Treatment with specific soluble factors promotes the functional maturation of transcription factor

26 Pages·2017·26.14 MB·English

RESEARCHARTICLE
Treatment with specific soluble factors
promotes the functional maturation of
transcription factor-mediated, pancreatic
transdifferentiated cells
HiroakiMotoyama*,AkiraKobayashi,TakahideYokoyama,AkiraShimizu,HiroshiSakai,
TsuyoshiNotake,KentaroFukushima,Shin-ichiMiyagawa
a1111111111 FirstDepartmentofSurgery,ShinshuUniversitySchoolofMedicine,Matsumoto,Nagano,Japan
a1111111111
*[email protected]
a1111111111
a1111111111
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Abstract
Pancreaticlineage-specifictranscriptionfactors(TFs)displayinstructiverolesinconverting
adultcellstoendocrinepancreaticcellsthroughaprocessknownastransdifferentiation.
OPENACCESS However,littleisknownaboutpotentialfactorscapableofacceleratingtransdifferentiation
Citation:MotoyamaH,KobayashiA,YokoyamaT, followingtransductiontoachievethefunctionalmaturationoftransdifferentiatedcells.Inthis
ShimizuA,SakaiH,NotakeT,etal.(2018) study,wedemonstrated,usingadultliver-derivedprogenitorcells,thatsolublefactorsuti-
Treatmentwithspecificsolublefactorspromotes
lizedinpancreaticdifferentiationprotocolsofpluripotentstemcellspromotefunctionalmatu-
thefunctionalmaturationoftranscriptionfactor-
rationofTFs-mediatedtransdifferentiatedcells.TreatmentwithanN2supplementin
mediated,pancreatictransdifferentiatedcells.PLoS
ONE13(5):e0197175.https://doi.org/10.1371/ combinationwiththreesolublefactors(glucagon-likepeptide-1[GLP-1]receptoragonist,
journal.pone.0197175 notchinhibitor,andtransforminggrowthfactor-β[TGF-β]inhibitor)enhancedliver-to-pan-
Editor:WataruNishimura,InternationalUniversity creastransdifferentiationbasedonthefollowingfindings:i)theincidenceofc-peptide-posi-
ofHealthandWelfareSchoolofMedicine,JAPAN tivecellsincreasedbyapproximately1.2-foldaftertheaforementionedtreatment;ii)thec-
Received:October22,2017 peptideexpressionlevelinthetreatedcellsincreasedbyapproximately12-foldascom-
paredwiththelevelintheuntreatedcells;iii)thetreatedcellssecretedinsulininaglucose-
Accepted:April28,2018
dependentmanner,whereastheuntreatedcellsdidnot;andiv)transplantationoftreated-
Published:May16,2018
transdifferentiatedcellsintostreptozotocin-inducedimmunodeficientdiabeticmiceledto
Copyright:©2018Motoyamaetal.Thisisanopen
theameliorationofhyperglycemia.Theseresultssuggestthattreatmentwithspecificsolu-
accessarticledistributedunderthetermsofthe
blefactorspromotesthefunctionalmaturationoftransdifferentiatedcells.Ourfindingscould
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and facilitatethedevelopmentofnewmodalitiesforcell-replacementtherapyforpatientswith
reproductioninanymedium,providedtheoriginal diabetes.
authorandsourcearecredited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation
files.
Funding:Thisworkwassupportedinpartby
Introduction
fundingfromGrant-in-AidforScientificResearch
toShin-ichiMiyagawa(No.21390375,https://
Allogeneicislettransplantationoffersaminimallyinvasiveoptionforβ-cellreplacementin
kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-
patientswithtype1diabetes(T1D).However,thewidespreadapplicationofthistreatmentis
21390375/)andtoAkiraKobayashi(No.22591516,
limitedbecauseofthescarcityofdonortissueandhealthconcernsassociatedwiththechronic
https://kaken.nii.ac.jp/en/grant/KAKENHI-
PROJECT-22591516/)(No,25462108,https:// useofimmunosuppressivedrugsintherecipient.Toovercometheselimitations,effortshave
PLOSONE|https://doi.org/10.1371/journal.pone.0197175 May16,2018 1/26
Cellulartransdifferentiationandsolublefactors
kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT- beenfocusedoninsulin-producingcellsderivedinvitrofromhumanpluripotentstemcells[1,
25462108/)fromtheJapanSocietyforthe 2].Inparticular,recentadvancesintheuseofhumaninducedpluripotentstemcells(hiPSCs)
PromotionofScience(Tokyo,Japan).
