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Lineageconversionofmousefibroblaststopancreaticα-TianjinLiu*1,LiangliangSun2,BeigeJiang3,JinCen1,XiaotaoChen1,ZhaoyunZhang4,QinghuaWang5,XinCheng1,LijianHui*11.InstituteofBiochemistryandCellBiology,InstituteofHealthSciences,ShanghaiInstitutesforBiologicalSciences(SIBS),AcademyofSciences(CAS)（200031）2.DepartmentofEndocrinologyandMetabolism,ChangzhengHospital,SecondMilitaryMedicalUniversity,Shanghai,PR.（200003）3.DepartmentofHepaticSurgery,EasternHepatobiliarySurgeryHospital,SecondMilitaryMedicalUniversity,Shanghai,PR.（200438）4.DepartmentofEndocrinologyandMetabolism,HuashanHospital, 5.DivisionofEndocrinologyandMetabolism,theKeenanResearchCentreintheLiKaShingKnowledgeInstitute,St.Michael’sHospital,Toronto,Canada.AbbreviatedTitle:InvitroreprogrammingoffibroblasttoendocrineKeyterms:αWordcount:Numberoffiguresandtables:TheseauthorscontributeequallytothisCorrespondingauthorand towhomreprintrequestsshouldbeaddressed:TianjinLiu，PhDInstituteofBiochemistryandCellBiology,InstituteofHealthSciences,ShanghaiforBiologicalSciences(SIBS),AcademyofSciencesNo.320,RoadYueyang,Shanghai,PR，200031ThelaboratoryofL.H.isfundedbyNationalKeyBasicResearchandDevelopmentProgramof（2013CB967103）,strategicPriorityResearchProgramoftheAcademyofSciences（XDA ）,theHundredTalentsProgram（ NaturalScienceFoundationof（ ）andNaturalScienceFoundationofShanghaiMunicipality，PR（13ZR α-cellsareresponsibleforsynthesizingandsecretingthepeptidehormoneglucagontoelevatetheglucoselevelintheblood.Hence,thedysfunctionofα-cellswillresulttoseverehypoglycemiaandshock.Furthermore,α-cellsrequiredforislettransntationtherapyfordiabetes.Recentlystudiesshowα-cellssupportβ-cellssurvivalandareevenabletotrans-differentiatetoβ-incaseofdiabetes.However,theunderstandingofα-cellsinpathologicalandclinicalapplicationsremainselusiveduetounavailableofmatureα-cells.Herewepresentanewtechniquetogeneratefunctionalα-cells-likecells(iAlphacells).iAlphacellswereconvertedfrommousefibroblastsbytransductionoftranscriptionfactors(TFs)includingHhex,Foxa3,Gata4,Pdx1andPax4,whichcanspecificallyexpressα-cellspecificgenesandsecreteglucagoninresponsetoKclandArgstimulation.iAlphacellspresenttheα-cellsfunctionsinvitroandareabletodisturbthebloodglucoselevelinvivo.TransntationofiAlphacellsledthenudemicetoinsulin-andincreaseβ-cellsproliferation.Ourstudydemonstratesanovelnewstrategytogeneratefunctionalα-likecellsforthepurposeofdiseasemodelingandregenerativeCurrenttherapiesforthetreatmentoftype1diabetesincludedailyadministrationofexogenousinsulinand,lessfrequently,whole-pancreasorislettransntation.However,insulininjectionsoftenresultininaccurateinsulindoses,andexposingthepatienttohypoand/orhyperglycemicepisodesthatleadtolong-termcomplications.Transntationofisolatedisletsorentirepancreasestorecoverdysfunctionofpatient’sislethasyieldedsomepromisingandbloodglucosebalancecontrolledbythemisessentialformaintainingenergyhomeostasis.However,limitedavailabilityofhigh-qualityisletdonorshasrestrictedclinicalapplicationsofisletstransntation(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.Ithasshownthatdexpressionoflineage-