(A) Schematic representation of the strategy to test candidate cardiomyocyte-inducing factors.
(B) Morphology and characterization of fibroblast-like cells migrating from αMHC-GFP heart explants. Phase contrast (left), GFP (middle), and Thy-1 immunostaining (right). Insets are high-magnification views. See also Figure S1.
(C) Thy-1⁺/GFP⁻ cells were FACS sorted from explant cultures for reprogramming.
(D) Summary of FACS analyses for α-MHC-GFP⁺ cells. Effect on GFP⁺ cell induction with 14 factors or the removal of individual factors from the pool of 14 factors (n = 3). Removal of Baf60c, Hand2, Hopx, Hrt2 or Pitx2c did not decrease the percent of GFP⁺ cells and were excluded for further analyses. See also Figure S2.
(E) FACS plots for analyses of GFP⁺ cells. GFP⁺ cells were analyzed 1 week after 14-factor transduction. The number of GFP⁺ cells were reduced by removal of Gata4, but increased by removal of Pitx2c from 14 factors.
(F–H) Effect on GFP⁺ cell induction of the removal of individual factors from the pool of 9 (F), 6 (G), or 5 (H) factors (n = 3). Factors that did not decrease efficiency upon removal were excluded from further study.
(I) 20% GFP⁺ cells were induced from fibroblasts by the combination of four factors, Gata4, Mef2c, Mesp1, and Tbx5. Representative data are shown in each panel. PI, propidium iodine. All data are presented as means ± SD. *, P<0.01; **, P<0.05 vs relevant control. Scale bars, 100 μm. A

(A) FACS analyses for α-MHC-GFP and cardiac Troponin T (cTnT) expression. Effects of the removal of individual factors from the pool of four factors on GFP⁺ and cTnT⁺ cell induction.
(B) Quantitative data of GFP⁺ cells and cTnT⁺ cells in (A) (n = 3).
(C) Effect of the transduction of pools of three, two, and one factors on GFP⁺ and cTnT⁺ cell induction (n = 3).
(D) FACS analyses for α-MHC-GFP and cTnT expression. Effects of GMT plus Nkx2.5 and Baf60c/Gata4/Tbx5 transduction are shown.
(E) The mRNA expression in GFP⁺ and GFP⁻ cells 7 d after GMT transduction was determined by qPCR (n = 3).
(F) Immunofluorescent staining for GFP, α-actinin, and DAPI. The combination of the three factors, GMT, induced abundant GFP and α-actinin expression in cardiac fibroblasts 2 weeks after transduction. High-magnification views in insets show sarcomeric organization. See also Figure S1 and S2.
(G) Induced cardiomyocytes expressed cTnT by immunocytochemistry with clear sarcomeric organization 4 weeks after transduction. Insets are high-magnification views.
(H) Induced cardiomyocytes expressed ANF at perinuclear sites 2 weeks after transduction. All data are presented as means ± SD. *, P<0.01 vs relevant control. Scale bars, 100 μm.

(A) Cardiac fibroblasts (CF) isolated by the conventional isolation method. Most cells were positive for Thy1, and Thy-1⁺/GFP⁻ cells were sorted by FACS for transduction.
(B) FACS analyses for αMHC-GFP and cTnT expression in cardiac fibroblasts isolated in (A) 1 week after transduction by GMT.
(C) Immunofluorescent staining for GFP, α-actinin, and DAPI in the GMT induced cardiomyocytes originated from (A).
(D) Cardiac fibroblasts isolated from adult αMHC-GFP hearts were transduced with three factors. See also Figure S2.
(E) Thy-1⁺/GFP⁻ tail-tip dermal fibroblasts (TTFs) were sorted by FACS for transduction.
(F) FACS analyses for GFP and cTnT expression in TTFs isolated in (E) one week after GMT transduction.
(G) Quantitative data of GFP⁺ cells and cTnT⁺ cells indicated in (F) (n = 3 in each group).
(H) Immunofluorescent staining for GFP, α-actinin, and DAPI in TTF-derived iCMs. See also Figure S3.
(I) Isl1-YFP⁻/Thy1⁺cells were sorted from Isl1-Cre/Rosa-YFP heart explants and transduced with GMT. See also Figure S3.
(J) The vast majority of cTnT⁺ cells induced from Isl1-YFP⁻/Thy1⁺cells was negative for YFP.
(K) Mesp1-YFP⁻/Thy1⁺cells were sorted from Mesp1-Cre/Rosa-YFP TTFs and transduced with GMT. See also Figure S3.
(L) All cTnT⁺ cells induced from Mesp1-YFP⁻/Thy1⁺cells were negative for YFP.
All data are presented as means ± SD. *, P<0.01 vs relevant control. Scale bars, 100 μm. See also Figure S3 for analyses of c-kit⁺ cells.

