The reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) raises the possibility that a somatic cell could be reprogrammed to an alternative differentiated fate without first becoming a stem/progenitor cell. A large pool of fibroblasts exists in the postnatal heart, yet no single “master regulator” of direct cardiac reprogramming has been identified. Here, we report that a combination of three developmental transcription factors (i.e., Gata4, Mef2c, and Tbx5) rapidly and efficiently reprogrammed postnatal cardiac or dermal fibroblasts directly into differentiated cardiomyocyte-like cells. Induced cardiomyocytes expressed cardiac-specific markers, had a global gene expression profile similar to cardiomyocytes, and contracted spontaneously. Fibroblasts transplanted into mouse hearts one day after transduction of the three factors also differentiated into cardiomyocyte-like cells. We believe these findings demonstrate that functional cardiomyocytes can be directly reprogrammed from differentiated somatic cells by defined factors. Reprogramming of endogenous or explanted fibroblasts might provide a source of cardiomyocytes for regenerative approaches.

In this study, we examined whether key developmental cardiac regulators could reprogram cardiac fibroblasts into cardiomyocytes. We found that out of a total of 14 factors, a specific combination of three transcription factors, Gata4, Mef2c, and Tbx5, was sufficient to generate functional beating cardiomyocytes directly from mouse postnatal cardiac or dermal fibroblasts and that the induced cardiomyocytes (iCMs) were globally reprogrammed to adopt a cardiomyocyte-like gene expression profile.

The generation of iPSCs suggests that a specific combination of defined factors, rather than a single factor, could epigenetically alter the global gene expression of a cell and allow greater plasticity of cell type than previously appreciated. Consistent with this, the bHLH transcription factor, Neurogenin 3, in combination with Pdx1 and Mafa, can efficiently reprogram pancreatic exocrine cells into functional β cells in vivo, although the exocrine cells were known to have some potential to become islet cells in vitro and share a common parent cell with islet cells (). A combination of three factors, Ascl1, Brn2, and Myt1l, converts dermal fibroblasts to functional neurons (), although the degree of global reprogramming of the neurons is unknown.

The human heart is composed of cardiomyocytes, vascular cells, and cardiac fibroblasts. In fact, cardiac fibroblasts comprise over 50% of all the cells in the heart (). Cardiac fibroblasts are fully differentiated somatic cells that provide support structure, secrete signals, and contribute to scar formation upon cardiac damage (). Fibroblasts arise from an extracardiac source of cells known as the proepicardium, and do not normally have cardiogenic potential (). The large population of endogenous cardiac fibroblasts is a potential source of cardiomyocytes for regenerative therapy if it were possible to directly reprogram the resident fibroblasts into beating cardiomyocytes. Unfortunately, although embryonic mesoderm can be induced to differentiate into cardiomyocytes (), efforts to accomplish this in somatic cells have thus far been unsuccessful, and to our knowledge, no “master regulator” of cardiac differentiation, like MyoD for skeletal muscle (), has been identified to date.

Heart disease is a leading cause of adult and childhood mortality. The underlying pathology is typically loss of cardiomyocytes that leads to heart failure or improper development of cardiomyocytes during embryogenesis that leads to congenital heart malformations. Because postnatal cardiomyocytes have little or no regenerative capacity, current therapeutic approaches are limited. Embryonic stem cells possess clear cardiogenic potential, but efficiency of cardiac differentiation, risk of tumor formation, and issues of cellular rejection must be overcome (). The ability to reprogram fibroblasts into induced pluripotent stem cells (iPSCs) with four defined factors might address some of these issues by providing an alternative source of embryonic-like stem cells (). However, generating sufficient iPSC-derived cardiomyocytes that are pure and mature and that can be delivered safely remains challenging ().

