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Conversion of human fibroblasts into functional cardiomyocytes by small molecules

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Science  03 Jun 2016:
Vol. 352, Issue 6290, pp. 1216-1220
DOI: 10.1126/science.aaf1502

Making cardiac cells from fibroblasts

Reprogramming noncardiac cells into functional cardiomyocytes without any genetic manipulation could open up new avenues for cardiac regenerative therapies. Cao et al. identified a combination of nine small molecules that could epigenetically activate human fibroblasts, efficiently reprogramming them into chemically induced cardiomyocytes (ciCMs). The ciCMs contracted uniformly and resembled human cardiomyocytes. This method may be adapted for reprogramming multiple cell types and have important implications in regenerative medicine.

Science, this issue p. 1216

Abstract

Reprogramming somatic fibroblasts into alternative lineages would provide a promising source of cells for regenerative therapy. However, transdifferentiating human cells into specific homogeneous, functional cell types is challenging. Here we show that cardiomyocyte-like cells can be generated by treating human fibroblasts with a combination of nine compounds that we term 9C. The chemically induced cardiomyocyte-like cells uniformly contracted and resembled human cardiomyocytes in their transcriptome, epigenetic, and electrophysiological properties. 9C treatment of human fibroblasts resulted in a more open-chromatin conformation at key heart developmental genes, enabling their promoters and enhancers to bind effectors of major cardiogenic signals. When transplanted into infarcted mouse hearts, 9C-treated fibroblasts were efficiently converted to chemically induced cardiomyocyte-like cells. This pharmacological approach to lineage-specific reprogramming may have many important therapeutic implications after further optimization to generate mature cardiac cells.

Advances in reprogramming enable the fate of a cell to be changed, with potential applications for regenerative therapy. Cardiomyocyte (CM)–like cells can be reprogrammed from somatic fibroblasts by overexpression of cardiac genes in vitro (16) and in vivo (5, 710). However, efficiently transdifferentiating human noncardiac cells into highly functional CMs has remained a major challenge (1, 4, 6). In contrast to conventional reprogramming by genetic methods, a chemical reprogramming approach introduces small molecules that interact with and modulate endogenous factors in the starting cell type (e.g., fibroblast) in the absence of target cell type–specific proteins. Small molecules have certain advantages over genetic methods: They are convenient to use, can be efficiently delivered into cells, provide greater temporal control, and are nonimmunogenic and more cost-effective. Moreover, their effects can be fine-tuned by varying their concentrations and combinations. Here we report the identification of a combination of small molecules that enables reprogramming of human fibroblasts into chemically induced functional CMs (ciCMs) that uniformly display contractile properties.

To induce cardiac reprogramming of human fibroblasts, we used an established human foreskin fibroblast (HFF) line that contains no cardiac cells, as assayed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), flow cytometry, and immunofluorescence analyses (fig. S1). HFFs were virally transduced with an alpha myosin heavy chain (αMHC)–green fluorescent protein (GFP) reporter (11) to specifically label CMs. Based on the cell activation and signaling-directed (CASD) reprogramming paradigm (12, 13), we treated cells with compounds that induce or enhance cellular reprogramming to alternative fates (cell activation) in conjunction with cardiogenic molecules (signaling-directed) to induce cardiogenesis. We initially screened a collection of 89 small molecules that are known to facilitate reprogramming (table S1). Each compound was added to a baseline cocktail containing SB431542, CHIR99021, parnate, and forskolin, which enabled cardiac reprogramming in an earlier study when combined with a single gene, Oct4 (13). Cells were treated with the various small-molecule cocktails for 6 days and then cultured for 5 days in an optimized cardiac induction medium (CIM) containing the cardiogenic molecules activin A, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor, and CHIR99021 (fig. S2A). Positive hits that enhanced cardiogenic gene expression (as assayed by qRT-PCR) after CIM treatment were identified and further tested in different combinations. Cells were then re-plated in human CM–conditioned medium, which may provide cardiac paracrine signals that mimic the in vivo environment for further maturation (14). After iterative rounds of testing, we found that a combination of 15 compounds (termed 15C) generated about five αMHC-GFP–positive clusters that beat spontaneously from 3 × 105 re-plated cells at day 30 (Fig. 1A), indicating reprogramming toward the cardiac fate.

Fig. 1 ciCMs from HFFs.