haveenabledtheproductionoffunctionalinsulin-producingcellswithmanycharacteristics
Competinginterests:Theauthorshavedeclared thatcloselyresemblethoseofbonafideβcellsinvitro[2].Thissuccessmarksthebeginningof
thatnocompetinginterestsexist. anoveltransplantationtreatmentfordiabetesusingpatient-derivedhiPSCsthatcouldelimi-
natetheneedforimmunosuppression.However,sincetheengraftmentoftransplantedislets
hasneverbeensatisfactoryinT1Dpatients(withaninsulinindependencerateoflessthan
50%at3yearsaftertransplantation[3]),theengraftmentpotentialmightbearate-limiting
stepinhiPSCs-basedcelltherapy,despitethefactthatcelltransplantationisindispensablefor
hiPSCs-basedcelltherapy.Furthermore,anotherconcernisthatpatientswithT1Dmaynot
benefitfrompersonalizedhiPSCs-derivedβ-cellsbecauseofautoimmunerejectionofthe
reconstitutedβ-cells.Hence,forclinicalapplicationofhiPSC-basedcelltherapy,development
ofanimmunoprotectivemethod(suchasmacro-ormicro-encapsulation[4])isrequired,in
parallelwithfurtherimprovementsinpancreaticinductionprotocols.
Alternatively,transdifferentiationisaprocessinwhichoneadultcelltypeisdirectlycon-
vertedintoanalternatecelltypewithadifferentfunction[5].Theectopicexpressionsofline-
age-specifictranscriptionfactors(TFs)havebeensuggestedtodisplayinstructiverolesinthese
processes[6,7].UnlikehiPSCs,althoughtheinducibilityofTFs-mediatedtransdifferentiation
variesaccordingtotheTFsthatarebeingtransduced,transdifferentiationoccurswithinafew
daysoncetheappropriateTFshavebeenadministered[8,9].Furthermore,thepotential
advantageofTFs-mediatedtransdifferentiationisthatfunctionaltransdifferentiatedcellscan
beinducedinsituintargetedtissues.Thus,TFs-mediatedtransdifferentiationcouldenablethe
establishmentofaregenerativetherapeuticmodalitywithouttheneedforcellular
transplantation.
Recently,Baeyensetal.showedthatitispossibletoinducepancreaticexocrine-to-endo-
crinetransdifferentiationartificiallyusingthetransientadministrationofepidermalgrowth
factorandciliaryneurotrophicfactor[10].Althoughthisapproachisexpectedtoestablisha
transgene-freecellulartransdifferentiationstrategy,itsimultaneouslyraisesthepossibilitythat
therearespecificsolublefactorsthatactsynergisticallywithtransducedTFstopromotecellu-
lartransdifferentiation.However,littleattentionhasbeenpaidtoexploringfactorsthatare
suitableforTFs-mediatedtransdifferentiation.
Inthecurrentstudy,weanalyzedtheinducibilityofTFs-mediatedcellulartransdifferentia-
tionusingportalbranchligation-stimulatedhepaticcells(PBLHCs),whichwerebipotent
hepaticprogenitorcellclonesthatwereisolatedfromtheportalbranch-ligatedliverlobesof
adultmiceatourinstitution[11],byculturingthecellsinaserum-freemediumcontaining
specificsolublefactorsusingathree-dimensional(3D)cellularaggregationtechnique.We
foundthattreatmentwithanN2supplementincombinationwiththreesolublefactors(gluca-
gon-likepeptide-1[GLP-1]receptoragonist,notchinhibitor,andtransforminggrowthfactor-
β[TGF-β]inhibitor)promotefunctionalmaturationofTFs-mediatedtransdifferentiatedcells.
Thisresultcouldleadtoanoveltherapymodalityfordiabetes.
Materialsandmethods
Portalbranchligation-stimulatedhepaticcells
Theportalbranchligation-stimulatedhepaticcells(PBLHCs)werebipotenthepaticprogeni-
torcellclonesthatwereisolatedfromtheportalbranch-ligatedliverlobesofadultmiceatour
institution.TheisolationofPBLHCswasperformedasdescribedpreviously[11].Briefly,8-to
12-week-oldC57Bl/6Jmice(CLEAJapan,Tokyo,Japan)wereusedfortheportalveinligation
experiments.Themiceweresacrificedonday7afterportalveinligation.Theanimalprotocol
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Cellulartransdifferentiationandsolublefactors
wasapprovedbytheAnimalExperimentationCommitteesofShinshuUniversity.Isolated
livercellswerepreparedusinga2-stepcollagenaseperfusiontechnique,asdescribedprevi-
ously[12].Thenon-parenchymalcellfractionwasplatedinalaminin-coatedplate(BDBiosci-
ences,FranklinLakes,NJ)andculturedinWilliam’sEmediumsupplementedwith5%fetal
bovineserum(Sigma-Aldrich,St.Louis,MO),1×penicillin/streptomycin/glutamine(Thermo
FisherScientific,Waltham,MA),recombinantmouseepidermalgrowthfactor(20ng/mL;
R&DSystems,Minneapolis,MN),nicotinamide(10mmol/L;Sigma-Aldrich),1×Insulin–
Transferrin–SodiumSeleniteSupplement(RocheDiagnostics,Mannheim,Germany),and
dexamethasone(0.1uM;Sigma-Aldrich)withahumidified5%CO /95%airatmosphereat
2
37˚C.Aforementionedculturemediumwasdenotedasserum-containingmedium(SCM)in
thiscontext.After2–4months,theproliferationofepithelial-likecolony-formingsubpopula-
tionswasobserved,andthecellswereclonedusingthelimitingdilutionmethod,accordingto
theproceduredescribedbyParketal.[13].Wenamedthesecolony-formingcellsasPBLHCs,
andthecellswereusedinsubsequentexperimentsasapossiblesourceofTFs-mediatedcellu-
lartransdifferentiation.