To investigate whether GMT-transduced cardiac fibroblasts can be reprogrammed to express cardiomyocyte-specific genes in their native environment in vivo, we harvested GFP/Thy1cardiac fibroblasts 1 day after viral transduction and injected them into immunosuppressed NOD-SCID mouse hearts. GMT-infected cells did not express GFP at the time of transplantation ( Figure 4 A). Cardiac fibroblasts were infected with either the mixture of GMT and DsRed retroviruses or DsRed retrovirus (negative control) to be readily identified by fluorescence. Cardiac fibroblasts infected with DsRed did not express α-actinin or GFP, confirming cardiomyocyte conversion did not happen in the negative control ( Figures 7 A and 7B ). Despite being injected into the heart only 1 day after viral infection, a subset of cardiac fibroblasts transduced with GMT and DsRed expressed GFP in the mouse heart within 2 weeks ( Figure 7 B). Importantly, the GFPcells expressed α-actinin and had sarcomeric structures ( Figure 7 C). These results suggested that cardiac fibroblasts transduced with Gata4, Mef2c, and Tbx5 can reprogram to cardiomyocytes within 2 weeks upon transplantation in vivo.

Representative data are shown in each panel (n = 4 in each group). Scale bars represent 100 μm. Note that GMT/DsRed or GMT-infected cells did not express GFP at the time of transplantation ( Figure 4 A).

(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.

(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.

(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).

In addition to the characteristic Caflux, cardiac fibroblast-derived iCMs showed spontaneous contractile activity after 4–5 weeks in culture (Movies S3 and S4 Figure S5 ). Single-cell extracellular recording of electrical activity in beating cells revealed tracings similar to the potential observed in neonatal cardiomyocytes ( Figure 6 F). Intracellular electrical recording of iCMs displayed action potentials that resembled those of adult mouse ventricular cardiomyocytes ( Figure 6 G). Thus, the reprogramming of fibroblasts to iCMs was associated with global changes in gene expression, epigenetic reprogramming, and the functional properties characteristic of cardiomyocytes.

To determine if iCMs possessed the functional properties characteristic of cardiomyocytes, we analyzed intracellular Caflux in iCMs after 2–4 weeks of culture. Around 30% of cardiac fibroblast-derived iCMs showed spontaneous Caoscillations and their frequency was variable, resembling what was observed in neonatal cardiomyocytes ( Figures 6 A, 6B, and 6D ; Movie S1 ). We observed that tail-tip dermal fibroblast-derived iCMs also exhibited spontaneous Caoscillations, but the oscillation frequency was lower than that of cardiomyocytes and cardiac fibroblast-derived iCMs ( Figures 6 C and 6E; Movie S2 ).

(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 Movies S1 S3 and S4

(E) Spontaneous Caoscillation observed in the TTF-derived α-MHC-GFPiCMs with Rhod-3 at Camax and min is shown. Fluorescent images correspond to the Movie S2

(D) Spontaneous Cawaves observed in CF-derived α-MHC-GFPiCMs (white dots) or neonatal cardiomyocytes (arrows) with Rhod-3 at Camax and min is shown. Fluorescent images correspond to the Movie S1

To further assess the stability of the reprogramming event, we generated a doxycycline-inducible lentiviral system in which transgene expression of the reprogramming factors was controlled by doxycycline administration. We first transduced wild-type tail-tip fibroblasts with a mixture of lentiviruses containing pLVX-tetO-GFP and pLVX-rtTA to determine the expression kinetics of this system ( Figure 5 C). We confirmed that the majority of fibroblasts infected with both viruses expressed GFP within 1 day after doxycycline induction, and the GFP expression was instantly diminished by withdrawal of doxycycline and disappeared within 6 days ( Figure 5 D). Thy1/GFPtail-tip fibroblasts were harvested from αMHC-GFP neonatal mice, transduced with a pool of lentiviruses containing inducible Gata4, Mef2c, and Tbx5, along with pLVX-rtTA, and subsequently treated with doxycycline ( Figure 5 E). We found that αMHC-GFP expression was induced from tail-tip fibroblasts after doxycycline administration and that the iCMs had well-defined sarcomeric structures marked with an anti-α-actinin antibody after 2 weeks of culture ( Figure 5 F). Doxycycline was withdrawn after 2 weeks of culture, and cells were subsequently cultured without doxycycline for 1 week to fully remove exogenous expression of the reprogramming factors ( Figure 5 E). The iCMs maintained αMHC-GFP expression and had sarcomeric structures after doxycycline withdrawal, suggesting that the fibroblasts were stably reprogrammed into iCMs after 2 weeks exposure to Gata4, Mef2c, and Tbx5 ( Figure 5 G).

