Direct reprogramming of human fibroblasts toward a cardiomyocyte-like state

Abstract

Direct reprogramming of adult somatic cells into alternative cell types has been shown for several lineages. We previously showed that GATA4, MEF2C, and TBX5 (GMT) directly reprogrammed nonmyocyte mouse heart cells into induced cardiomyocyte-like cells (iCMs) in vitro and in vivo. However, GMT alone appears insufficient in human fibroblasts, at least in vitro. Here, we show that GMT plus ESRRG and MESP1 induced global cardiac gene-expression and phenotypic shifts in human fibroblasts derived from embryonic stem cells, fetal heart, and neonatal skin. Adding Myocardin and ZFPM2 enhanced reprogramming, including sarcomere formation, calcium transients, and action potentials, although the efficiency remained low. Human iCM reprogramming was epigenetically stable. Furthermore, we found that transforming growth factor β signaling was important for, and improved the efficiency of, human iCM reprogramming. These findings demonstrate that human fibroblasts can be directly reprogrammed toward the cardiac lineage, and lay the foundation for future refinements in vitro and in vivo.

Figure 1

Identifying a Minimal Cocktail of Transcription Factors to Reprogram Human Fibroblasts toward CM-like Cells (A) Screen results of αMHC-mCherry⁺ cell induction in H9Fs with 19 candidate reprogramming factors, and the effects of removing individual factors from the 19-factor (19F) pool on days 4, 7, and 10 after retroviral infection (n = 3). (B) Effects of removing individual factors from the 15F pool on αMHC-mCherry⁺ cell induction, as assessed by FACS (n = 3). (C–E) Effects of removing individual factors from the nine-factor (9F) (C, n = 4), 7F (D, n = 5), or 5F (E, n = 6) pool on αMHC-mCherry⁺ (top) and cTNT⁺ (bottom) cell induction by FACS. (F) Removal of both MYOCD and ZFPM2, but not either one alone, from the 7F pool significantly decreased the induction of αMHC-mCherry⁺ cells by FACS (n = 6). (G) Summary of results of αMHC-mCherry⁺ and cTNT⁺ induction 2 weeks after 5F (n = 11) or 7F (n = 13) retroviral infection. (H) Representative FACS plots of αMHC-mCherry⁺ cells 2 weeks after infection with the indicated factors. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 compared with dashed lines. Data represent the mean ± SD from independent experiments. See also Figures S1 and S2.

Figure 2

Human Fibroblasts Reprogrammed by Seven Factors Express Cardiac Genes and Form Sarcomere-like Structures (A) Immunocytochemistry of αMHC-mCherry H9-CMs and H9Fs 10 weeks after retroviral infection with five or seven factors using mCherry, cTNT, or α-actinin antibodies, revealing partial sarcomeric organization in the cells indicated (highlighted in insets). (B and C) Immunocytochemistry of HDFs and HCFs 6 weeks after 7F retroviral plus αMHC-mCherry lentiviral infection reveals sarcomeric gene expression in mCherry⁺ cells. (D and E) Electron microscopic images of αMHC-mCherry⁺ 7F-iCMs (D, day 45 postinduction) reveal enriched mitochondria (star) and sarcomeres (dashed area) with Z lines (arrow) in 7F reprogrammed cells, which were similar to H9-CMs (E, day 22 postdifferentiation). Scale bars, 20 μm (A–C) and 1 μm (D and E). Nu, nucleus. See also Figure S3.

Figure 3

Human iCMs Are Transcriptionally Reprogrammed toward the CM State (A) Heatmap image of microarray data illustrating gene expression profiles for the panel of genes that were differentially expressed between H9Fs and human fetal CMs evaluated in H9Fs, HDFs, human fetal CMs, H9-CMs, 5F- and 7F-iCMs, and HDF-derived iCMs (4 and 12 weeks postinduction); range of ±256-fold (log₂8) changes. The average level in three H9F samples was used as a baseline for comparison. Groups 1 and 2 include the genes that were upregulated or downregulated in CMs and iCMs compared with H9Fs. (B) Heatmap of gene expression profiles for a panel of cardiac genes in group 1 of (A) that were more highly upregulated in 7F-iCMs compared with 5F-iCMs at 4 and 12 weeks after reprogramming. (C) Correlation heatmap of orthologous gene expression for the panel of genes that were differentially expressed between fibroblasts and CMs evaluated in human iCMs reprogrammed by 5F or 7F and GMT-reprogrammed mouse (m) in vitro and in vivo iCMs. The correlation is indicated by color range; the scale along the values −1, 0, and 1 indicates perfect anticorrelation, no correlation, or perfect correlation, respectively. (D) Heatmap of single-cell gene expression by qPCR using microfluidics (Fluidigm) in the cell types indicated. Each horizontal row represents an individual cell, and each vertical column represents the levels of a single gene. Gene-expression level is indicated by the color range. See also Figures S4 and S5.