(A) The percent of αMHC-GFP⁺ cells after GMT transduction (n = 3). The number of GFP⁺ cells was counted by FACS at each time point and divided by the number of plated cells.
(B) FACS analyses of percent of cells with cTnT expression among αMHC-GFP⁺ iCMs. Note that cTnT⁺ cell number and cTnT intensity were both increased over time (n = 4).
(C) Quantitative data of cTnT intensity in (B) (n = 4).
(D) Actc1, Myh6, Ryr2, Gja1, and Col1a2 mRNA expression in cardiac fibroblasts (CF), induced cardiomyocytes (iCMs) (1 week (W), 2 W, 4 W after transduction), and neonatal cardiomyocytes (CM), determined by qPCR (n = 3).
(E) Heatmap image of microarray data illustrating differentially expressed genes among CF, α-MHC-GFP⁻, iCMs (FACS sorted 2 and 4 weeks after transduction), and CM (n = 3 in each group). The scale extends from 0.25- to 4-fold over mean (−2 to +2 in log2 scale). Red indicates increased expression, whereas green indicates decreased expression. Group 1 includes the genes upregulated only in CM, and group 2 includes the genes upregulated in CM and 4W-iCMs compared to CF. Lists of genes are shown in Table S1 and S2. All data are presented as means ± SD. *, P<0.01; **, P<0.05 vs. relevant control. See also Figure S4 for endogenous and exogenous expression of reprogramming factors and Tables S1 and S2 for differentially expressed genes.

(A) The promoters of Actn2, Ryr2, and Tnnt2 were analyzed by ChIP for trimethylation status of histone H3 of lysine 27 or 4 in cardiac fibroblasts (CF), CF-derived iCMs, tail-tip fibroblasts (TTF), TTF-derived iCMs, and neonatal cardiac cells. Data were quantified by qPCR.
(B) The promoters of Nppa and Myh6 were analyzed with bisulfite genomic sequencing for DNA methylation status in CF, α-MHC-GFP⁻ cells, α-MHC-GFP⁺ iCMs (FACS sorted 4 weeks after transduction), and neonatal CM. Open circles indicate unmethylated CpG dinucleotides, while closed circles indicate methylated CpGs.
(C) Schematic representation of the strategy to test expression kinetics of the Doxycycline (Dox)-inducible lentiviral system.
(D) Wild-type TTFs were infected with pLVX-tetO-GFP and pLVX-rtTA and imaged before (off Dox), 1 day after Dox addition, and at time points after Dox withdrawal (− Dox). All images were taken using constant exposure times and identical camera settings.
(E) Schematic representation of the strategy to determine temporal requirement of Gata4/Mef2c/Tbx5 in reprogramming. Thy1⁺/GFP⁻ TTF were infected with the pLVX-tetO-GMT and pLVX-rtTA lentiviruses, and Dox was added for 2 weeks and thereafter withdrawn for 1 week.
(F) Immunofluorescent staining for GFP, α-actinin, and DAPI in iCMs 2 weeks after lentiviral infection and Dox induction.
(G) Immunofluorescent staining for GFP, α-actinin, and DAPI 1 week after Dox withdrawal. iCMs maintained α-MHC GFP expression and had α-actinin positive sarcomeric structures. High-magnification views in insets show sarcomeric organization. Representative data are shown in each panel. All data are presented as means ± SD. *, P<0.01; **, P<0.05 vs. relevant control. Scale bars, 100 μm.

(A, B) Cardiac fibroblast (CF)–derived iCMs showed spontaneous Ca²⁺ oscillation with varying frequency (A), similar to neonatal cardiomyocytes (B). Rhod-3 intensity traces are shown.
(C)Tail-tip dermal fibroblast (TTF)–derived iCMs showed spontaneous Ca²⁺ oscillation with lower frequency. The Rhod-3 intensity trace is shown.
(D) Spontaneous Ca²⁺ waves observed in CF-derived α-MHC-GFP⁺ iCMs (white dots) or neonatal cardiomyocytes (arrows) with Rhod-3 at Ca²⁺ max and min is shown. Fluorescent images correspond to the Movie S1.
(E) Spontaneous Ca²⁺ oscillation observed in the TTF-derived α-MHC-GFP⁺ iCMs with Rhod-3 at Ca²⁺ max and min is shown. Fluorescent images correspond to the Movie S2.
(F) Spontaneously contracting iCMs had electrical activity measured by single cell extracellular electrodes. Neonatal cardiomyocytes showed similar electrical activity.
(G) Intracellular electrical recording of CF-derived iCMs cultured for 10 weeks displayed action potentials that resembled those of adult mouse ventricular cardiomyocytes. Representative data are shown in each panel (n = 10 in A–F, n = 4 in G). See also Figure S5 and Supplementary Movies.

(A) DsRed infected cardiac fibroblasts (DsRed-cell) were transplanted into NOD-SCID mouse hearts 1 day after infection and cardiac sections were analyzed by immunocytochemistry after 2 weeks. Transplanted fibroblasts marked with DsRed did not express α-actinin (green).
(B) Cardiac fibroblasts infected with DsRed or Gata4/Mef2c/Tbx5 with DsRed (GMT/DsRed-cell) were transplanted into NOD-SCID mouse hearts 1 day after infection and visualized by histologic section. Note that a subset of GMT/DsRed cells expressed α-MHC-GFP. Data were analyzed 2 weeks after transplantation.
(C) Gata4/Mef2c/Tbx5-transduced cardiac fibroblasts (GMT-cell) were transplanted into mouse hearts and histologic sections analyzed. A subset of induced GFP⁺ cells expressed α-actinin (red) and had sarcomeric structures. Insets are high-magnification views of cells indicated by arrows. Data were analyzed 2 weeks after transplantation.
Representative data are shown in each panel (n = 4 in each group). Scale bars, 100 μm. Note that GMT/DsRed or GMT-infected cells did not express GFP at the time of transplantation (Figure 4A).