The DNA methylation status of specific loci also reflects the stability of the reprogramming event and we therefore investigated such changes during reprogramming from cardiac fibroblasts to iCMs. We performed bisulfite genomic sequencing in the promoter regions of Nppa and Myh6 in cardiac fibroblasts, 4 week GFPcells, iCMs, and neonatal cardiomyocytes. Both promoter regions were hypermethylated in cardiac fibroblasts and GFPcells, as expected from the cardiomyocyte-specific expression of these genes, but were comparatively demethylated in iCMs, similar to neonatal cardiomyocytes ( Figure 5 B). These results indicated that reprogramming by Gata4, Mef2c, and Tbx5 induced epigenetic resetting of the fibroblast genome to a cardiomyocyte-like state.

To determine if iCMs have gained a cardiomyocyte-like chromatin state, we analyzed the enrichment of histone modifications in the promoter regions of the cardiac-specific genes Actn2, Ryr2I, and Tnnt2. We analyzed the enrichment of trimethylated histone H3 of lysine 27 (H3K27me3) and lysine 4 (H3K4me3), which mark transcriptionally inactive or active chromatin, respectively (), in cardiac fibroblasts, 4 week iCMs, and neonatal cardiac cells by chromatin immunoprecipitation, followed by qPCR ( Figure 5 A ). After reprogramming, H3K27me3 was significantly depleted at the promoters of all the genes analyzed in iCMs, reaching levels comparable to those in cardiac cells, whereas H3K4me3 increased on the promoter regions of Actn2 and Tnnt2 in iCMs, as compared with cardiac fibroblasts. Ryr2 had similar levels of H3K4me3 in iCMs as in fibroblasts, suggesting that its activation reflects the resolution of a “bivalent” chromatin mark (). These results suggested that cardiac fibroblast-derived iCMs gained a chromatin status similar to cardiomyocytes at least in some cardiac specific genes. Intriguingly, H3K27me3 levels were higher in tail-tip fibroblasts than cardiac fibroblasts on all three genes analyzed and, despite a significant reduction upon reprogramming to iCMs, remained somewhat higher than in cardiac cells and cardiac fibroblast-derived iCMs.

(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 versus relevant control. Scale bars represent 100 μm.

(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.

(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.

(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; closed circles indicate methylated CpGs.

(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.

We next compared the progressive global gene expression pattern of iCMs, neonatal cardiomyocytes, and cardiac fibroblasts by mRNA microarray analyses. We sorted GFPcells and GFPcells 2 and 4 weeks after GMT transduction. The iCMs at both stages were similar to neonatal cardiomyocytes, but were distinct from GFPcells and cardiac fibroblasts in global gene expression pattern ( Figure 4 E). We found that functionally important cardiac genes were upregulated significantly more in 4 week iCMs than in 2 week iCMs, including Pln (phospholamban), Slc8a1 (sodium/calcium exchanger), Myh6, Sema3a (semaphorin 3a), Id2 (inhibitor of DNA binding 2), and Myl2 (myosin, light polypeptide 2, regulatory, cardiac, slow, also known as MLC2v) ( Table S1 ). Conversely, some genes were downregulated more in 4 week iCMs than in 2 week iCMs ( Table S1 ). The array analyses also identified genes that were upregulated more in neonatal cardiomyocytes than in 4 week iCMs or cardiac fibroblasts (group 1 in Figure 4 E), including Bmp10 (bone morphogenetic protein 10), Erbb4 (v-erb-a erythroblastic leukemia viral oncogene homolog 4), Irx4 (Iroquois related homeobox 4), and Atp1a2 (ATPase, Na/Ktransporting, α 2 polypeptide) ( Table S2 ). We also identified genes that were expressed to a greater extent in both cardiomyocytes and 4 week iCMs than in fibroblasts (group 2 in Figure 4 E), including Actc1, Myl7 (myosin, light polypeptide 7, regulatory, also known as MLC2a), Tnnt2 (troponin T2, cardiac), Tbx3 (T-box 3), and Srf (serum response factor) ( Table S2 ). Thus, iCMs were similar, but not identical, to neonatal cardiomyocytes, and the reprogramming event was broadly reflected in global gene expression changes.

