepaticglucoseproduction.Furthermore,generatedcellsexhibitmatureα-cellsphenotyperesemblingthatofprimarycells.Moreover,thesecellsacquireafullyfunctionalandmatureprofileafterengraftmentintothehosttissue.Oneinterestingfindingisthatgraftdisturbstheinsulin/glucagonequilibriumandaffectsthestabilityofbloodglucoselevels.Glucagonandinsulinmayconstituteafeedbacksystemthatstabilizing/maintainingbloodglucoselevel.thehigherglucagoniniAlphacellsrecipientsenhancedtheplasmainsulinlevels,whichmaybecausedbytheproliferationofβ-cellsorenhancedsecretionofinsulin.Andwefoundtheproliferationofβ-cellsintheiAlphacellsDuringembryogenesis,Gcgexpressioncanbedetectedatearlieststageintheendocrinecells(25).Basedonrecentstudies,α-cellshavebeenassignedanewroleintheisletsasdirectprogenitorsofβ-cells(26).Lineage-tracingstudiesshowedthatα-cellscanserveasprogenitorsofβ-cellsandpresentedahypotheticalmodelthatinjuredβ-cellsmightactivateα-cellsinadultisletstopromoteβ-cellregeneration(27,28).Inlightofthesefindings,β-cellsmaybegeneratedfromiAlphacellsbydirectconversion.Suchtechniquemayprovideanewplatformformoleculescreeningonβ-cellsconversion.Moreover,micetransplantedwithiAlphacellscanbeservicedasadiseasemodelofinsulintoidentifydrugsforthetreatmentofFinally,thesestudiesshowthatreplacementofendocrinetissuebytransplantationofinsulin-producingcellsthatderivedfromembryonicstemcellsisnottheonlyfeasibleapproachtoapermanenttreatmentfordiabetes.Tothecontrary,itisnowpossibletocompletetreatmentapproachesthatconvertα-cellstomakenewβ-cells.Therefore,Ourstudydemonstratesanovelnewstrategytogeneratefunctionalα-likecells.iAlphacellspresenttheα-cellsfunctionsinvitroandareabletodisturbthebloodglucoselevelinvivo,whichcanledtoinsulin-andincreasesβ-cellsproliferationinrecipents.AndiAlphacellsmaytakeimportantrolesindiabetesdiseasemodelingandregenerativeShapiro,A.M.,Lakey,J.R.,Ryan,E.A.,Korbutt,G.S.,Toth,E.,Warnock,G.L.,Kneteman,N.M.,andRajotte,R.V.(2000)TheNewEnglandjournalofmedicine343,230-238Turgeon,N.A.,Avila,J.G.,Cano,J.A.,Hutchinson,J.J.,Badell,I.R.,Page,J.,Adams,A.B.,Sears,M.H.,Bowen,P.H.,Kirk,A.D.,Pearson,T.C.,andLarsen,C.P.(2010)Americanjournaloftransplantation:officialjournaloftheAmericanSocietyofTransplantationandtheAmericanSocietyofTransplantSurgeons10,2082-2091Ryan,E.A.,Paty,B.W.,Senior,P.A.,Bigam,D.,Alfadhli,E.,Kneteman,N.M.,Lakey,J.R.,andShapiro,A.M.(2005)Diabetes54,2060-2069Ashcroft,F.M.,andRorsman,P.(2012)Cell148,1160-Xie,R.,Everett,L.J.,Lim,H.W., 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iAlphacells transducedby 5TFstablyexpressαcellsfunctionalgenesandαcellspecifictranscriptionfactors.a,OverlapofisletandTTF-iaenrichedgenes,selectedlevelwereislet/TTF>5andTTF-ia/TTF>5.Therewassignificantoverla