Differentiation of spontaneously contracting cardiomyocytes from non-virally reprogrammed human

12 Pages·2017·10.39 MB·English

RESEARCHARTICLE
Differentiation of spontaneously contracting
cardiomyocytes from non-virally
reprogrammed human amniotic fluid stem
cells
AaronJ.Velasquez-Mao1☯,ChristopherJ.M.Tsao1☯,MadelineN.Monroe1,
XavierLegras2,BeatriceBissig-Choisat2,Karl-DimiterBissig2,RodrigoRuano3,Jeffrey
G.Jacot1,4,5*
a1111111111
a1111111111 1 DepartmentofBioengineering,RiceUniversity,Houston,TX,UnitedStatesofAmerica,2 Departmentof
MolecularandCellularBiology,CenterforCellandGeneTherapy,StemCellsandRegenerativeMedicine
a1111111111
Center,BaylorCollegeofMedicine,Houston,TX,UnitedStatesofAmerica,3 DepartmentofObstetricsand
a1111111111
Gynecology,MaternalFetalMedicineTexasChildren’sHospital,Houston,TX,UnitedStatesofAmerica,
a1111111111 4 CongenitalHeartSurgeryService,TexasChildren’sHospital,Houston,TX,UnitedStatesofAmerica,
5 UniversityofColoradoDenver,DepartmentofBioengineering,Aurora,CO,UnitedStatesofAmerica
☯Theseauthorscontributedequallytothiswork.
*[email protected]
OPENACCESS
Citation:Velasquez-MaoAJ,TsaoCJM,Monroe Abstract
MN,LegrasX,Bissig-ChoisatB,BissigK-D,etal.
(2017)Differentiationofspontaneouslycontracting
Congenitalheartdefectsarethemostcommonbirthdefect.Thelimitingfactorintissueengi-
cardiomyocytesfromnon-virallyreprogrammed
humanamnioticfluidstemcells.PLoSONE12(5): neeringrepairstrategiesisanautologoussourceoffunctionalcardiomyocytes.Amnioticfluid
e0177824.https://doi.org/10.1371/journal. containsanidealcellsourceforprenatalharvestanduseincorrectionofcongenitalheart
pone.0177824
defects.Thisstudyaimstoinvestigatethepotentialofamnioticfluid-derivedstemcells(AFSC)
Editor:AustinJohnCooney,UniversityofTexasat toundergonon-viralreprogrammingintoinducedpluripotentstemcells(iPSC)followedby
AustinDellMedicalSchool,UNITEDSTATES
growth-factor-freedifferentiationintofunctionalcardiomyocytes.AFSCfromhumansecond
Received:February2,2017 trimesteramnioticfluidweretransfectedbynon-viralvesiclefusionwithmodifiedmRNAof
Accepted:May3,2017 OCT4,KLF4,SOX2,LIN28,cMYCandnuclearGFPover18days,thendifferentiatedusing
inhibitorsofGSK3followed48hourslaterbyinhibitionofWNT.AFSC-derivediPSChadhigh
Published:May17,2017
expressionofOCT4,NANOG,TRA-1-60,andTRA-1-81after18daysofmRNAtransfection
Copyright:©2017Velasquez-Maoetal.Thisisan
andformedteratomascontainingmesodermal,ectodermal,andendodermalgermlayersin
openaccessarticledistributedunderthetermsof
theCreativeCommonsAttributionLicense,which immunodeficientmice.ByDay30ofcardiomyocytedifferentiation,cellscontractedspontane-
permitsunrestricteduse,distribution,and ously,expressedconnexin43andβ-myosinheavychainorganizedinsarcomericbanding
reproductioninanymedium,providedtheoriginal
patterns,expressedcardiactroponinTandβ-myosinheavychain,showedupregulationof
authorandsourcearecredited.
NKX2.5,ISL-1andcardiactroponinTwithdownregulationofPOU5F1,anddisplayedcalcium
DataAvailabilityStatement:Allrelevantdataare
andvoltagetransientssimilartothoseindevelopingcardiomyocytes.Theseresultsdemon-
withinthepaperanditsSupportingInformation
stratethatcellsfromhumanamnioticfluidcanbedifferentiatedthroughapluripotentstateinto
files.
functionalcardiomyocytes.