We next analyzed the time course of cardiomyocyte induction from cardiac fibroblasts. GFPcells were detected 3 days after induction and gradually increased in number up to 20% at day 10 and were still present after 4 weeks ( Figure 4 A ). GFPcells were less proliferative than GFPcells and, over time, decreased in percentage relative to the total number of cells. Importantly, the percentage of cTnTcells among the α-MHC-GFPiCMs and the intensity of cTnT expression increased significantly over time ( Figures 4 B and 4C). We sorted GFPcells at 1, 2, and 4 weeks after transduction with GMT and compared cardiac gene expression with cardiac fibroblasts and neonatal cardiomyocytes. The cardiomyocyte-specific genes, Actc1, Myh6, Ryr2 (ryanodine receptor 2), and Gja1 (connexin43), were significantly upregulated in a time-dependent manner in GFPcells, but were not detected in cardiac fibroblasts by qPCR ( Figure 4 D). Col1a2 (collagen 1a2), a marker of fibroblasts, was dramatically downregulated in GFPcells from 7-day culture to the same level as in cardiomyocytes. These data indicated that the three factors induced direct conversion of cardiac fibroblasts to cardiomyocytes rapidly and efficiently, but full maturation was a slow process that occurred over several weeks. Total gene expression of the three reprogramming factors was upregulated 6- to 8-fold in iCMs over neonatal cardiomyocytes. However, only endogenous expression of Gata4 was upregulated in iCMs to the same level as in neonatal cardiomyocytes, whereas endogenous Mef2c and Tbx5 expression was lower in iCMs than in cardiomyocytes, potentially reflecting negative autoregulatory loops ( Figure S4 ).

mRNA expression levels of endogenous or retrovirally introduced Gata4, Mef2c or Tbx5 in 4 week iCMs compared to cardiac fibroblasts (CF) or neonatal cardiomyocytes (CM). Data are shown relative to levels in CM. RNA levels were determined using primers specific for endogenous transcripts (white bars) and those common for both endogenous and transgene transcripts (white and black bars) by qRT-PCR.

(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 Table S2 . All data are presented as means ± SD.p < 0.01;p < 0.05 versus relevant control. See also Figure S4 for endogenous and exogenous expression of reprogramming factors and Table S1 and Table S2 for differentially expressed genes.

(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).

(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.

Whereas Isl1 marks most early cardiac progenitors, a subpopulation of cardiac progenitors remains Isl1 negative. Mesp1 is the earliest pan-cardiovascular progenitor cell marker that is transiently expressed in nascent mesoderm before further cardiovascular differentiation ( Figure S3 ) (). We therefore generated Mesp1-YFP mice by crossing Mesp1-Cre and R26R-EYFP mice to determine if iCMs were reprogrammed into early cardiac mesoderm before further differentiation. We isolated Mesp1-YFP/Thy1tail-tip dermal fibroblasts by FACS and transduced the cells with GMT ( Figures 3 K and 3L). The resulting cTnTcells did not express YFP, suggesting that the iCMs were not converted into the cardiac mesoderm cell state for reprogramming, but rather they were directly reprogrammed into differentiated cardiomyocytes by the three factors ( Figure 3 L).

MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube.

We also determined whether the reprogramming of fibroblasts to differentiated cardiomyocytes was a direct event or if the fibroblasts first passed through a cardiac progenitor cell fate before further differentiation. To distinguish between these two possibilities, we used mice expressing Isl1–yellow fluorescent protein (YFP) obtained by crossing Isl1-Cre mice and R26R-EYFP mice () ( Figure S3 ). Isl1 is an early cardiac progenitor marker that is transiently expressed before cardiac differentiation. If iCMs generated from fibroblasts passed through a cardiac progenitor state, they and their descendants would permanently express YFP (). We isolated Isl1-YFP/Thy1cells from Isl1-YFP heart explants by FACS and transduced the cells with GMT. The resulting cTnTcells did not express YFP in significant numbers, suggesting that the iCMs were not first reprogrammed into Isl1cardiac progenitor cells ( Figures 3 I and 3J). Moreover, these results provided supportive evidence that the iCMs did not originate from a rare population of cardiac progenitor cells that might exist in neonatal hearts.

We then sought to more definitively exclude the possibility of rare cardiac progenitors giving rise to iCMs. We tested the potential of mouse tail-tip dermal fibroblasts to generate iCMs. We found that sorted Thy1/GFPtail-tip dermal fibroblasts transduced with GMT expressed GFP at the same level as GMT-transduced cardiac fibroblasts, although the percentage of cTnTcells was less than cardiac fibroblast-derived iCMs ( Figures 3 E–3G). Like cardiac fibroblasts, tail-tip fibroblast-derived GFPcells expressed α-actinin and had well-defined sarcomeric structures ( Figure 3 H; Figure S3 ), suggesting noncardiac fibroblasts can also be reprogrammed into cardiomyocytes by GMT induction. These results excluded the possibility that the iCMs arose from contamination of cardiomyocytes or cardiac progenitors before cardiac induction in the fibroblast population.

To determine if the induced cardiomyocyte-like cells (iCMs) were arising from a subpopulation of stem-like cells, we analyzed c-kit expression () in the Thy1/GFPcells. Most c-kitcells coexpressed Thy1, whereas 15% of Thy1cells expressed c-kit, which is consistent with a previous report of cardiac explant-derived cells (). We isolated GFP/Thy1/c-kitcells and GFP/Thy1/c-kitcells by FACS and transduced each population of cells with GMT. We found 2–3-fold more cardiomyocyte induction in GFP/Thy1/c-kitcells than in GFP/Thy1/c-kitcells ( Figure S3 ). These results suggest that most of the iCMs originated from a c-kit-negative population.

(F) Mesp1-Cre/Rosa-YFP hearts were stained with YFP, α-actinin and DAPI. YFP was diffusely expressed in the heart and also in vascular cells. Insets are high-magnification views showing coronary vasculature.

(E) Isl1-Cre/Rosa-YFP hearts were stained with YFP, α-actinin and DAPI. YFP was diffusely expressed in RV and IVS, and in some part of LV.

We next isolated neonatal cardiac fibroblasts by the conventional fibroblast isolation method in which hearts were digested with trypsin and plated on plastic dishes (). More than 85% of the cells expressed Thy1, and we isolated Thy1/GFPcells by FACS to exclude cardiomyocyte contamination ( Figure 3 A ). Fibroblasts transduced with GMT expressed GFP, cTnT, and actinin after 1 week at the same level as fibroblasts isolated from explant cultures ( Figures 3 B and 3C). Similar results were obtained on introduction of GMT into adult cardiac fibroblasts, with full formation of sarcomeric structures ( Figure 3 D; Figure S2 ).

All data are presented as means ± SD.p < 0.01 versus relevant control. Scale bars represent 100 μm. See also Figure S3 for analyses of c-kitcells.

