JoseFranciscoIslas1*,MohamedA.Mohamed2*,CliffordDacso3,VladimirN.Potaman1,RezaAbbasgholizadeh4,StephenNavran5,RichardBond6,RaviBirla2#andRobertJ.Schwartz1,4

1TexasHeartInsNtute,TexasMedicalCenter,HoustonTexas,2DepartmentofBiomedicalEngineering,UniversityofHouston,Houston,Texas,3DepartmentofMolecularandCellularBiology,BaylorCollegeofMedicine,TexasMedicalCenter,Houston,Texas,4DepartmentofBiologyandBiochemistry,UniversityofHouston,Houston,Texas.

5Synthecon,Inc.,Houston,Texas,6CollegeofPharmacy,UniversityofHouston,Houston,Texas.

Figure1.Athreestageprotocolforconver>nghAdMSCsintocardiacprogenitorsandhighlyconduc>vemyocytes.A. SchemaNc diagram shows 3 stages of reprogrammingand maturaNon. Stage 1 consNtutes protocol used toreprogram human fibroblasts using TAT-ETS2 and TAT-MESP1 proteins. Stage 2 β-adrenergic sNmulaNon withepinephrine to enhance cardiac myocyte Ca2+management.Stage 3 Use Synthecon RCCS bioreactor toform3Dcardio-spheres.B.CellmorphologyshownbytheappearanceofNKX2.5 red td-Tomato reporter.C,D.FACSanalysis for cardiac markers KDR/VEGFR (CD309) andPDGFRa (CD140a) and E. Appearance of Nkx2.5, andGATA4, HAND2 and TBX5 contracNle proteins genes,MLC2V,andcardiacα-acNn.

Figure2.MyofibrilogenesiswiththeappearanceofthinfilamentI-Z-Istaining(anNa-acNninanNbody)andexpressionofRYR2,L-andT-TypeCa2+channels:CAV1.2andCAV3.1.A.ShowscellmorphologyforStage2.(Dapi/α-AcNnin).B.ShowsupregulaNonofcoreCa2+managementgenes.C.CalciumanalysisusingIC200,showscalciumfluctuaNonduringdifferenNaNon.

A.  B.C.D.Figure 3. Converted hAdMSCs form 3D cardio-spheres in the SyntheconRCCS.A. Lef. Slow turning lateral vessal RCCS reactor (Syntecon ®), right,ETS2/MESP1stage3cells.B.Cellgrowthover6days.C..RNAexpressionanalysis showed up-regulaNon of a hypoxic program. ContracNleproteins including adult myosin heavy chain genes MYH7 and thinfilament proteins cardiac a-acNn,MLC2v and TNNT2 and ion channelgenes,CACNA1,SCN4A,SCN7A,SCN8A,KCNC1,KCND3,KCNQ3,KCNQ5,and SRand t-tubule genes, CASQ, JPH,ASPH,PLN, TRDN,BIN1,CALR,NCX1 and CASQ2. D SR and T-tubule markers correlated withappearance of immuno-stained RYR2 adjacent to nascent caveloaestained with anN-Cav3 anNbody. Electron microscopic images alsodisplayedcaveloaevesiclesthatwillcoalescetoformt-tubules.

Molds 3DPatches Confocal

EKGData

Figure 4. Electrocardiogram of highly conduc>vecardiomyocytes.Cellswereculturedin3Dpatches(NssuesimulaNon)andEKGmeasurementwasperformed. DataindicatesconNnuouselectricalacNvity.ArrowsdepictQRScomplexappearance.

Figure 5. Biopoten>als and velocity. A, B. RepresentaNon of a 32electrodesystemusedtomeasurebiopotenNalandvelocityrecordings..MaturaNon coincideswithdevelopmentof higher amplitude andmorerhythmic biopotenNal peaks. B. Bulk conducNon velociNes, measuredfrom the amplitude lags between rhythmic waveform pamernscoincident in mulNple electrodes and electrode distances, increasedduringthethreestagesofmaturaNon.C)UpregulaNonofkeyelectricalconducNvity genes Ned to HCN channels coincides with measuredimprovementinbiopotenNalandincreasedconducNonvelociNes.

