Fig 2: CM characterization. (A) Light-microscope image of hiPS-CMs (scale bar 25 µm), immunofluorescence staining of hiPS-CMs with alpha-actinin, MYL7 and cTnT (scale bar 20 µm). (B) Images at lower magnification illustrate the purity of the CM population (scale bar 50 µm). (C) Light microscope image of hiPS-EC and hiPS-CM co-culture (scale bar 200 µm), immunofluorescence staining hiPS-EC and hiPS-CM co-culture with alpha-actinin and VE-cadherin (scale bar 20 µm).

Fig 3: Assaying the level of cardiac protein expression in reprogrammed MEFs and following genetic selection A–C. GFP(+) cells were readily detected in MEFs transduced with either G4T5MCMDSF, or G4T5MCMDSFM1S3 within 2 days of induction of TF expression. Only rare GFP(+) cells were detected in MEFs transduced with either G4T5MC or G4T5MCM1S3. Using immunofluorescence analysis (day 7) we detected cells staining positive for cardiac antigens Actn2 or Tnnt2 although GFP expression was not always co-localized with these two proteins. We also detected co-localization of GFP expression with Acta2. The GTM label refers to the TF module containing GATA4, TBX5, and MEF2C. D–F. Puromycin was used to positively select for cells activating the transgenic cardiac promoter element in isolated MEFs following transduction and induction of expression of the four TF module combinations. We observed significant death of non-GFP expressing cells following 3 days of low-level puromycin selection (induction day 7+3). In MEFs transduced with either G4T5MCMDSF or G4T5MCMDSFM1S3 GFP(+) cells remained alive but did not proliferate. Following 7 days of puromycin selection, GFP(+) cells for those two TF module combinations peeled off the plastic surface and subsequently underwent apoptosis (D). Rare GFP(+) cells were detected in MEFs transduced with G4T5MCM1S3. The GFP(+) cells proliferated in the presence of puromycin (E). Relative gene expression analysis performed on puromycin-selected surviving GFP(+) cells in MEFs transduced with G4T5MCM1S3 (F). (G–I). Cocultures of transgenic MEFs transduced with the four combinations of TF modules established with freshly isolated neonatal rat ventricular myocytes (NRVMs). Following induction of TF expression for 7 days we assayed cardiac protein expression (Actn2, Tnnt2) and gap junction formation (Gja1). In cocultures where MEFs were transduced with either G4T5MCMDSF or G4T5MCMDSFM1S3 we detected three distinct staining patterns. Firstly, we detected GFP(+) cells with Actn2 or Tnnt2 co-localization. Importantly the cardiac proteins in these cells formed cross-striations (G). Secondly, we detected GFP(+) cells with Actn2 or Tnnt2 co-localization, where however the two cardiac proteins remained unorganized and no cross-striations where detected (H). Thirdly, we detected GFP(+) cells which were negative for either Actn2 or Tnnt2 expression (I). Gja1 staining did not indicate noticeable gap junction formation between the GFP-expressing cells and NRVMs.

Fig 4: Characterization of intracellular Ca2+ activity.iCML cells and control HDF were transduced with a vector allowing the expression of GCaMP3 under the control of the TNNT2 promoter. (A,B) Fluorescence intensity imaging of temporal Ca2+ transients in an iCML or control cell (M2rtTA only). Black arrows denote the area of a cell at which data acquisition was performed (fluorescence intensity) and blue arrows denote a control area within the same frame. Acquired data was normalized and relative fluorescence intensity is presented in relation with elapsed time. Black and blue line correspond to the arrows in panel A. (C,D) Fluorescence intensity imaging of an iCML cell group being stimulated with epinephrine (100 µM). Number coding corresponds to areas within individual cells at which data acquisition was performed (fluorescence intensity). Top graph: Acquired data of individual iCML cells was normalized and relative fluorescence intensity is presented in relation with elapsed time. Bottom graph: The mean value of acquired data from multiple iCML cells (n = 12) is presented as a black line and the standard deviation is presented as the gray lines.

Fig 5: Determination of transcriptional cardio-inducing effect through detection of reporter vector activity and relative quantification of endogenous gene expression levels. A–D. Prior to transduction and induction of TF module expression, MEFs were transduced with four different reporter vectors (Nkx2-5.Hsp68.eGFP, Myl2.mCherry, Myh6.eGFP, TNNT2.copGFP). MEFs were subsequently transduced with G4T5MC, G4T5MCM1S3, G4T5MCMDSF, or G4T5MCMDSFM1S3. Negative control MEFs were only transduced with reporter vector and the M2rtTA-expressing lentivirus. The gates were defined using MEFs that were only transduced with the M2rtTA expressing lentivirus and no reporter vector. Following 7 days of induction of TF module expression, the fraction of cells expressing either GFP or mCherry was determined by FACS analysis. The relative gene expression level of the gene used in each of the reporter vectors was determined using quantitative RT.PCR analysis (endogenous locus). Results for both FACS and RTPCR are based on biological triplicates. Error bars represent calculated standard deviation (One *for p-value <0.05, Two *for p-value <0.01). E. Live imaging of cells transduced with either Myh6.eGFP, or TNNT2.copGFP. White arrows point to examples of brightest and elongated cells detected in MEFs transduced with either G4T5MCMDSF, or G4T5MCMDSFM1S3. F. Detection of cardiac protein expression (Actn2, Tnnt2, Myh6, Myl2, Acta2) in MEFs transduced with G4T5MCMDSFM1S3 using immunofluorescence.