(A) A representative contracting cluster induced by 15C at reprogramming day 30 (D30). Scale bars, 100 μm. (B to E) Number of contracting clusters (B to D) and flow cytometry analyses of cTNT (E) under the indicated conditions (n = 3). A minus (plus) sign indicates removal from (addition to) the small-molecule combination. The red dashed line indicates values of 15C (B), 7C [(C) and (D)], and 9C (E). Plotted are means with standard errors. *P < 0.05 versus 15C (B), 7C (C and D), or 9C (E). #P < 0.05 versus +SU16F/JNJ.

To identify indispensable factors in 15C, we removed compounds one by one and treated cells with the remaining compounds. The number of beating clusters was significantly reduced by removal of CHIR99021, A83-01, BIX01294, AS8351, SC1, Y27632, or OAC2 (Fig. 1B). Together, these seven compounds (7C) were sufficient and necessary to efficiently induce cardiac reprogramming (Fig. 1C). We then screened a larger in-house–generated library containing ~300 modulators of signaling pathways, including known inhibitors of kinases, phosphatases, and other signaling receptors, with 7C as the baseline. Two inhibitors of the platelet-derived growth factor pathway, SU16F and JNJ10198409 (JNJ), accelerated the down-regulation of fibroblast genes (fig. S3) and increased the yield of beating clusters (Fig. 1D). These two inhibitors were added to 7C (9C) for subsequent assays after optimization of dosage and treatment duration (table S2 and fig. S4). At day 30, 6.6 ± 0.4% of the 9C-treated cells expressed the CM marker cardiac troponin T (cTNT), with a yield of up to 1.2 cTNT+ CMs per input HFF. Removal of any of the nine compounds markedly reduced the induction efficiency (Fig. 1E and fig. S2B). 9C reprogrammed human fetal lung fibroblasts (HLFs) into ciCMs with comparable efficiency (fig. S5).

Cardiogenesis involves sequential induction of mesoderm, cardiac progenitor cells (CPCs), and CMs (15). This developmental sequence, which is apparent in the differentiation of human pluripotent stem cells (hPSCs) (1618), was observed during 9C-induced conversion of HFFs into ciCMs. After exposure to CIM, up to 27.9 ± 4.0% of the 9C-treated cells started to express a key mesoderm marker, KDR (fig. S6). These cells initiated a cardiogenic program by sequentially expressing mesoderm, CPC, and CM genes (figs. S7 and S8) and ultimately became beating ciCMs (fig. S6). Expression of CPC genes, particularly markers of second but not first heart field progenitors, was further confirmed by qRT-PCR and immunofluorescence analysis in 9C-treated cells (figs. S8A and S9). Small contracting clusters of ciCMs began to appear around day 20, continued to beat in culture (movies S1 and S2), and expressed specific CM markers (figs. S5 and S8B). Thus, they closely resembled CMs derived from hPSCs (hPSC-CMs) (fig. S1G).

Although new reprogramming protocols are improving cell yield, cardiac cells generated by genetic methods are heterogeneous, with only about 0.1% of genetically reprogrammed human iCMs achieving a high degree of reprogramming, as characterized by beating spontaneously, displaying cardiac action potentials, and expressing multiple cardiac genes uniformly in vitro (10). In contrast, we found that more than 97% of the ciCMs spontaneously beat and uniformly expressed multiple cardiac structural proteins, similar to hPSC-CMs (fig. S10). The homogeneity of ciCMs was further confirmed by single-cell qRT-PCR analyses, which revealed little, if any, difference in the expression levels of cardiac genes among individual ciCMs (fig. S11). Collectively, these results confirm that ciCMs are highly reprogrammed and largely homogeneous.

Next, we further characterized ciCMs that were reprogrammed from HFFs (HFF-CMs) and HLFs (HLF-CMs). Immunofluorescence and transmission electron microscopy revealed that ciCMs exhibited well-organized sarcomeres, closely resembling those of hPSC-CMs (Fig. 2A and fig. S12). ciCMs also highly expressed genes involved in CM function, including those that encode atrial natriuretic factor, connexin 43, Cav3.2, HCN4, and Kir2.1 (Fig. 2A and fig. S13). Intracellular electrical recordings from ciCMs at reprogramming days 45 to 50 revealed robust action potentials that were synchronized (1:1) with rhythmic Ca2+ transients (Fig. 2B and fig. S14, A and B), similar to hPSC-CMs (19); this suggests that ciCMs, in addition to being highly reprogrammed, are comparable in maturity to hPSC-CMs. Most ciCMs exhibited ventricular-like action potentials and expressed ventricular but not atrial CM markers (Fig. 2B and fig. S14). Moreover, ciCMs responded to caffeine (a ryanodine receptor agonist), isoproterenol (a β-adrenergic agonist), and carbachol (a muscarinic agonist) (fig. S15). Thus, ciCMs are functional in vitro and possess electrophysiological features that are similar to those of hPSC-CMs.