References:Islas,J.F.etal.TranscripNonfactorsETS2andMESP1transdifferenNatehumandermalfibroblastsintocardiacprogenitors.Proc.Natl.Acad.Sci.109,13016–13021(2012).Liu,Y.&Schwartz,R.J.AdriverofcardiaccellfatedeterminaNonTranscrip>on.4,92–96(2013)Michel,M.C.,Harding,S.E.&Bond,R.a.AretherefuncNonalβ₃-adrenoceptorsinthehumanheart?Br.J.Pharmacol.162,817–822(2011).Cerignoli,F.etal.HighthroughputmeasurementofCa2+dynamicsfordrugriskassessmentinhumanstemcell-derivedcardiomyocytesbykineNcimagecytometry.J.Pharmacol.Toxicol.Methods66,246–56(2012).Bers,D.M.AdrenergicFight-or-Flight:S-NOFallsonPKATargets.Circ.Res.GenesDev.117,747–9(2015).Birla,R.K.,Borschel,G.H.,Dennis,R.G.&Brown,D.L.Myocardialengineeringinvivo:formaNonandcharacterizaNonofcontracNle,vascularizedthree-dimensionalcardiacNssue.TissueEng.11,803–13Salazar,B.,Reddy,A.,Tao,Z.,Madala,S.&Birla,R.32-ChannelSystemtoMeasuretheElectrophysiologicalProperNesofBioengineeredCardiacMuscle.IEEETrans.Biomed.Eng.(2015).Synthecon.TheRotaryCellCultureSystemTMRCCS-1/RCCS-4ThePremierTissueEngineeringCultureSystem.UserGuide

ABSTRACT.InauniquemannerhumantranscripNonfactorsETS2andMESP1were sufficient to convert human adipogenic mesenchymal stem cells(hAdMSC)intocardiacprogenitorcells(CPCs).Thesetwofactorsupregulatea cadre of cardiac regulatory factors, Nkx2.5, Tbx5, Mef2C, dHAND andGATA4.Yet, theyareunable toproduce theappearanceofmaturemyosinheavy chains and many calcium-handling proteins. Nevertheless, theaddiNon of epinephrine was capable of promoNng maturaNon of theelectrophysiological and Ca2+ handling properNes of hAdMSC convertedCPCs.AdrenergicsignalingthroughBetaAdrenergic2receptorreposiNonedconvertedCPCsintomorematuremyocytescells,alongwiththeappearanceof RYR2, CAV2.1, CAV3.1, Nav1.5, SERCA2 and CX45 gene transcripts.Following treatment with epinephrine, acNon potenNals were observed in(Nkx2.5-puromycin) drug selected myocytes. Further improvement wasfostered by 3D cardio-spheroids formed in a Synthecon, Inc. rotaNngbioreactor (RCCS). These cardio-spheroids induced the appearance ofhypoxic genes: HIF1a/b, PCG1a/b and iNOS2. InducNon of the hypoxicprogram coincided with the robust acNvaNon of adult contracNle genes,MYH6, MYH7 and TNNI3, ion channel genes, CACNA1C, SCN8a, KCNQ5,KCNQ3 and t-tubule genes, CASQ, JPH, ASPH PLN, TRDN, BIN1 and CALR.Thesemyocyteswere found tobeelectricallycoupledandconductathighrates. AddiNonal in-depth studies demonstrated the appearance ofhyperpolarizaNon-acNvated and cyclic nucleoNde-gated channels (HCN1-4).OurexperimentalparadigmcontributestonovelregeneraNvestrategiesthatenhancedmaturaNonofconvertedhAdMSC’stoelectricalacNvemyocytes.