βTC6cells
MouseβTC6cells[14],purchasedfromATCC,weremaintainedinDulbecco’sModified
Eagle’smedium(ThermoFisherScientific)supplementedwith15%fetalbovineserum
(Sigma-Aldrich),1×penicillin/streptomycin/glutamine(ThermoFisherScientific),and1×
MEMNon-EssentialAminoAcidsSolution(ThermoFisherScientific).Allcellsweregrownat
37˚Cinahumidifiedairincubatorinthepresenceof5%CO .
2
Adenovirusvectors
RecombinantadenovirusesexpressingpancreaticTFsofmousetogetherwithnGFPwere
kindlyprovidedbyDouglasMelton.Thedetailsoftheplasmidswereasfollows:pAdPdx1-I-
nGFP(Addgeneplasmid#19411),pAd2BNgn3-I-nGFP(#19410),pAdNeuroD-I-nGFP
(#19414),andpAdMafA-I-nGFP(#19412)[7].Adenovirusconstructedforthe2Apeptide-
mediatedpolycistronicexpressionoffourgenes(Pdx1,Ngn3,MafA,andmonomericcherry
fluorescentprotein[mCherry])wasalsoprovidedbyAddgeneasagenerousgiftfromQiao
Zhou(pAd-M3cherry,#61041)[15].pAd-M3cherrywasdenotedaspAd-PNMcherry
(PNMc)inthiscontexttoclarifythedifferenceintransducedTFs.Theviraltiterswereesti-
matedusingtheAdeno-XqPCRTitrationKit(Clontech)accordingtothemanufacturer’spro-
tocol.TheoptimalMOIwasdeterminedasatiterachievinganinfectionrateofabove80%in
culturedPBLHCsat24hoursafterinfectionwiththemanualcountingofnGFP-positivecells.
SincemCherryfluorescencewashardlyvisibleat24hoursafterinfection,theexperiments
wereconductedusingaviraltitersimilartothatofthenGFPexpressionvector.TheMOIsof
thevirusesusedintheexperimentswereasfollows:pAdPdx1-I-nGFP,5MOI;pAd2B
Ngn3-I-nGFP,5MOI;pAdNeuroD-I-nGFP,5MOI;pAdMafA-I-nGFP,50MOI;andpAd-
M3cherry,5MOI.
Serum-freedifferentiationmediumcontainingspecificcompounds
(SFDM)forinvitrotransdifferentiation
Toanalyzewhetherliver-to-pancreasdirecttransdifferentiationisfacilitatedbyspecificcom-
pounds,wepreparedaserum-freedifferentiationmediumcontainingspecificsolublefactors
(SFDM).SFDMwaspreparedbyreferringtotheprotocolsforgeneratingpancreaticendo-
crinecellsfromhiPSCs/humanembryonicstemcells(hESCs),especiallythemoleculesusedin
theprocessofendocrinelineagecommitment[1,16,17].Inbrief,SFDMwasamedium
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Cellulartransdifferentiationandsolublefactors
supplementedwithbelow-mentionedmoleculesandsupplementsforbasalmediummainly
composedofIMDMandHamm’sF12(IHmedium).Theselectedmoleculesandsupplements
wereasfollows:epidermalgrowthfactor(EGF),exendin-4(GLP-1receptoragonist),forskolin
(adenylatecyclaseactivator),L685,458(notchinhibitor),nicotinamide,noggin(inhibitorof
bonemorphogeneticprotein[BMP]pathway),SB431542(inhibitorofTGF-βtype1receptor),
N2,andB27supplement.ThedetailedformulationsofSFDMaredescribedintheTable1.
Cultureformatsforinvitrotransdifferentiation
PBLHCsobtained1dayafterthetransductionofTFswithadenovirusvectorweretrypsinized
andre-seededeitherforadherentculture(AC)(2.5×104cells/cm2)orsuspensionculture
(SC)(5.0×105cells/mL).ToformcellularaggregatesinSC,thedetachedcellswerealiquoted
into15-mLconicaltubesandpreincubatedstaticallyinahumidifiedatmosphereof5%CO /
2
95%airat37˚Cfor2hours.Then,thecellswerere-seededinto24-wellultra-lowattachment
culturedishes(Corning,Corning,NY)withappropriatemediums.