Fig. 2 Structural and functional characterization of ciCMs.

(A) Immunofluorescence analyses of CM markers. Insets show boxed areas at higher magnification. Scale bars, 25 μm. (B) Representative traces of ventricular-like action potentials and Ca2+ transients. Em, membrane potential. Dashed lines indicate 0 mV. F/F0, fluorescence relative to the baseline.

We next compared the transcriptomes of ciCMs, their parental fibroblasts, hPSC-CMs, purified human fetal CMs (1), and primary human heart tissue (20) by microarray analyses. ciCMs, hPSC-CMs, human fetal CMs, and primary heart tissue displayed transcriptional profiles that differed from those of the fibroblasts (Fig. 3 and fig. S16, A and B). The genes that were most significantly up-regulated in ciCMs were related to CM function and heart development; genes for fibroblast function, such as cell proliferation and motility, were down-regulated in ciCMs (fig. S16, C and D, and fig. S17).

Fig. 3 ciCMs acquire transcriptional signatures of normal CMs.

(A) Hierarchical clustering analysis of genes that were differently expressed in fibroblasts and fetal CMs, across all tested cell types. GO, gene ontology. On the horizontal axis, 1 and 2 indicate duplicate samples. (B) Cell and tissue classification heatmap of all tested cell types, generated by CellNet analyses. Higher classification scores indicate a higher probability that a query sample (vertical axis) resembles the training sample (horizontal axis).

We examined the expression of several maturation-related genes that are distinctly expressed during cardiogenesis (20). ciCMs closely resembled hPSC-CMs in a hierarchical clustering analysis (fig. S18A), and both CM types had a similar expression pattern of α-smooth muscle actin and ventricular myosin light chain 2v, two maturation-related markers (21) (fig. S14, C and D, and fig. S18B), indicating that ciCMs acquire maturity similar to that of hPSC-CMs. To determine whether the ciCMs had an established CM-like chromatin state, we analyzed histone and DNA methylation status in the promoter regions of several fibroblast and cardiac genes and found that ciCMs gained key epigenetic features similar to those of hPSC-CMs (fig. S19). Furthermore, ciCMs were directly reprogrammed without going through a PSC-like state (fig. S20) and maintained genomic stability relative to their parental fibroblasts (fig. S21).

Next, we investigated whether the diseased-heart niche would support the generation of ciCMs. HFFs harboring the αMHC-GFP reporter were treated with 9C for 6 days and then with CIM for 5 days and transplanted into the infarcted hearts of immunodeficient mice. HFFs not treated with 9C served as the negative control. Two weeks after transplantation, 9C-treated HFFs (identified by human-specific lamin A/C staining) robustly expressed CM markers, exhibited well-organized sarcomeres, and partially remuscularized the infarcted area (Fig. 4). These results suggest that 9C-treated cells are compatible with the diseased-heart environment and can further mature into CMs in vivo under these conditions.

Fig. 4 9C-treated fibroblasts convert into CMs in vivo.

Immunofluorescence analyses of heart sections after transplantation of control (left) or 9C-treated (right) HFFs. Insets show boxed areas at higher magnification. Scale bars, 100 μm.

We hypothesized that 9C promotes an epigenetic state characterized by open chromatin, which renders cells responsive to extrinsic cardiogenic signals. We observed a decrease in the number of heterochromatin foci (densely stained for H3K9me3 and HP1γ) in 9C-treated HFFs (Fig. 5A and fig. S22). Thus, 9C treatment appears to decondense closed chromatin regions in HFFs, possibly creating a more euchromatic structure at loci that are important for cardiogenesis. To assess this mechanism further, we analyzed the genome-wide epigenetic changes by chromatin immunoprecipitation–sequencing (ChIP-seq) analysis of H3K4me3 and H3K27ac (active chromatin marks) and H3K27me3 (inactive chromatin mark) at reprogramming days 6 and 11. We observed a dynamic loss of most of the H3K27me3 and specific gain of H3K4me3 among a subset of genomic loci during reprogramming (Fig. 5B). These genes were frequently related to developmental processes and cell differentiation (fig. S23). More specifically, we observed a significantly increased enrichment of H3K4me3 and H3K27ac on a cohort of heart developmental genes during reprogramming, whereas the deposition of H3K27me3 was down-regulated (Fig. 5C and fig. S24). Consistently, 9C enabled the binding of β-catenin and Smad1 (effectors of the major cardiogenic signals Wnt and BMP, respectively) to core promoters and enhancers of key heart development genes (Fig. 5, D and E). Thus, 9C appeared to convert the passive chromatin state of fibroblasts into a more euchromatic state, increasing chromatin accessibility on core cardiogenesis gene loci and thereby facilitating cardiac reprogramming (Fig. 5F).