Funding:Thisworkwassupportedinpartby
grantsfromtheAmericanHeartAssociation
(14BGIA18750004toJ.Jacot),theNational
ScienceFoundation(CBET-1547838toJ.Jacot),
theNationalInstitutesofHealth(1R01HL130436-
01toJ.Jacot),andTexasChildren’sHospital.K.D.
B.issupportedbytheNationalHeartLungand
PLOSONE|https://doi.org/10.1371/journal.pone.0177824 May17,2017 1/12
Cardiacdifferentiationofreprogrammedhumanamnioticfluidstemcells
BloodInstitute(NHLBI)grantR01HL134510,the Introduction
TexasHepatocellularCarcinomaConsortium
Congenitalheartdefects(CHD)arethemostcommonbirthdefectsandtheleadingcauseof
(THCCC)(CPRIT#RP150587)andtheDianaHelis
HenryandAdrienneHelisMalvinMedicalResearch infantdeathintheUnitedStates[1].Autologouslyderivedcontractilecardiaccellscanbe
Foundations. appliedtopatchesforstructuraldefectrepair[2],engineeredhearttissue[3],cellsforcardio-
myoplasty[4],andgeneeditingcorrectionofspecificdefects[5].With80%ofCHDsdiagnosed
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist. inthesecondtrimester[6],amnioticfluidpresentsanidealsourceforautologouscellsforuse
inneonatalCHDtreatment[4,7].
Amnioticfluidstemcells(AFSC)arebroadlymultipotent,butdonotdirectlydifferentiate
intocontractilecardiomyocytes(CM).Specifically,AFSCexpressmesenchymalstemcell
markers(CD29,CD44,CD90,andCD105),certainpluripotentmarkers(SOX2),andarecapa-
bleofdifferentiatingintoallthreegermlayers[8].Whileattemptsatdirectcardiacdifferentia-
tionhaveshowngeneandproteinlevelsimilarities(GATA4,Nkx2.5,α-actinin,cTnT),
resultingcellsultimatelylackcontractility[8,9].
Inducedpluripotentstemcells(iPSC)canbedifferentiatedintoforce-generatingCM[3,4,
10],andstudiesshowthatiPSCcanbegeneratedfromAFSC[11,12].However,nostudyhas
investigatedthetransformationofAFSCintoCMusingnon-virallyattainediPSCasan
intermediary.
TheobjectivesofthisstudyweretotestwhetherAFSCcanbereprogrammedtoiPSCby
mRNAdeliveryandwhethernon-virallyattainedAFSC-iPSCarecapableofcardiacdifferenti-
ation.ReprogrammedAFSCwereevaluatedforpluripotencybyproteinexpressionandtera-
tomaformation.CMderivedfromAFSC-iPSCwereevaluatedforexpressionofcardiacgenes
andproteins,membranepotentialfluctuation,calciumhandling,andcontractilefunction.
Materialsandmethods
AFSCcultureisolationandexpansion
AFSCwereisolatedbasedonpreviouslypublishedmethodsfromourgroup[8,13].Primary
humanamnioticfluidwasobtainedfrompatientsintheirsecondtrimesterundergoing
plannedamnioreductionaspartofatherapeutictreatmentfortwin-twintransfusionsyn-
drome(TTTS).Amnioticfluidwascentrifugedat1200rpmfor10min,andcollectedcells
wereplatedat2500cells/cm2onstandardplasticPetridishesandculturedinamodifiedα-
MinimumEssentialMedia:63%αMEM(Invitrogen,Carlsbad,CA),18%ChangBasal
Medium(IrvineScientific,SantaAna,CA),2%ChangCsupplement(IrvineScientific),15%
fetalbovineserum(PAALaboratories,Dartmouth,MA),andGlutaMAX(Invitrogen)at37˚C
and5%CO2inahumidifiedenvironment.Mediawaschangedeverytwotothreedays,and
cellswerepassagedat60–70%confluence.Atthefirstpassage,asubpopulationofprogenitor
cellswasisolatedthroughfluorescence-activatedcellsortingforexpressionofthemembrane
receptorCD117/c-kit(BDBiosciences,Bedford,MA).Cellcoloniesweredetachedintosingle
cells(Accutase;Sigma-Aldrich,St.Louis,MO;37˚C,10min),andc-kit+cellswerecollected
usingaDakoMoFlosterilecellsorter.Allstudiesofprimaryhumancellswereapprovedby
theInstitutionalReviewBoardsofbothBaylorCollegeofMedicineandRiceUniversity,and
subjectsgaveinformedconsent.