Semi-quantitativeRT-PCRandreal-timequantitativeRT-PCR(qRT-PCR)
TotalRNAwasisolatedfromatleasttriplicatesamplesinthreeindependentexperimentsusing
anRNeasyplusminikit(Qiagen,Venlo,Netherlands),followedbycDNAsynthesisusingstan-
dardprotocols.Semi-quantitativeRT-PCRwasperformedasdescribedpreviously[9].The
primersequencesusedforsemi-quantitativeRT-PCRareavailableuponrequest.qRT-PCRwas
performedintriplicateusingEppendorfMastercyclerepGradientS(Eppendorf,Hamburg,
Germany)withtheTaqManGeneExpressionAssay(AppliedBiosystems,Waltham,MA).The
cDNAsamplessubjectedtoqRT-PCRweresynthesizedfrom500ngoftotalRNAusingPrime-
ScriptRT-PCRKit(TaKaRaBio).Theassaymixesthatwereanalyzedwereasfollows:insulinI
(Mm01259683_g1),insulinII(Mm00731595_gH),glucagon(Mm00801714_m1),somatostatin
Table1. Formulationofserumfreedifferentiationmediumcontainingspecificcompounds(SFDM).
Reagent Company Workingconcentration
Basalmedium(IHmedium)
IMDM ThermoFisherScientific 75%
Ham’sF12 ThermoFisherScientific 25%
Penicillin/Streptomycin/Glutamine ThermoFisherScientific 1%
Ascorbicacid Sigma-Aldrich 50μg/mL
BSA Sigma-Aldrich 0.05%
Glucose Wako 40mmol/L
Molecules(mixtureof7molecules,M7)
Epidermalgrowthfactor R&DSystems 50ng/mL
Exendin-4 Sigma-Aldrich 5nmol/L
Forskolin Wako 10μmol/L
Nicotinamide Sigma-Aldrich 10mmol/L
Noggin TONBOBioscience 50ng/mL
L685,458 PeptideInstitute 1μmol/L
SB431542 CaymanChemical 6μmol/L
Supplements
B27supplement ThermoFisherScientific 1%
N2supplement ThermoFisherScientific 1%
EGF,epidermalgrowthfactor;BSA,bovineserumalbumin.
https://doi.org/10.1371/journal.pone.0197175.t001
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Cellulartransdifferentiationandsolublefactors
(Mm00436671_m1),pancreaticpolypeptide(Mm01250509_g1),proproteinconvertasesubtili-
sin/kexintype1(Mm00479023_m1),proproteinconvertasesubtilisin/kexintype2(Mm00500981_
m1),ATP-bindingcassette,sub-familyC(CFTR/MRP),member8(Mm00803450_m1),solute
carrierfamily2(facilitatedglucosetransporter),member2(Mm00446229_m1),pancreatic
andduodenalhomeobox1(Mm00435565_m1),neurogenin3(Mm00437606_s1),neurogenic
differentiation1(Mm01946604_s1),v-mafmusculoaponeuroticfibrosarcomaoncogene
family,proteinA(avian)(Mm00845206_s1),pairedbox4(Mm01159036_m1),pairedbox6
(Mm00443081_m1),ISL1transcriptionfactor,LIM/homeodomain(Mm00517585_m1),arista-
lessrelatedhomeobox(Mm00545903_m1),NK6homeobox1(Mm00454961_m1),albumin
(Mm00802090_m1),cytochromeP450,family1,subfamilya,polypeptide2(Mm00487224_
m1),CCAAT/enhancerbindingprotein(C/EBP),beta(Mm00843434_s1),transferrin
(Mm00446715_m1),keratin19(Mm00492980_m1),alphafetoprotein(Mm00431715_m1),
andglyceraldehyde-3-phosphatedehydrogenase(Gapdh;Mm99999915_g1).Theexpression
levelsofeachgenewerenormalizedtothoseofGapdhinthesamesamples.
Measurementofintracellularcontentofc-peptide
ThesamplesofinfectedPBLHCswerewashedfourtimeswithPBSandthentreatedwithacid-
ethanol(0.18mol/Lhydrochloricacidin95%ethanol)at4˚Covernight.Theclearsuperna-
tantswereusedtoassaytheintracellularinsulinandc-peptidecontents,andthevalues
obtainedwerenormalizedrelativetothetotalproteincontent.Thec-peptidecontentswere
measuredusinganenzyme-linkedimmunosorbentassay(MouseC-peptideELISAKit,Shi-
bayagi,Gunma,Japan).ThetotalproteincontentwasmeasuredusingProteinAssay(Bio-Rad,
Hercules,CA).
Glucose-stimulatedinsulinsecretionassay
Transdifferentiatedcellularaggregates(almost200aggregates/assay)treatedwithSCMor
SFDMweresampled.TheaggregateswerewashedwithKrebs-RingerbicarbonateHEPES
buffer(KRBH:116mmol/LNaCl,4.7mmol/LKCl,2.5mmol/LCaCl ,1.2mmol/LKH PO ,
2 2 4
1.2mmol/LMgSO ,24mmol/LHEPES,25mmol/LNaHCO ,and0.1%BSA)andwerethen
4 3
preincubatedinlow(2.5mmol/L)glucoseKRBHfor2hourstoremoveresidualinsulin.