(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.

To determine if other cardiac genes were enriched in GFPcells, we sorted GFPcells and GFPcells 7 days after transduction with GMT and compared gene expression of cardiomyocyte-specific genes, Myh6 (α-myosin heavy chain), Actc1 (cardiac α-actin), Actn2 (actinin α2), and Nppa (natriuretic peptide precursor type A) by quantitative RT-PCR (qPCR). We found that these cardiac genes were upregulated significantly more in GFPthan in GFPcells ( Figure 2 E). Next, we used immunocytochemistry to determine if cardiac proteins were expressed in GFPcells. Despite the detection of cTnT in only 30% of GFPcells, most GFPcells induced with the three factors expressed sarcomeric α-actinin (α-actinin) and had well-defined sarcomeric structures, similar to neonatal cardiomyocytes ( Figure 2 F; Figure S1 ). In addition to α-actinin, some GFPcells also expressed cTnT and ANF (atrial natriuretic factor), indicating GFPcells expressed several cardiomyocyte-specific markers ( Figures 2 G and 2H). We also confirmed that neither GFPnor GFPcells expressed smooth muscle or endothelial cell markers ( Figure S2 ), suggesting specificity of GMT effects.

We found that 30% of GFPcells expressed cTnT 1 week after the three-factor transduction. Next, to confirm our screening results, we transduced cardiac fibroblasts with three factors (Gata4, Mef2c, and Tbx5, hereafter referred to as GMT) plus Nkx2-5, a critical factor for cardiogenesis but excluded by our initial screening. Surprisingly, adding Nkx2-5 to GMT dramatically inhibited the expression of GFP and cTnT in cardiac fibroblasts. We also transduced cardiac fibroblasts with the combination of Baf60c, Gata4, and Tbx5, which can transdifferentiate noncardiogenic mesoderm to cardiomyocytes in mouse embryos (). We found that this combination did not efficiently induce cTnT or GFP expression above that of Tbx5 alone, confirming our screening results ( Figure 2 D).

Next, we examined the expression of cTnT by FACS. We found that 20% of GFPcells expressed cTnT at high enough levels to detect by FACS 1 week after the four-factor transduction. Again removing individual factors from the four-factor pool in transduction, we found that Mesp1 was dispensable for cTnT expression ( Figures 2 A and 2B ). In contrast, we did not observe cTnTor GFPcells, when either Mef2c or Tbx5 was removed. Removal of Gata4 did not significantly affect the number of GFPcells, but cTnT expression was abolished, suggesting Gata4 was also required. Whereas the combination of two factors, Mef2c and Tbx5, induced GFP expression but not cTnT, no combination of two factors or single factor induced both GFP and cTnT expression in cardiac fibroblasts ( Figure 2 C). These data suggested that the combination of three factors, Gata4, Mef2c, and Tbx5, is sufficient to induce cardiac gene expression in fibroblasts.

(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

(C) Effect of the transduction of pools of three, two, and one factors on GFP + and cTnT + cell induction (n = 3).

(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.

To determine which of the 14 factors were critical for activating cardiac gene expression, we serially removed individual factors from the pool of 14. Pools lacking five factors (Baf60c, Hand2, Hopx, Hrt2, and Pitx2c) produced an increased number of GFPcells, suggesting they are dispensable in this setting ( Figures 1 D and 1E). Of note, removing Gata4 decreased the percentage of GFPcells to 0.5%, and removing Pitx2c increased it to 5%. Removal of the five factors listed above resulted in an increase in the percentage of GFPcells to 13% ( Figure 1 F). We conducted three further rounds of withdrawing single factors from nine-, six-, and five-factor pools, removing those that did not decrease efficiency upon withdrawal, and found that four factors (Gata4, Mef2c, Mesp1, and Tbx5) were sufficient for efficient GFPcell induction from cardiac fibroblasts ( Figures 1 F–1H). The combination of these four factors dramatically increased the number of fibroblasts activating the αMHC-GFP reporter to over 20% ( Figure 1 I).