iPSCgenerationandculture
AFSCweretransfectedwithmRNAtogenerateaniPSstateusingtheStemgentmRNARepro-
grammingSystem(Lexington,MA)[14].Briefly,frozenc-kit+passage2AFSC,werethawed
andplatedonto100mmpetridishes.Thecellswereallowedtoexpandto80%confluencyand
thenplatedin6wellplatescontainingafeederlayerofmitomycin-treatednewbornhuman
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Cardiacdifferentiationofreprogrammedhumanamnioticfluidstemcells
foreskinfibroblasts(NuFF,Stemgent,Inc.,Cambridge,MA).Afterattachment,transfectionof
theAFSCwascarriedoutbyexposuretoreprogrammingfactors(Oct4,Klf4,Sox2,c-Myc)for
4hourseachdayfor18days.Briefly,AFSCwereplatedonafeederlayerofNuFFinPluriton
ReprogrammingMedium(Stemgent)supplementedwith4ng/mLbFGF(Stemgent)andB18R
recombinantprotein(eBioscience,Inc.,SanDiego,CA).AFSCwereexposedfor4hoursper
daytoanmRNAcocktailcomprisedofOCT4,SOX2,KLF4,c-Myc,LIN28,andnGFP(Tri-
LinkBiotechnologiesInc.,SanDiego,CA)complexedwithLipofectamine(RNAiMAX,
ThermoFisherScientific,Carlsbad,CA)for18consecutivedays.Attheendofthe18-day
transfection,cellcolonieswereselectedbasedonmorphologyandthepluripotencyexpression
markerTRA-1-81.Eachwellyieldedapproximately10iPSCcoloniesperwellthatwereeach
1–2mmindiameter.Colonieswerethencontinuouslysplitandpassagedevery5–7daysonto
mouseembryonicfibroblastfeeder(MEF,GlobalStem,Inc.,Rockville,MD)ontreated6-well
plates,andmaintainedusingCDF12mediumasdescribedbyWarrenetal18:DMEM/F-12
(Invitrogen,Carlsbad,CA),knockoutserumreplacement(LifeTechnologies,Carlsbad,CA),
non-essentialaminoacids(LifeTechnologies),Glutamax(LifeTechnologies),2-mercap-
toethanol(Gibco,Carlsbad,CA),20ng/mLbFGF(Stemgent),and1xpenicillin-streptomycin.
Cellswerepassagedusing0.1%collagenasetype4(WorthingtonBiochemicalCorp.,Lake-
wood,NJ)inDMEM/F-12medium(Invitrogen).Eachwellofthenewpassagewasseeded
with4coloniesthatwerebrokenupphysicallybyscrapingwitha10mLpipetandgentletritu-
ration,andyieldedapproximately30–40newcoloniesthatgrewto2–5mmindiameterbefore
thenextpassage.
Teratomaformation
AnimalexperimentswereapprovedbytheInstitutionalAnimalCareandUseCommittee
(IACUC)ofBaylorCollegeofMedicineandconformedtotheGuidefortheCareandUseof
LaboratoryAnimalsasstatedbytheNIH.InordertocharacterizetheAFSC-iPSCself-renewal
andpluripotencyproperties,ateratomastudywasconductedtoassesstheirabilitytodifferen-
tiateintoderivativesofthethreeembryonicgermlayers.ThisworkwasdonebytheBisseglab
atBaylorCollegeofMedicine.Briefly,approximately1x10^6AFSC-iPSCwereinjectedsubcu-
taneouslyintoSCIDmice(8–12weeksofage)andmonitoredfor8–10weeksforteratomafor-
mation.TeratomaevaluationwasdonebyhistologyandH&Estaining.
Immunocytochemistry
Cellcultureswerefixedin4%paraformaldehyde(AlfaAesar,WardHill,MA)at4˚Cfor20
minutes.Fixedcellswerethenpermeatedwith0.5%TritonX100(Sigma-Aldrich)inPBSfor
5minatroomtemperature.Nextcellswereincubatedwithspecificantibodies(Abcam,Cam-
bridge,UK)forpluripotency(Oct4,Nanog,Tra-1-81.Tra-1-60)andcardiacmarkerlineage
(myosinheavychain,connexin43)ata1:100dilution,theninDyLight-conjugatedsecondary
antibodiesata1:500dilution(JacksonImmunoResearchLaboratories)andDAPIwithVecta-
Shield(VectorLaboratories,Burlingame,CA).Thecellwereimagedusinganepifluorescence
microscope(DMI6000B,LiecaMicrosystems,Wetzlar,DE).