AggregateswerewashedtwotimesinKRBH,incubatedinlow-glucoseKRBHfor60min,and
thesupernatantwascollected.Then,theaggregateswerewashedtwotimesinKRBH,incu-
batedinhighglucose(22.5mmol/L)KRBHfor60min,andthesupernatantwascollected
again.ThesupernatantsamplescontainingsecretedinsulinwereprocessedusingtheMouse
InsulinELISAKit(Shibayagi).
Immunohistochemicalanalysis
TheinfectedPBLHCstreatedwithACorSCwerepreparedindividuallyforimmunohisto-
chemicalanalysisasfollows:samplestreatedwithACwerefixedwith4%paraformaldehyde/
PBS(PFA/PBS)for20minandthenpermeabilizedwith0.1%TritonX/PBSfor10minat
roomtemperature.ThecellularaggregatesformedbySCwerecollectedandembeddedin
iPGell(Genostaff,Tokyo,Japan)accordingtothemanufacturer’srecommendations.The
embeddedsampleswerefixedwith4%PFA/PBSovernightatroomtemperatureandsectioned
forhistologicalanalysis.Immunohistochemicalstainingwasperformedaspreviously
described[9,11];theantibodiesaredetailedinSupplementalTable1.Representativeimages
weretakenusinganBZ-8000FluorescenceMicroscope(Keyence,Osaka,Japan)orOlympus
BX60FluorescenceMicroscope(Olympus,Tokyo,Japan).
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Cellulartransdifferentiationandsolublefactors
Measurementofc-peptide-positivecells
ThecellularaggregatesformedbySCweredispersedintoasingle-cellsuspensionbyincuba-
tioninTrypLEExpress(ThermoFisherScientific)at37˚C,then3μLofthecellsuspensionat
aconcentrationof2.5×107cells/mLweresmearedusingSmearGell(Genostaff),accordingto
themanufacturer’sprotocol.Subsequently,c-peptideimmunostainingwasperformed,andthe
rateofc-peptide-positivecellswasdeterminedbymanuallycounting50randomfieldsat200×
magnificationin5independentsamples.
Streptozotocin(STZ)-induceddiabeticmousemodelandtransplantation
analysis
Male9-to12-week-oldBALB/cAJcl-nu/numice(CLEAJapan)weremaintainedunderspe-
cificpathogen-freeconditionsat22±2˚Cwith12hoursofartificiallightingfrom9AMand
werefedchowandwateradlibitum.Diabeteswasinducedviaasingleintraperitonealinjec-
tionofSTZ(160μg/gin0.01mol/Lcitratebuffer,pH4.6).Non-fastingbloodglucoselevels
weremeasuredusingaMedisafeMiniGlucometer(Terumo,Tokyo,Japan).Thefollowing
week,micewithnon-fastingbloodglucoselevelsof300–600mg/dLon2consecutivedays
wereconsideredtohavediabetes.Forthetransplantationanalysis,themiceunderwentleft
renalsubcapsulartransplantationwithcellularaggregatescollectedfrom12wellsina24-well
plateunderanesthesiausingamixtureofmedetomidine(0.3mg/kg;WakoChemicals,Osaka,
Japan),midazolam(4mg/kg;WakoChemicals)andbutorphanoltartrate(5mg/kg;Wako
Chemicals)administeredbyintraperitonealinjectionaccordingtotheinstitutionalrecom-
mendationtoobtainthedesiredlevelsofsedation,analgesiaandskeletalmusclerelaxation.
Themicewerehousedinatemperature-andhumidity-controlledroomandallowedaccessto
acommercialdietandwateradlibitum.Aleftnephrectomywasperformedinallthetrans-
plantedmiceat28daysafterthetransplantation.Bloodglucoselevelswererecordeduntil35
daysaftertransplantation,thenthemiceweresacrificedbycervicaldislocationundersodium
pentobarbitalanesthesia(100mg/kg;Dainabot,Osaka,Japan).Tocomparethefunctionality
ofthetransdifferentiatedcellswiththatofcellsactuallyhavinginsulin-secretingability,we
alsocreatedtheβTC6-transplantedmodelbytheaforementionedanestheticandsurgicalpro-
cedure(1.0×104cells/body,n=5).Transplantedmiceweresacrificedhumanelytoalleviate
sufferingunderthefollowingconditions:
i. sustaineddecreaseofthebodyweightto80%orlowerthanthatrecordedbeforetheinduc-
tionofdiabetes,indicativeofmalnutrition.
ii. reductionofthenon-fastingbloodglucoselevelsto60mg/dLorlower,indicativeofsevere
hypoglycemia.
Ethicsstatements
AllexperimentsusinganimalswereapprovedbytheShinshuUniversityAnimalCareCom-
mittee(ApprovalNumber:270022)andalltheexperimentsusinganimalwereperformed
accordingtotheguidelinepresentedbythecommittee.