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.WefounddHhex,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-positivecellsbydexpressionofPdx1invivo(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Fig1b（与空白对照Gfp组相比，Hhex组p＜0.01，Pdx1组0.01，Pax4组p＜0.01；Islet组为阳性对照组indicatingthatiHepcellscanbeconvertedtoglucagon-producingcells.Next,iHepcellsweretransducedwiththecombinationofPdx1,HhexandPax4toenhancesuchconversion.ExpressionsofGcgweresignificantlyincreasedupontransductionofPdx1,HhexandPax4.Moreover,anotherα-cell-markergene,Arx,wasalsoincreased(Fig.1c).见下图，即原图中Fig1c（0d组相比，D2-9组Arxp＜0.01，D5-9组Gcgp＜0.01；Islet组为阳性对照组Morphologically,iHepcellsalsotransferedfromtypicalepithelial-likemesenchymal-likewithPdx1,HhexandPax4over-expression(Fig.1d).图，即原图中FigSinceiHepcellswerederiveddirectlyfromTTFs,wedeterminedwhetherGcg-producingcellscouldbeacquireddirectlyfromTTFs.Wetransducedp19Arf-/-TTFswithiHepinductionfactors,Gata4,Hnf1aandFoxa3,andPdx1,Pax4(collectivelyreferredas6TF),whichcouldformclones14dayslater1e).见下图，即原图中FigGcgmRNAexpressionweredetectedafter6TFsinfection(Fig1f).原图中Fig1gWenextremovedindividualfactorsfromthecombinationofGcgexpressionincreaseduponremovingofHnf1afrom6TF(Fig1f).Fig1g（6TFHnf1ap＜0.01，Islet组Hence,wetransducedTTFsbythecombinationtoGata4,Pdx1,Hhex,andFoxa3(collectivelyreferredtoas5TF).Removinganyfactorfrom5TFreducetheexpressionofGcg(Fig.1g,Supplementaryfigure1a).图中Fig1h（与5TF组相比，移除Hhex/Foxa3组的p＞0.01，移除1/Gata4/Pax41/Gata4/Pax4组的p＞0.05，Gfp Markedly,GcgexpressionwasnotinduceduponwithdrawalofHhexorFoxA3,Incontrast,over-expressionofHhexandFoxa3werealreadysufficientinduceGcgexpression(SupplementaryfigureHowever,mRNAlevelsofGcgandArxin,HhexandFoxa3inducedcellssignificantlylowerthanthatin5TFtransducedcells(Supplementaryfigure见下图，即原图中supplementaryFig1b（与5TFs相比，过表达HhexFoxa3组，Gcg和ArxmRNA5TFsProteinlevelsofglucagonwerealsoexpressedatalowlevelinHhexFoxa3inducedcellscomparewithiAlphacells(SupplementaryfigurePreviousstudieshaveshownthatcultureconditioniscriticalfordifferentiationofendocrinecellsinvitro(20).WenextimprovedGcginductionbyoptimizingtheculturemedium.Wefoundoneconditionalmedium,whichcontainedneuralbasalmedium,N-2supplement,B-27supplement,nicotianamineandbFGF,wasmorepotentininducingGcgexpressionthanDMEM,RPMI1640,DMEM-F12media(Fig.1h).见下图，即原图Fig2a（DMEM件培养基组的GcgmRNA表达显著增高，p＜0.01，Islet是阳性对照组TheexpressionofGcgincreasedgraduallyduringtheinduction,thattheconversionisaprogressiveprocess(Fig.1i).见下图，即原图Fig（0d组相比，7d、14d、28d组的GcgTakentogether,weconcludedthatTTFareconvertedintoGcg-producingcellswith5TFoverexpressionandneural-basalmedium-basedconditional(Fig.1j5Gata4Pdx1HhexPax4，Foxa3理的成纤维细胞（5TF），能够转化成胰岛α样细胞（iAlphacells），Todeterminetheindividualfunctionof5TFininduction,wemonitoredtheexpressionsoflineagespecificmarkersafterremovingindividualfactorsfrom5TF,includingendodermmarker(Emoes,Wnt3a,Gata6),pancreaticprogenitormarker(Sox9),liverspecificmarker(Alb),βcellspecificmarker(Mafa),δcellspecificmarker(sst)andexocrinecellspecificmarker(Amy2)withdrawalanyfactorfrom5TFswillinducetheup-regulationofdifferent(Supplementaryfigure(Supplementaryfigure2).这里应该 的Fig2blineagemarkers做修改。