Cardiacdifferentiation
Byadaptingpreviouslypublishedprotocols[10],AFSCderivediPSCweredifferentiatedinto
cardiaccellsbysmallmoleculeinhibitionoftheGSK3andWntsignalingpathways.Briefly,
reprogrammedAFSCcoloniesweremaintainedonafeederlayerconsistingofirradiated
mouseembryonicfibroblastwithdailychangesofmTeSR1media(Stemcelltechnologies,
Vancouver,BC).Oncesufficientcellnumberswereobtained,undifferentiatedcolonieswere
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Cardiacdifferentiationofreprogrammedhumanamnioticfluidstemcells
dissociatedincollagenasetype2(WorthingtonBiochemicalCorp.,Lakewood,NJ)for5min
thenmanuallydislocatedfromthefeederlayer,dispersedintosinglecellsuspension,then
platedasamonolayerofcellsontoMatrigel(BDBiosciences,SanJose,CA)atadensityof
approximately260,000cells/cm2.Cellswereexpandedfor4daysinmTeSR1media,which
thencorrespondedtoday0.Atthispoint,themediawaschangedtoRPMImediawithB27
supplementwithoutinsulinandtheGSK3inhibitor,CHIR99021,wasexposedtothecellsfor
24hoursataconcentrationof12μM.Attheendof24hours,mediawasreplacedwithfresh
RPMI/B27withoutinsulin.Atday3,theWntinhibitor,IWP2,wasaddedtoRPMI/B27with-
outinsulinataconcentrationof5μM.Atday7,insulinwasaddedtotheRPMI/B27media.
Theoccurrenceofbeatingcolonieswasmonitoredthroughphasecontrastmicroscopyafter
day7.
Flowcytometry
CellsweredetachedintosuspensionwithAccutase(ThermoFisher)andstainedwithafluores-
centlyconjugatedantibodyforcardiactroponinT(BDBiosciences)withdilutionspermanu-
facturerrecommendedconcentrations.FACSDivasoftware(BDBiosciences)wasusedforall
flowcytometrydatacollection.FlowJosoftware(TreeStar,Inc.,Ashland,OR)wasusedfor
dataanalysis.
Westernblot
WesternblotantibodieswerepurchasedfromAbcamInc.,electrophoresisandtransfermaterials
werepurchasedfromBio-Rad(Hercules,CA),anddevelopingmaterialswerepurchasedfromLi-
Cor(Lincoln,NE).After30daysofdifferentiation,totalproteinlysateswereisolatedfromdifferen-
tiatedAFSC-iPSCandanalyzedusingabicinchoninicacidkit(BCA;ThermoScientific,Rockford,
IL).Extractsweredenaturedusingβ-mercaptoethanolandboilingfor5min,thendilutedtoequal
concentrationsoftotalprotein.Thesampleswereelectrophoresedby0.1%sodiumdodecylsulfate-
polyacrylamidegelelectrophoresis(SDS-PAGE)andblottedontonitrocellulosemembranesat100
Vfor1.5and1.0hrs,respectively.Membraneswerewashedintris-bufferedsalinewith0.05%
Tween-20(TBST),thenblockedwithOdysseyblockingbuffer(Li-Cor)for1hour.Membranes
wereincubatedovernightat4˚CwithmousemonoclonalantibodiesagainstMHCandcTnT
(1:300dilutioninOdysseyblocker)andmousemonoclonalantibodiesagainstGAPDH(1:1000
dilutioninOdysseyblocker).MembraneswerewashedandsubmergedinIRDYE800CWGoat
anti-MouseIgGsecondaryantibodywithadilutionat1:1000inOdysseyblockerfor60minwith
gentleshaking.MembraneswerewashedandscannedusinganOdysseyCLxscannersettodetect
an800nmwavelength.WesternblotswerenormalizedtoGAPDHexpression.Westernblotana-
lyzewasperformedusingImageJ(NIH,Bethesda,MD).