Statistics
Theresultswereexpressedasthemean±standarddeviation(S.D.).Astatisticalanalysisofthe
dataamongthegroupswasperformedusingat-test,Mann–WhitneyUtest,oraone-way
analysisofvariance(ANOVA)followedbyDunnett’stestformultiplecomparisons,asappro-
priate.Thegroupsusedasacontrolforeachofthemultiplecomparisonsweredenotedas
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Cellulartransdifferentiationandsolublefactors
such.AlltheanalyseswereperformedusingJMPsoftwareversion10.0(SASInstitute,Cary,
NC).DifferencesofP<0.05wereconsideredstatisticallysignificant.
Results
AtriplecombinationofTFsefficientlyinducespancreatic
transdifferentiationinvitro
Fouradenovirusvectorsco-expressingoneofeachoftheexaminedTFstogetherwithnuclear-
localizedGFP(nGFP),namelypAd-Pdx1,pAd-Ngn3,pAd-NeuroD1,andpAd-MafA,were
prepared.TheratiosofnGFP-positivecellsat24hoursafterinfectionwiththeindicatedvector
atanoptimalmultiplicityofinfection(MOI)wereasfollows:94.1%±4.5%forpAd-Pdx1;
88.8%±7.6%forpAd-Ngn3;88.6%±3.1%forpAd-NeuroD1;and87.7%±4.2%forpAd-
MafA.Wedefinedatotalof15patternsofgenetransferusingthe4aforementionedexpression
vectorsasfollows:singlefactor(4patterns),doublecombination(6patterns),triplecombina-
tion(4patterns),andquadruplecombination(1pattern)(Fig1A).Onthedayafterviralinfec-
tion,numerousnGFP-positivecellsappeared(Fig1B).Immunostainingoftheco-infected
PBLHCsrevealedthenuclearlocalizationofeachectopicallyexpressedTFandnGFP(S1A
andS1BFig).At3daysafterinfection,selectiveincreasesinthemRNAlevelsofinsulinI(Ins1)
andinsulinII(Ins2)wereobservedinthecellsthathadbeeninfectedwithatriplecombination
ofadenovirus(Pdx1andMafA,witheitherNgn3orNeuroD1)inasemiquantitativeRT-PCR
analysis(S1CandS1DFig).IntheqRT-PCRanalyses,theinsulingeneexpressionlevels(Ins1
andIns2)werethemostenhancedinthecellsexpressingthetriplecombinationofPdx1,Ngn3
andMafA(PNM)(Fig1Cand1D).Inparticular,theexpressionofIns2wassignificantly
enhanced(Fig1D)ascomparedtothatinthecellsexpressingthequadruplecombinationof
Pdx1,Ngn3,NeuroD1andMafA(PNDM).Althoughnosignificantintergroupdifferencesin
theexpressionoftheglucagongene(Gcg)wereobserved,Gcgexpressionwasmostenhanced
inthecellsexpressingthetriplecombinationofNgn3,NeuroD1andMafA(NDM)(Fig1E).
Expressionofsomatostatin(Sst)wasthehighestinthecellsexpressingthetriplecombination
ofPdx1,Ngn3andNeuroD1(PND)(Fig1F).Expressionofpancreaticpolypeptide(Ppy)was
thehighestinthenon-transgeniccontrol,andtheexpressionlevelsweredecreasedinallthe
transgenicgroups(Fig1G).Theintracellularc-peptidecontentsweresimilaramongthecells
transfectedwithtriplecombinations(PND,PNMandPDM[Pdx1,NeuroD1,andMafA])as
comparedwiththoseinthecellstransfectedwiththequadruplecombination(Fig1H).
AlthoughtherelativeexpressionlevelsofIns1,Ins2,andc-peptideproteininthePBLHCs
expressingPNMweremuchlowerascomparedtothoseintheβTC6cells(1/43000th,1/
120000th,and1/250000th,respectively),thecombinationofPdx1,Ngn3,andMafAwascon-
sideredastheoptimalforinducingselectivetransdifferentiationofthePBLHCstoinsulin-pro-
ducingcells.Therefore,weusedthiscombinationofTFsinsubsequentanalyses.