见下图：此处可 Supplementaryfigure3的结果Overall,5TFinfectioninwildtypeTTFandpancreaticfibroblasttriggeredandArxexpression(SupplementaryFigure3a,b)见下图，即原图supplementaryFig2b,5TFsGcgArx表达明显升高,（GFP组相比，5TFs组中的Gcg和ArxmRNA表达明显升高，p＜0.01，Islet阳性对照组）indicatingthepossibilitytoinduceα-cellfromfibroblastinp19Arf-/-TTFswereconvertedintoαcell-likeWecomparedtheglobalexpressionprofilesamongiAlphacells,TTFs,andislet. upregulatediniAlphacellscomparedtoTTFs(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)wereundetectableorexpressedatlowlevel(Fig2b见下图，即原图Fig2d，但并未检测Amy2a做修改。（与TTF组相比，iAlpha组的α细胞特 表达明显高于TTF组PP＜0.01，β、δ细胞特异表 与TTF组类似，p＞0.01，islet组是阳性照组TheculturedisletsandiAlphacellsshowsimilarpatternofglucagon(Fig2c).见下图，即原图Fig2cThedatasuggestedthatTTFswereconvertedintoα-cellslikecells(iAlphacells),butnototherpancreaticlineagecells.Toassaythestabilityofconversion,wedetecteddynamicchangesofexpression,andnoticedthat5TFsweresilencediniAlphacellsFigure5a-e)见下图，即原图Fig3a-e（14diAlphacells相比，Gata4/Pax4/Hhex/FoxA3/Pdx1随着时间的延长表达逐渐下降，至时上述mRNA表达与0d时无明显差异，p＞0.05）.TheprogenitorGata6andWnt3awereup-regulatedatday5(SupplementaryFigure5f,g图，即原图Fig3f,g（0d相比，Gata6/Wnt3a5d0.01）,whileα-cellspecificmarkerIrx2maintainhighexpressionduringcourse(SupplementaryFigure5h)见下图，即原图Fig3h（0d时间的延长，αmarkerIrx2mRNA14d高水平，P＜0.01）.Hence,α-cellconversionmayundertakeaprocessofreversiontoprogenitors.CharacterizationofiAlphacellsThereceptorsofglucose,insulinandglucagoninα-cellarecriticalforglucagonsenseandsecreting(21).Therefore,wedetectedandfoundthesewereup-regulatediniAlphacells(Fig.3a),见下图，即原图Fig4a（与TTF比，iAlphacells中GcgR、InsR、Glut2、TtrmRNAIslet是阳性对照组GlucagoncontentiniAlphacellswere50percentofthatinislets(Fig.3b),insulinproteincouldnotbedetectediniAlphacells(Fig3c).BiologicallyactiveglucagonsecretionwasdetectediniAlphacells,iAlphaexhibitedsimilarglucagonsecretionpropertyinresponsetoKclandArgstimulationasislets,suggestingthatglucagonbutnotinsulinsecretioncanphysiologicallyregulatedinthesecells(Fig3d,e)见下图，即原图Fig4b-Notably,iAlphacellsestablishedα-cellfunctionsinToverifythephysiologicfunctionofiAlphacells,sensitivitytostimuluswastested.Nudemiceweretransntedwith1*106iAlphacellsorTTFsinkidneycapsule.Bodyweights,insulintolerancetest(ITT)andglucosetolerance(GTT)weremeasuredat2,4and8weeksaftertransntation.At1-2weeks,therewasnosignificantdifferencebetweenbodyweightsofnudetransntedwithTTFsandiAlphacells(Fig.4a,SupplementaryFigure6a).即原图Fig5a（P＞0.05）.iAlphacellrecipientsexhibitedsimilarresponseITTandGTTatfirstweek(SupplementaryFigure6b,c即原图Fig5b-cat2weeks,wefoundadelayedbloodglucoselevelsreduction(Fig4b)Fig5b,andthebehaviorofiAlphacellrecipientsaremoreactivethanrecipients(supplementarymovie1),Movie1未提供indicatingthatiAlphacellswasnosignificantdifferencebetweennudemicetransntedwithTTFsoriAlphacellsat2weeks(Fig.4c)即原图Fig5cAt4weeks,thebodyweightsofiAlphacellstransntednudemicesignificantlydecreasedcomparedTTFstransntedones(Fig.4d)即原图Fig5d.Therewassignificantdifferenceofbloodglucoseinfastandnormalstatecomparedwiththatoftransntedanimals(Fig.4e,f)即原图Fig5e-f.ITTresultsshowthattherehavesignificantdifferenceiniAlphaandTTFtransntedmice(Fig.4e)即原图Fig5e.At8weeksaftertransntation,bodyweightsofiAlphacellstransntedmicerecovered(Fig.4g)即原图Fig5g,however,therewasnosignificantdifferenceofITTandGTTcomparedwithcontrolanimals(Fig.4h,i即原图Figi.Therefore,iAlphacellspresenttheα-cellsfunctionsinvitroandareabledisturbthebloodglucoseregulationinvivo.