We transduced Thy1/GFPneonatal mouse cardiac fibroblasts with a mixture of retroviruses expressing all 14 factors or with DsRed retrovirus (negative control) (). We did not observe any GFPcells in cardiac fibroblasts 1 week after Ds-Red retrovirus infection or 1 week of culture without any viral infection. In contrast, transduction of all 14 factors into fibroblasts resulted in the generation of a small number of GFPcells (1.7%), indicating the successful activation of the cardiac-enriched αMHC gene in some cells ( Figures 1 D and 1E).

To select potential cardiac reprogramming factors, we used microarray analyses to identify transcription factors and epigenetic remodeling factors with greater expression in mouse cardiomyocytes than in cardiac fibroblasts at embryonic day 12.5 (). Among them, we selected 13 factors that exhibited severe developmental cardiac defects and embryonic lethality when mutated ( Figure S2 ). We also included the cardiovascular mesoderm-specific transcription factor Mesp1 because of its cardiac transdifferentiation effect in Xenopus (). We generated individual retroviruses to efficiently express each gene in cardiac fibroblasts ( Figure S2 ).

(D) Immunofluorescent staining for GFP, α-actinin and DAPI in induced cardiomyocytes derived from adult cardiac fibroblasts from explants as indicated in Figure 3 D.

(C) Induced cardiomyocytes were stained with SM-MHC and CD31 antibodies. No expression was detected by either staining.

(B) Cardiac fibroblasts were transduced with retrovirus expressing Nkx2-5. Cells were immunostained with anti-Nkx2-5 antibody (green) and DAPI (blue). Note that most cells expressed Nkx2-5, detected in nuclei.

(A) Candidate factors upregulated in embryonic day (E) 12.5 cardiomyocytes compared to cardiac fibroblasts by microarray are listed along with their fold enrichment (n = 3). Mesp1 expression was not detected in either cell type (ND).

To have enough cardiac fibroblasts for FACS screening, we obtained GFPcardiac fibroblasts from neonatal αMHC-GFP hearts by explant culture. Fibroblast-like cells migrated from the explants after 2 days and were confluent after 1 week. The migrating cells did not express GFP, but expressed Thy1 and vimentin, markers of cardiac fibroblasts ( Figure 1 B and data not shown) (). To avoid contamination of cardiomyocytes, we filtered the cells by cell strainers to remove heart tissue fragments and isolated Thy1/GFPcells by FACS ( Figure 1 C). Using FACS, we confirmed that Thy1/GFPcells did not express cardiac troponin T (cTnT), a specific sarcomeric marker of differentiated mature cardiomyocytes ( Figure S1 ) (). With these procedures, we had no cardiomyocyte contamination in the fibroblast culture and could generate greater than twice the number of cardiac fibroblasts than by conventional fibroblast isolation techniques ().

We developed an assay system in which the induction of mature cardiomyocytes from fibroblasts could be analyzed quantitatively by reporter-based fluorescence-activated cell sorting (FACS) ( Figure 1 A ). To accomplish this, we generated αMHC promoter-driven EGFP-IRES-puromycin transgenic mice (αMHC-GFP), in which only mature cardiomyocytes expressed the green fluorescent protein (GFP) (). We confirmed that only cardiomyocytes, but not other cell types such as cardiac fibroblasts, expressed GFP in the transgenic mouse hearts and in primary cultured neonatal mouse cardiac cells ( Figure S1 available online).

(E) GFP/Thy1cells from Figure 1 C were analyzed by FACS for cTnT and GFP expression, confirming no contamination of cardiomyocytes.

(D) Dissociated αMHC-GFP cardiac cells were immunostained with GFP, α-actinin, cTnT or ANF (red) antibodies, and DAPI (blue). Note that GFP was expressed only in α-actinin + and cTnT + cells, and a subset of GFP + cells expressed ANF (a marker of atrial myocytes).