TreatmentwithSFDMand3Dculturearenecessaryforefficient
transdifferentiation
Next,weanalyzedwhethercellulartransdifferentiationisaffectedbythedifferenceofculture
medium.Simultaneously,weanalyzedthedifferencesincultureformat,thatis,thedifference
betweenanadherentculture(AC)andasuspensionculture(SC).Sincetheexpressionlevelsof
individualTFsmaydifferincaseswiththeco-infectionofmultiplevectors,weprepared
anotheradenovirusvectortomediatethepolycistronicexpressionofthe3TFsandmCherry,
namelyPNMc(Fig2A).WeculturedtheinfectedPBLHCsusing4differentculturesystems:
anACwithSCM(AC-SCM),anACwithSFDM(AC-SFDM),aSCwithSCM(SC-SCM),and
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Cellulartransdifferentiationandsolublefactors
Fig1.Atriplecombinationoftranscriptionfactors(TFs)inducestransdifferentiationintoinsulin-producingcellsinvitro.(A)Schematic
diagramoftheexperimentalstrategyusedtooptimizethetransgene.AdenovirusesencodingbicistronicTFsandnuclearlocalizedGFP(nGFP)
linkedbyanIRESelement(I)wereused.EachvectorcontaininggenesencodingPdx1,Ngn3,NeuroD1,andMafAisabbreviatedasP,N,D,andM,
respectively.ThecombinationofP,N,D,andMmeanstheco-infectionofeachvector.(B)AppearanceofnGFP-positivecellsonthedayafter
transfection.Scalebar,200μm.(C,D,E,FandG)Quantitativereal-timepolymerasechainreaction(qRT-PCR)analysesstratifiedaccordingtothe
combinationofTFsthatwereusedwereperformedusingprimersetsforIns1(C),Ins2(D),Gcg(E),Sst(F)andPpy(G).“Pre”representsPBLHCs
withouttransfection.Thedataarepresentedasfold-changesinthegeneexpressionsrelativetothevaluesinthecellstransfectedwithPNDM(n=4).
(cid:3)P<0.05(one-wayANOVAfollowedbyDunnett’stest.ThevaluesinthecellstransfectedwithPNDMwereusedasthecontrol.);errorbars,S.D.
(H)Intracellularc-peptidecontentsinthetransfectedcells(n=4).(cid:3)P<0.05(one-wayANOVAfollowedbyDunnett’stest.Thevaluesinthecells
transfectedwithPNDMwereusedascontrol.);errorbars,S.D.TheresultsobtainedfromβTC6,amouseinsulinomacellline,wereexcludedfromall
thestatisticalanalyses,becauseofthemarkeddeviationsfromothervalues.CMV,cytomegaloviralpromoter;Ins1,insulinI;Ins2;insulinII;Gcg,
glucagon;Sst,somatostatin;Ppy,pancreaticpolypeptide.
https://doi.org/10.1371/journal.pone.0197175.g001
aSCwithSFDM(SC-SFDM)(Fig2B).Fig2Cshowsrepresentativemicroscopicimagesofthe
infectedPBLHCstreatedwithSFDMinthetwodifferentcultureformats.TheSCcellshad
formedaggregatesonthedayafterre-seeding,butnomorphologicalchangeswereobserved
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Cellulartransdifferentiationandsolublefactors
Fig2.Theassessmentofthesynergisticeffectsbetweena3Dculturesystemandserum-freedifferentiationmediumforcellular
transdifferentiation.(A)Adenoviralconstructsforthepolycistronicexpressionofthreetranscriptionfactors(TFs).Darkgraybar,2Apeptide.(B)
SchematicdiagramoftheproceduresusedtoanalyzethedifferencesamongculturesystemsforTFs-mediatedtransdifferentiation.(C)Representative
microscopicimagesofinfectedcellstreatedwithserum-freedifferentiationmedium(SFDM)intwodifferentcultureformats.Arrow,cellular
aggregate.Scalebars,200μm.(DandE)Theanalysesofinsulingeneexpressivitybasedonthedifferenceinculturesystem.TheexpressionofinsulinI
(Ins1:D)andinsulinII(Ins2:E)wasanalyzedbyquantitativereal-timepolymerasechainreaction(qRT-PCR)(n=4).(cid:3)P<0.05(one-wayANOVA
followedbyDunnett’stest.ThevaluesinthecellstreatedwithAC-SCMwereusedascontrol);errorbars,S.D.(F)Intracellularc-peptidecontentsin
infectedcells(n=4).(cid:3)P<0.05(one-wayANOVAfollowedbyDunnett’stest.ThevalueofcellstreatedwithAC-SCMwasusedasacontrol.);error
bars,S.D.TheresultsobtainedfromtheβTC6cellswereexcludedfromallthestatisticalanalyses,becauseofthemarkeddeviationsfromothervalues.
CMV,cytomegaloviruspromoter;mCherry,monomericcherryfluorescentprotein;AC-SCM,adherentculturewithserum-containedmedium;
AC-SFDM,adherentculturewithserum-freedifferentiationmedium;SC-SCM,suspensionculturewithserum-containedmedium;SC-SFDM,
suspensionculturewithserum-freedifferentiationmedium.