这一段就是在讲FigiAlphacellsinducetheregenerationofisletcellsinThefunctionofiAlphacellsweredecreasedat8weeksaftertransntation,sowedetectthesurvivalofiAlphacellsintherecipients,andfoundgraftcanbeseeninthecapsuleofrecipient’skidney(Fig.6a),andthesecellsglucagon-positivebutnotinsulin-positive(Fig6b,SupplementaryFigure7c).里是指里是指Fig6a-b，需 Confusingly,at8weeks,survivediAlphacellsdon’ttakeimportantrolesregulatingglucose.Wedetectedthesecretionofinsulinandglucagon smaofrecipients,andfoundthatbothglucagonandinsulininrecipientswerehigherthanthoseinTTFsrecipientsat8weeks(Fig6c,d).ThesedatadeterminethatiAlphacellstransntationdisturbthefunctionofislet.ThenwedetecttheisletchangesagainsttokidneytransntediAlphacells.Interestingly,ProportionofKi67positivecellsintheisletofiAlpharecipients,aswellasβ-cells,werehigherthanthatinTTFrecipients,proportionofα-cellsinisletweredecreased(Fig.6d).见下图，即原图FigTheseresultsdemonstrateiAlphacellsdecreasetheratioofα/βcellsinrecipients’islet.Thesemaybecausedbytheαtoβcellstrans-differentiationinislet.Remarkably,iAlphacellsdonotformtumors8weeksafterxeno-graftnudemice,while,α-TC,aαtumorcellline,formtumors(supplementfigure见下图，应该为supplementfigureMoreover,iAlphacellsingraftisPCNAnegative(supplementfigure6b),datasuggestediAlphacellsarenottumor-prone.此段内容见下图，即原图supplementfigure6b.Therehasbeenreportthatα-cellscanserveasprogenitorsofβ-cells,Basedontheabovedata,wefoundtheenhancedproliferationofislets’cellsandincreasedβ-cellsiniAlphacellsrecipients’islet,whichenhancedratioofβ/αwewhichmaybecausedbydedifferentiationofαcells,ifso,thisnewroleofα-cellstogeneratenewβ-cellsmightbetheirmostimportantone.Thedefectinα-cellsthatoccursintypeIIdiabetescausesimpairedglucosesensing.Abnormalglucagonsecretionisinvolvedinbothhypoglycemiaandhyperglycemiaindiabetes.Glucagonsecretionisregulatedbyavarietyofnutrient,neuralandhormonalfactors(22).Followinganovernightfast,smaglucagonrisesonceglucosefallsbelowathreshold(23)anddecreasesprogressivelyuntilsmaglucoserisesabovethenormalrange(24).Duetothelimitedaccessibilityandtyofα-cellspopulations,thefunctionsofα-cellsinthepancreaticisletsremainasanenigma.Exceptmaintainingsmaglucoselevels,α-cellshavealsobeensupposedtoprotectandtogeneratenewβ-cells.However,thepossibilityandmechanismhasremainedcontroversialfortheabsenceofinvitroevidence,asitisnoteasytopurifyα-cellsinvitro.Inthisstudy,weexperimentallydemonstratetheprotocoltogeneratedα-cellslikecells.Byoverexpression5transcriptionfactors(TFs),mousetail-tipfibroblasts(TTFs)canbeconverteddirectlytomaturepancreaticcells(iAlpha)inconditionalculture.Inducedcellsexpresstheα-cellsmarkersGcgandArx.TransntediAlphacellsinthekidneycapsuledisturbmetabolismofrecipient,i.e.inducinginsulin