Figure 4

Human Fibroblasts Are Stably Reprogrammed into iCMs (A) Bisulfite genomic sequencing for MYH6, MYH7, and MYL7 promoter methylation status in H9Fs, 5F- and 7F-iCMs (2 weeks postinfection), and H9-CMs. Open circles indicate unmethylated CpG dinucleotides; closed circles indicate methylated CpGs. (B) The promoters of ACTC1, ACTN2, RYR2, TNNT2, PLN, and COL1A1 were analyzed by ChIP for trimethylation status of histone H3 of lysine 27 or 4 (H3K27me3 or H3K4me3) in H9Fs, 7F-iCMs, and H9-CMs. n = 3, ^∗p < 0.05, ^∗∗p < 0.01 versus H9Fs. Data represent the mean ± SD from independent experiments. (C) Strategy to determine the temporal requirement of reprogramming factors for human cardiac reprogramming. (D) FACS plots of αMHC-mCherry⁺ cells after retroviral infection without Dox (no Dox) or with 2-week Dox induction (+Dox 2W) followed by Dox withdrawal (−Dox) for 1, 3, and 5 weeks. See also Figure S6.

Figure 5

Human iCMs Exhibit Ca²⁺ Flux and Action Potentials (A) Top: Electrical field stimulation induces Ca²⁺ transients, as shown by Ca²⁺ imaging (minimal (Min) and maximal (Max) levels), in αMHC-mCherry⁺ 7F-iCMs 4 weeks after retroviral infection. Bottom: A representative trace of Ca²⁺ transients recorded from reprogrammed αMHC-mCherry⁺ 7F-iCMs (left, n = 9) is similar to the trace recorded from 3-week-old H9-CMs (right). (B) A representative trace of caffeine (20 mM)-induced Ca²⁺ transients recorded from reprogrammed 8-week αMHC-mCherry⁺ 7F-iCMs (top, n = 15) is similar to the trace recorded from 4-week-old H9-CMs (below). (C) Electrical field stimulation induces Ca²⁺ transients, as shown by Ca²⁺ imaging (Min and Max level, top) and a representative trace (bottom), in HDF-derived iCMs 6 weeks after 7F retroviral infection. (D) Typical action potential measured in 7F-iCMs (n = 3) 10 weeks after induction in comparison with H9-CMs (right). Scale bars, 20 μm (A and C). See also Movies S1 and S2.

Figure 6

Human iCM Reprogramming Involves TGF-β1 Signaling (A) Results from exposure to eight small molecules show the relative degree of αMHC-mCherry⁺ cell induction 2 weeks after 7F retroviral infection (n = 3). Cells were exposed to individual compounds from days 1–14 after 7F infection at the following doses: A-83-01 (0.5 μM), Chir99201 (3 μM), forskolin (5 μM), LY-2157299 (300 nM), PMA (100 nM), SIS3 (10 μM), SB415286 (10 μM), and SB431542 (10 μM). Data represent the mean ± SD from independent experiments. (B) Effects of activin A (20 ng/ml) and TGF-β1 (20 ng/ml) on αMHC-mCherry⁺ cell induction 2 weeks after retroviral infection with five or seven factors (n = 3). The inset panel represents the effects of TGF-β1 at different doses. ^∗p < 0.001. The SIS3 dose was 10 μM. Data represent the mean ± SD from independent experiments. (C) A cell proliferation assay using CFSE revealed that activin A and TGF-β1 did not increase the proliferation of iCMs. Noninfected H9Fs (black line) were used as a positive control of cell division, as indicated by decreased fluorescence intensity. Mitomycin C (MC)-treated H9Fs (gray line) were used as negative controls for proliferation and demonstrated high fluorescence intensity. H9Fs were treated with activin A or TGF-β1 from days 1–14 after 5F or 7F infection. Images are representative of three independent experiments.