QuantitativeRT-PCR
TotalRNAwasextractedandpurifiedusingaPrepEaseRNAspinkit(Affymetrix)andquanti-
fiedbyNanoDrop1000Spectrophotometer(ThermoScientific).cDNAwassynthesizedfrom
2μgofpurifiedtotalRNAwithrandomprimersbyMultiscribeRevereseTranscriptase(Ther-
moFisher).Quantitativereal-timePCRwasperformedusingtheStepOnePlussystem(Ther-
moFisher)andVeriquestFastProbeqPCRMasterMix(Affymetrix),accordingtothe
manufacturer’sinstructions.AlldatawerenormalizedtomRNAlevelofhousekeepinggene
usingthe2ΔΔmethod.
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Cardiacdifferentiationofreprogrammedhumanamnioticfluidstemcells
Calciumandvoltagetransientanalysis
Theelectricalbehaviorofthespontaneouslycontractingcellswasmeasuredthroughvoltage-
sensitivedye(Di-8-ANEPPS)andcalcium-sensitivedye(Indo-1)withanepifluorescence
microscope(Olympus,CenterValley,PA)andphotomultipliertubesdetectionsystem.Stain-
ingproceduresforcellswasthesameforeitherdye.CellswerefirstwashedwithPBSwarmed
to37oCtoremoveanyserumcontainedinthemedia.Then2μlofeitherDi-8-ANEPPSor
Indo-1ataconcentrationof2mMwasaddedto2mlofTyrode’ssolutionwarmedto37oC.
Thesolutionwasthenaddedtowashedcellsandallowedtositatroomtemperaturefor30min
protectingthesamplefromlight.ThecellswerethenwashedthreetimeswithfreshTyrode’s
solutionandimagedusingtheepifluorescencemicroscope.ForIndo-1thedetectionwave-
lengthswere405nmand485nmdependingonthebindingorcalciumions.Di-8-ANEPPS
detectionwavelengthswere560nmand620nmdependingoftheshiftsinmembranepoten-
tials.EmissiondatawascollectedandanalyzedusingIonOptixsoftware(Westwood,MA).
Results
Non-viralreprogrammingofAFSC
mRNAreprogrammingofPassage3AFSCseededat2.6x104cellspercm2yieldedtransfected
andhighlyproliferativecells.Each9.6cm2wellyieldedapproximately10colonies.Repro-
grammedcolonieswereexpanded5–8passagesandthenfixedandstainedforpluripotent
markersOCT4,NANOG,TRA-1-60,andTRA-1-81,allofwhichwereexpressedinallcells
(Fig1A).Cellswerethensubcutaneouslyinjectedintoimmunodeficientmice(NSGstrain).
Tumorsformedafter8to12weeks.AnalysisbyH&Estainingexhibitedtissuefrommeso-
dermal,ectodermal,andendodermaloriginandconfirmedpluripotencyofinjectedAFSC
iPSC(Fig1B).
SmallmoleculedifferentiationandgeneticanalysisofAFSC-iPSC
derivedCM
GeneticexpressionofISL1,NKX2.5,TNNT2,andPOU5F1assessedateachtimepointwas
comparedtoexpressionatDay0(Fig2).Upregulationoccurredincardiacprogenitorgenes
ISL1andNKX2.5betweenDay0andDay8ofdifferentiationandinlatestagecardiacmarker
TNNT2betweenDay3andDay8ofdifferentiation.Amongthedayscollected,peakexpres-
sionwasobservedtobesignificantatDay8forNKX2.5andTNNT2.Day8,Day15,and
Day21datashowsadecreaseinexpressionofISL1,NKX2.5,andTNNT2betweenDay8and
Day15.Nosignificantstatisticaldifferencewasobservedinthefoldchangeinexpressionof
POU5F1betweenDay3andDay21ofdifferentiation.
Expressionoflatestagecardiacmarkers
DifferentiatedCMcultureswerestainedforvisualizationofMHCandCx43.Immunohisto-
chemicalanalysisshowscontractileregionsinFig3Aand3B.Highermagnificationrevealsa
distinctrepetitivebandingpatternofMHCincellularextensions,indicatingimmaturesarco-
mericcytoskeletalstructure(Fig3C).Connexin43wasnotorganizedbutexpressionwascyto-
plasmic(S1Fig).Differentiationefficiencywasmeasuredbyflowcytometryexpressionof
cTnTatDay15.TheaveragecTnTexpressionwasshowedtobe42.8±12.3%(Fig3D).