andpromotingβ-cellproliferation.iAlphacellsshowthecharacteristicofprimaryα-cells.Formanyyears,thedogmawasupheldthatterminallydifferentiatedcellswerecommittedtoaspecificfunctionandcouldnolongerchangetheiridentity.Incontrast,progenitor/stemcellsareexpectedtoretainsomemultipotencyHowever,theES/iPSderivedendocrinecellsusuallyshowbi-function(coexpressinsulinandglucagon)andimpaireglucosesensing.LimitedavailabilityoftheisletsandES/iPSderivedendocrinecellsrestrictthedevelopmentofcellrecementtherapyfordiabetes.Thus,findinganewresourceofisletcellswhichincludingα-andβ-cellsisdemanded.Reprogramingofisletcellsfromsomaticcellsmaybeanalternativeway.Nowadays,moreandmoreexamplesofefficient“transdifferentiation”havebeenreportedintheabsenceofapluripotentstate.However,reprogrammedendocrinecellscannotbegeneratedinvitropreviously.Here,wesuccessfullyconvertfibroblastsintoendocrinecellsbyoverexpressionofendocrinelineage-specifictranscriptionfactors.Suchconversionavoidsanintermediatepluripotentstateandessentiallyremovestheriskoftomaformation.α-cellsspecificmarkerGcgexpressionwasdrasticallyinducedinTFstransducedcells.Thefunctionofα-cellsinthepancreaticisletsiswellknowntoproduceglucagoninthepost-absorptivestatetomaintainsmaglucoselevelsbystimulatinghepaticglucoseproduction.Furthermore,generatedcellsexhibitmatureα-cellsphenotyperesemblingthatofprimarycells.Moreover,thesecellsacquireafullyfunctionalandmatureprofileafterengraftmentintothehosttissue.Oneinterestingfindingisthatgraftdisturbstheinsulin/glucagonequilibriumandaffectsthestabilityofbloodglucoselevels.Glucagonandinsulinmayconstituteafeedbacksystemthatstabilizing/maintainingbloodglucoselevel.thehigherglucagoniniAlphacellsrecipientsenhancedthesmainsulinlevels,whichmaybecausedbytheproliferationofβ-cellsorenhancedsecretionofinsulin.Andwefoundtheproliferationofβ-cellsintheiAlphacellsrecipents.Duringembryogenesis,Gcgexpressioncanbedetectedatearlieststageinendocrinecells(25).Basedonrecentstudies,α-cellshavebeenassignedanewroleintheisletsasdirectprogenitorsofβ-cells(26).Lineage-tracingstudiesshowedthatα-cellscanserveasprogenitorsofβ-cellsandpresentedahypotheticalmodelthatinjuredβ-cellsmightactivateα-cellsinadultisletstopromoteβ-cellregeneration(27,28).Inlightofthesefindings,β-cellsmaybegeneratedfromiAlphacellsbydirectconversion.Suchtechniquemayprovideanewtformformoleculescreeningonβ-cellsconversion.Moreover,micetransntedwithiAlphacellscanbeservicedasadiseasemodelofinsulintoidentifydrugsforthetreatmentofFinally,thesestudiesshowthatrecementofendocrinetissuebytransntationofinsulin-producingcellsthatderivedfromembryonicstemcellsisnottheonlyfeasibleapproachtoapermanenttreatmentfordiabetes.Tothecontrary,itisnowpossibletocompletetreatmentapproachesthatconvertα-cellstomakenewβ-cells.Therefore,Ourstudydemonstratesanovelnewstrategytogeneratefunctionalα-likecells.iAlphacellspresenttheα-cellsfunctionsinvitroandareabletodisturbthebloodglucoselevelinvivo,whichcanledtoinsulin- 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iAlphacells transducedby 5TFstablyexpressαcellsfunctionalgenesandαcellspecifictranscriptionfactors.a,OverlapofisletandTTF-iaenrichedgenes,selectedlevelwereislet/TTF>5andTTF-ia/TTF>5.Therewassignificantoverlapbetweenisletandb,Pathwayysis.Forthe20pathwayswhichmostlyactiveinislet,therewere17overlappathwaysinTTF-ia,