(C) αMHC-GFP hearts were immunostained with GFP (green), α-actinin or vimentin (red) antibodies, and DAPI (blue). Arrows indicate valve apparatus, immunostained with vimentin but not with GFP antibodies.

(I) GFP + (20%) 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 versus relevant control. Scale bars represent 100 μm.

(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.

(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.

(D) Summary of FACS analyses for α-MHC-GFPcells. Effect on GFPcell 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 GFPcells and were excluded for further analyses. See also Figure S2

(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

Discussion

Here we demonstrated that the combination of three transcription factors, Gata4, Mef2c, and Tbx5, can rapidly and efficiently induce cardiomyocyte-like cells from postnatal cardiac and dermal fibroblasts. iCMs were similar to neonatal cardiomyocytes in global gene expression profile, electrophysiologically, and could contract spontaneously, demonstrating that functional cardiomyocytes can be generated from differentiated somatic cells by defined factors. Although much refinement and characterization of the reprogramming process will be necessary, the findings reported here raise the possibility of reprogramming the vast pool of endogenous fibroblasts that normally exists in the heart into functional cardiomyocytes for regenerative purposes.

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et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. + cells appearing at day 3, in contrast to iPSC reprogramming, which typically takes 10–20 days and occurs with much lower efficiency (<0.1%) ( Takahashi and Yamanaka, 2006 Takahashi K.

Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Several lines of evidence suggest that the iCMs we describe here originated from differentiated fibroblasts. We found that any potential rare cardiac “progenitor-like” cells, marked by c-kit or Isl1, were dispensable for cardiomyocyte induction (). Furthermore, the high efficiency of cardiac induction (up to 20%) does not favor the interpretation that rare stem or progenitor cells were the origin of induced cardiomyocytes. Most importantly, the ability to reprogram dermal fibroblasts into iCMs supports the conclusion that cardiac progenitors are not the target cells for the reprogramming factors. Remarkably, reprogramming of cardiac fibroblasts to myocytes occurred in a relatively short period, with the first GFPcells appearing at day 3, in contrast to iPSC reprogramming, which typically takes 10–20 days and occurs with much lower efficiency (<0.1%) (). Despite the early initiation of reprogramming, the process appears to continue for several weeks, with progressive changes in gene expression, contractile ability, and electrophysiologic maturation.

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Takagi A.

Kitajima S.

Miyazaki J.

Inoue T. MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube. Although many questions remain regarding the mechanisms of reprogramming, we were able to genetically test the “route” of cell fate alteration. Our findings suggest that cardiomyocytes were directly induced from cardiac fibroblasts without reverting to a cardiac progenitor cell state, which may explain the rapid early reprogramming process. This conclusion was supported by the absence of Isl1-Cre-YFP or Mesp1-Cre-YFP activation during the process of reprogramming, which would have marked any cells that transiently expressed Isl1 or Mesp1 ().

The ability to reprogram endogenous cardiac fibroblasts into cardiomyocytes has many therapeutic implications. First, the avoidance of reprogramming to pluripotent cells before cardiac differentiation would greatly lower the risk of tumor formation in the setting of future cell-based therapies. Second, large amounts of an individual's own fibroblasts can be grown from a cardiac biopsy or skin biopsy in vitro for transduction with the defined factors, followed by delivery of cells to damaged hearts. Third, and most promising, is the potential to introduce the defined factors, or factors that mimic their effects, directly into the heart to reprogram the endogenous fibroblast population, which represents more than 50% of the cells, into new cardiomyocytes that can contribute to the overall contractility of the heart. Our observation that injection of fibroblasts into the heart only 1 day after induction of Gata4/Mef2c/Tbx5 resulted in reprogramming of the transplanted cells suggests that this may be possible. Future studies in human cells and advances in safe delivery of defined factors will be necessary to advance this technology for potential regenerative therapies.