GeneanalysisatDay30ofdifferentiationbyqRT-PCRshowedsignificantupregulationof
PLN,TNNT2,andMHC,genesforcontractilemachinery,andsignificantdownregulationof
pluripotenttranscriptionfactorPOU5F1comparedtoexpressionatDay0(Fig3E).The
humannon-musclealpha-actinin1wasshowntobedownregulatedatDay30.Nosignificant
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Cardiacdifferentiationofreprogrammedhumanamnioticfluidstemcells
Fig1.PluripotentcharacterizationofreprogrammedAFSC-iPSC.(A)Immunostainingforthehuman
pluripotencymarkersOCT4,NANOG,TRA-1-60,andTRA-1-81.Scalebars,100μm.(B)AFSC-iPSC-derived
teratomaexhibitingneuralepithelium(ectoderm,right)andgutepithelium(endoderm,bottom)surroundedby
muscleandadiposetissue(mesoderm,topleft)Scalebar,100μm.
https://doi.org/10.1371/journal.pone.0177824.g001
differencewasobservedinexpressionofsarcomericarchitectureregulatordesminandgap
junctionformationregulatorCx43,thoughspontaneouscontractionincultureswasobserved
priortocollection.ProteinanalysisatDay30ofdifferentiationbyWesternblotshowedupre-
gulationofcTnTandMHC,confirmingthepresenceofboththinandthickfilamentcontrac-
tilemachinery(Fig3F).
ElectrophysiologyofcontractileAFSC-iPSCderivedCM
SpontaneouslycontractingCMculturesgeneratedcalciumandvoltagefluorescentwaveforms
thatdemonstratefluctuationintheratioofextracellulartointracellularcalcium(Fig4A)and
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Cardiacdifferentiationofreprogrammedhumanamnioticfluidstemcells
Fig2.Geneticexpressionofcardiacdifferentiationmarkers.qRT-PCRassessmentofgenesforcommittedcardiaclineage(ISL1andNKX2.5),
cardiomyocytes(TNNT2),andpluripotency(POU5F1).Errorbarsrepresentstandarddeviationsofmeanvalues(n=3,p<0.05,*significancecomparedtoD0
othertimepoints).
https://doi.org/10.1371/journal.pone.0177824.g002
myocardialmembranedepolarization(Fig4B)uponcontraction.Filteredandamplifiedfluo-
rescentrecordingshadaverageperiodsof3.04s(SD=0.36,n=5)and3.83s(SD=0.15,n=3)
forthedifferentbeatingsamplesrecordedinFig4Aand4B,respectively.Comparisonshowsa
significantdifferenceinbeatfrequency.Infact,ahighdegreeofvariabilitywasobservedin
spontaneouslycontractingculturessubjectedtoidenticaldifferentiation,culturing,andenvi-
ronmentalconditions.
Discussion
AmnioticfluidistheidealsourceofautologouscellsforuseinneonatalCHDtreatment
becauseofitscapacitytobeharvestedduringCHDdiagnosisandthehighlyproliferative
natureofAFSC[7]thatenabletheexternaldevelopmentoftissueinparallelwithgestation.
PreviousstudiesraiseconcernsofAFSCimmunerejectionupontransplantationhowever,
becausecellsareinducedtopluripotency,theantigenicprofileisyettobediscoveredbut
hypothesizedtobeoflowimmuno-stimulatorynature,similartoESCanddifferentiatedderiv-
atives[15].TheseexperimentsverifyrecentfindingsthatiPSCcanbegeneratedfromAFSC
[12]andthatAFSC-derivediPSCcanbeusedtogenerateCM[16].However,wearethefirst
todemonstrateCMgenerationfromAFSCwithouttheuseofviruses.Ourstudiessupportthe
feasibilityofusingamnioticfluidforCHDrepairbyshowingthatiPSCcanbegeneratedfrom
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Cardiacdifferentiationofreprogrammedhumanamnioticfluidstemcells
Fig3.CharacterizationofspontaneouslycontractingCMculturesfromAFSC-iPSCdifferentiatedfor30days.(A,B)Immunofluorescentstainingof
acontractileregionforexpressionofsarcomereproteinMHC.Scalebar,20μm.(C)Magnifiedareashowsstriationpatternofsarcomericcytoskeleton
(whitearrows).Scalebar,10μm.(D)Day15cTnTexpressionanalyzedbyflowcytometry.(E)qRT-PCRassessmentofupregulatedcardiomyocytegenes
DES,PLN,TNNT2,MYH7,andGJA1anddownregulatedpluripotentgenePOU5F1.(F)WesternBlotanalysisshowingcardiacproteinexpressionofcTnT
andMHC.