https://doi.org/10.1371/journal.pone.0197175.g002
untilday8.Incontrast,theACcellsfirstformedmonolayers,andthecellnumberthengradu-
allydecreased.Onday8,althoughtheACcellshadalsoformedaggregates,theaggregates
weresmallerthanthoseoftheSCcells.Toexaminethedifferencesininducibilityamongthe4
differentculturesystems,wefirstexaminedtheexpressionsofinsulingenesundertheculture
conditions(Fig2Dand2E).Theinsulingeneexpressionsweremarkedlyincreasedinthe
SC-SFDM-treatedcells.Inparticular,theIns2expressionlevelintheSC-SFDMcellswas
increasedby180-fold,comparedwiththatintheAC-SCMcells.Differencesintheinsulin-
producingabilityofthetransfectedcellsdependingontheculturesystemswerealsoanalyzed
basedonquantitativeevaluationofthec-peptideproteinlevel.Asaresult,theintracellular
contentsofc-peptideintheSC-SFDMcellswerefoundtobesignificantlyelevatedamongall4
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Cellulartransdifferentiationandsolublefactors
culturesystems.Theamountofintracellularc-peptideintheSC-SFDMcellswasprominent,
witha60-foldincreasecomparedwiththeamountinAC-SCMcells(Fig2F).Therelative
expressionlevelsofIns1,Ins2,andc-peptideproteininthePNM-transfectedPBLHCscultured
inSC-SFDMwere1/129th,1/56th,and1/626th,respectively,ascomparedwiththoseinthe
βTC6cells.TheseresultssuggestedthatboththeutilizationofSFDManda3Dculturesystem
contributedtoefficienttransdifferentiation.
Comprehensiveanalyzesabouttheeffectsofsolublefactorson
transdifferentiatedcells
Basedontheresultthattheinductionoftransdifferentiationispromotedbyaserum-free3D
culture,wefurtherinvestigatedtheeffectsofsolublefactorsontransdifferentiatedcells.Auni-
fied3Dcultureformatwasused.SincethefactorsaddedtoSFDMwerecategorizedaseithera
supplementcomposedofseveralfactors(B27andN2supplement)orasinglemolecule(EGF,
exendin-4,forskolin,L685,458,nicotinamide,noggin,andSB431542)(Table1andFig3A),we
analyzedthemindividually.Firstly,weanalyzedthenecessityofB27andN2supplements.As
showninFig3Band3C,Ins2geneexpressionwassignificantlyincreasedinthecellstreated
withouttheB27supplement.Asfortheintracellularc-peptidecontents,thehighestc-peptide
levelwasobservedinthecellstreatedwithN2supplementalone,althoughthedifferenceswere
notstatisticallysignificant(Fig3D).TheseresultindicatedthattheadditionofB27supplement
wasnotessentialfortheinductionofcellulartransdifferentiation.Next,weanalyzedtheneces-
sityofmoleculesaddedtoSFDM.Wedesignatedthemixtureofthe7aforementionedmole-
culesasM7(mixtureof7molecules)andcomparedtheexpressionlevelsofinsulingenes
wheneachmoleculewasindividuallyremoved.Asaresult,insulingeneexpressionwassignifi-
cantlyincreased,comparedwiththeadditionofM7,bytheremovalofEGF,nicotinamide,or
SB431542(Fig3Eand3F).Wefurtherdesignatedthemixtureofexendin-4,forskolin,L685,
458,andnogginasM4(mixtureof4molecules)andconductedasimilaranalysis.Asaresult,
theexpressionlevelsofbothIns1andIns2weresignificantlyincreasedbytheremovaloffor-
skolin,whereastheadditionofM7significantlydecreasedtheexpressionlevelsofbothinsulin
genes,ascomparedtoadditionofM4(Fig3Gand3H).Theintracellularc-peptidecontent
wasalsohigherinthecellstreatedwithexendin-4,L-685,458ornoggin,thaninthecells
treatedwithM7.ThesefindingsindicatedthatamongthesevenmoleculesaddedtoSFDM,
exendin-4,L-685,458andnoggin(amixtureof3molecules,M3)wereimportantforenhanc-
inginsulingeneexpression.
SimultaneousadministrationofN2andM3acceleratedfunctional
maturationoftransdifferentiatedcells
Basedontheseresults,wefurtherexaminedthenecessityofN2andM3individually.Asshown
inFig4Aand4B,theexpressionsofbothIns1andIns2weregreatlyenhancedwhenthecells
weretreatedwithN2plusM3.Wefurtheranalyzedtheexpressionoftheproproteinconvertase
subtilisin/kexintype1(Pcsk1)andtype2(Pcsk2)genes,whichencodeproproteinconvertase1/3
(PC1/3)and2(PC2),respectively.Inmatureβcells,bothPC1/3andPC2areinvolvedinthe
processofgenerationofinsulinandc-peptidefromproinsulin.Inthepredominantprocessing
pathway,proinsulinisfirstconvertedtotheintermediatedes-31,32-proinsulinfollowingcleav-
agebythePC1/3,andsubsequentlytoinsulinandc-peptidefollowingcleavagebythePC2[18].
Asaresult,theexpressionsofbothPcsk1andPcsk2wereenhancedbyN2+M3treatment;in
particular,expressionoftheformerwassignificantlyenhancedascomparedwiththatinthe
untreatedcells(Fig4Cand4D).TheseresultsindicatethattheconcurrentadministrationofN2
andM3mightgenerateinsulin-producingcellsinwhichprohormoneprocessingsimilartothat
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