https://doi.org/10.1371/journal.pone.0177824.g003
AFSCusingnon-viralreprogramming,andthatiPSCderivedfromAFSCnon-virallyarecapa-
bleofcardiacdifferentiation.PreviousstudiesfromourgrouphaveverifiedthathumanAFSC
expressMSCmarkersCD29,CD44,CD73,CD90,andCD105,donotexpressendothelial
markerCD31orhematopoieticmarkerCD45,andexpressHLA-ABCbutnotHLA-DR[17].
TheworkofotherinvestigatorshasshownthatAFSCcanbereprogrammed[11,18]andthat
pluripotentcellsderivedfromothersourcescanbedifferentiatedtoCM[4,10,19].Thisstudy
integratestheseobservations,whilevalidatingtheuseofanon-viralmethodofgenerating
iPSCfromAFSCandconnectingthefindingsofotherstodemonstrateadirectlinkfordiffer-
entiatingCMfromiPSCderivedfromAFSC.
Whileotherwell-establishedmethodsincludetheuseofretroviruses,lentivirus,adenovirus,
andSendaivirusasDNAdeliveryagents,mRNAdeliverybylipofectionwasusedfortransfec-
tiontogenerateiPSCinthisstudy.LipofectiondeliveryofmRNAishighlyefficient,hasno
riskoftransgeneincorporation,anddoesnotinvolvetheuseofviruses,reducingpotentialfor
complicationinclinicaltranslation[20].Thisstudyisthefirsttodemonstratetheuseoflipo-
fectionforthetransformationofAFSCtoapluripotentstate.Theachievementofpluripotency
inthisstudy,evidentbyexpressionofpluripotentmarkersOCT4,Tra-1-60,andTra-1-81in
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Fig4.Waveformsgeneratedfromcontractingregionsasthechangeinratiooffluorescenceintensityreadingsoverbase-levelfluorescence.(A)
Calciumtransient-basedfluorescenceusingIndo1,and(B)Voltagetransient-basedfluorescenceusingDi-8-ANEPPS.
https://doi.org/10.1371/journal.pone.0177824.g004
vitroandsubcutaneousteratomaformation,issignificantbecauseitverifiestheuseofnon-
viraltransfection.invitroexpressionofNANOGsupportsdedifferentiation,thoughnotmain-
tenanceofpluripotency[21,22],whilecytoplasmiclocalizationiscomparabletopatternsin
otherstudies[23].
TheuseofGSK3andWntinhibitioninthisstudyvalidatescardiacdifferentiationforiPSC
derivedAFSC,butsuggestsadifferenceinitsefficiency.Oftheexistingmethodsofdifferenti-
atingpluripotentcells,GSK3andWntinhibitionhasshowntoproducethegreatestyieldeffi-
ciencyofcardiomyocytesinmonolayerculture[24].However,theseprotocolsweredeveloped
usingembryonicstemcells(ESC)andiPSCderivedfromfibroblasts[25,26].Ithasbeen
shownthattheiPSCoriginmayinfluencethedifferentiationpotential,specificallyinregards
totheefficiencyandmaturity[27].Inthisstudy,spontaneouscontractioninCMculturesdif-
ferentiatedfromAFSC-iPSCwasobservedatapproximately21daysofdifferentiation,com-
paredto7–12daysasreportedinotherstudies[4,10,19],thoughexpressionofISL1,NKX2.5,
andTNNT2overthetimecourseofdifferentiationresemblesthatofotherstudies[25,28].
CardiacimmaturityisfurthersupportedbylowupregulationofDesminandindistinctinter-
calateddiscgapjunctionformationsmarkedbyCx43,inspiteofclearcytoskeletalsarcomeric
bandingmarkedbyMHC,obviousupregulationofcardiacencodinggenesPLN,TNNT2,and
MYH7,downregulationofpluripotentgenePOU5F1,andtranscriptionofcardiacmachinery,
evidencedbyproteinscTnTandMHC.Alpha-actinin1isshowntohavelittletonoactivityin
laterstagesofdifferentiation,eludingtothedownregulationofnon-muscularencodinggenes.
Immaturityisalsoevidencedbytherecordedcalciumandvoltage-mediatedfluorescence
waveformsofcontractileregions,exhibitingactionpotentialssimilartoimmatureCMbecause
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Cardiacdifferentiationofreprogrammedhumanamnioticfluidstemcells
oftheslowupstrokesduringdepolarizationandtheobserveddelayedafterdepolarizations
[29],thoughthesecouldalsobeexplainedbyimpedancemismatchfromstructuraldiscontinu-
ities.DelayedspontaneouscontractiondespiteordinaryupregulationofNKX2.5andISL1,
incompletelyformedcommunicationstructures,andlimitedupregulationofthesarcomeric
architectureregulatorDESmayimplyadelayeddevelopmentalprogressioninthetranscrip-
tionandtranslationprocessesinvolvedinthedifferentiationofCMfromAFSC-iPSCusing
thisprotocol.
Amajorlimitationduringthisstudywasthehighdegreeofvariabilityincontractile
strengthbyday30ofdifferentiation.Beatingcultureswereobservedtobecomesparser,less
frequent,weakerorstoppedaltogether.Fromthistherecanbespeculationofdecreasingvia-
bilityofpacemakercellsorthegrowthofdiscontinuousstructuresduetodeathorfibrosis.
QuantificationofcTnTatDay15doesshowasubstantialpopulationoflatestagecardiaccells
howeverthereisstillvariabilityinobserveddifferentiationefficiency,delayedspontaneous
contraction,andcontractileviability.Thisstudysuggeststhatanalternativeormodifieddiffer-
entiationprocedureisneededforAFSCtobeaviablecellsourceforderivingCM.Although
thisstudyisthefirsttouseiPSCnon-virallyderivedfromAFSC,possibleoptionsforoptimiz-
ingCMyieldanddifferentiationconsistencyincludeinhibitiontimings,mediacomponents
[25],andcelldensity.
Conclusions
Inthepresentstudy,AFSCwerereprogrammedtoiPSCbymRNAtransfection,andAFSC-
derivediPSCweredifferentiatedintofunctionalCM.ThoughdifferentiatedCMwereimma-
ture,asevidencedbydelayedcontraction,incompletegapjunctionformation,andpoorupre-
gulationofDesmin,thisstudyisthefirsttoachievefunctionalCMfromAFSCbynon-viral
means,asevidencedbysarcomereformationwithincellularcytoskeleton,upregulationof
NKX.2.5,ISL1,andcTnT,expressionofcTnTandMHC,andclearcalciumhandlingand
membranevoltagepropagation.Inconclusion,whileAFSCcannowdefinitivelybesaidto
presentafeasiblesourceoffunctionalCMgeneration,workisneededtoimprovedifferentia-
tionefficiencyandcardiacmaturation.
Supportinginformation
S1Fig.Immunofluorescentexpressionofconnexin43inspontaneouslycontractingCM
culturesfromAFSC-iPSCdifferentiatedfor30days.Imageoverlay,fromlefttoright:DAPI,
Cx43,Combined.Scalebar,20μm.
(TIFF)
S1File.Supportinginformationfiles.DatasetsforqRT-PCR,calciumandvoltagesensitive
dyes.
(ZIP)
Acknowledgments
ThisworkwassupportedinpartbygrantsfromtheAmericanHeartAssociation
(14BGIA18750004toJ.Jacot),theNationalScienceFoundation(CBET-1547838toJ.Jacot),
theNationalInstitutesofHealth(1R01HL130436-01toJ.Jacot),andTexasChildren’sHospi-
tal.K.D.B.issupportedbytheNationalHeartLungandBloodInstitute(NHLBI)grant
R01HL134510,theTexasHepatocellularCarcinomaConsortium(THCCC)(CPRIT
#RP150587)andtheDianaHelisHenryandAdrienneHelisMalvinMedicalResearch
PLOSONE|https://doi.org/10.1371/journal.pone.0177824 May17,2017 10/12