Fig. 5 9C-treated fibroblasts have a globally more open chromatin structure and higher chromosome accessibility on core cardiogenesis genes.

(A) Heterochromatin foci shown by immunostaining for H3K9me3. Insets show a single cell nucleus at higher magnification. Scale bars, 100 μm. On the right, the graph shows the number of heterochromatin foci per nuclei. (B) Tracing of gene status at days 6 and 11, relative to HFFs. Percentages of genes that showed dynamic changes are indicated in color-coded text. The number of genes is indicated for each bar. (C) Levels of H3K4me3, H3K27ac, and H3K27me3 enrichment in genes in the GO “heart development” category. Black and blue bands inside the boxes represent median and mean values, respectively. Boxes represent the interquartile ranges, and whiskers represent the minimum and maximum values. RPKM, reads per kilobase of peak per million reads. (D and E) ChIP analyses of β-catenin (D) or Smad1 (E) binding on promoters and enhancers of cardiogenic genes (n = 3). IgG, immunoglobulin G. (F) A model for 9C-induced cardiac reprogramming. Red, blue, and green rectangles on the genes are transcription factor (TF) binding sites. *P < 0.05; n.s, not significant (P > 0.05).

We next investigated the role of the reprogramming compound AS8351. AS8351 and its functional analogs affect epigenetic modifications (22, 23) by competing with α-ketoglutarate (α-KG) for chelating iron [Fe(II)] in certain epigenetic enzymes, such as the JmjC domain–containing histone demethylases (JmjC-KDMs) that require α-KG and iron as co-factors (24). We hypothesized that AS8351’s effects on cardiac reprogramming might in part be mediated through modulation of a specific JmjC-KDM. To test this hypothesis, we abrogated each of the 22 genes in the JmjC-KDM family by small hairpin RNAs and found that only knocking down KDM5B or using a KDM5B inhibitor (PBIT) could phenocopy AS8351 in generating ciCMs (fig. S25), suggesting that KDM5B might be a target of AS8351. KDM5B catalyzes the demethylation of tri-, di-, and monomethylation states of H3K4 and facilitates heterochromatin formation (24). Consistent with the overall effect of 9C on reopening the closed chromatin structure, inhibition of KDM5B may facilitate this process and sustain the active chromatin marks (i.e., H3K4 methylation) at specific genomic loci.

Our study shows that reprogrammed and functional lineage-specific cells can be generated from human fibroblasts by defined small molecules and growth factors. This study not only achieved considerably higher-quality human iCMs than those previously reported but also provides a chemical approach, free of foreign genetic material, that may be adapted to generate multiple cell types. ciCMs are functionally comparable to PSC-CMs, although generating fully mature CMs remains a critical challenge for cardiac regenerative therapies. An important feature of ciCM induction is that, in response to signals mimicking the paracrine environment in the in vivo heart, 9C-treated cells are induced to become CMs. This finding may ultimately provide a foundation for in situ repair of the heart by targeting endogenous cardiac fibroblasts with small molecules. However, many challenges (e.g., reprogramming efficiency and tissue-specific delivery of multiple drugs in an efficient and controllable manner) need to be resolved before this strategy can be considered for in vivo therapeutic applications. Additional studies are needed to determine whether unintended genomic changes occur in ciCM subpopulations and to investigate how to increase the maturity of ciCMs. Furthermore, a better understanding is needed of the underlying mechanisms for this reprogramming.

Supplementary Materials

www.sciencemag.org/content/352/6290/1216/suppl/DC1

Materials and Methods

Figs. S1 to S25

Tables S1 to S6

References (25, 26)

Movies S1 and S2

References and Notes

Acknowledgments: N.C. is supported by the California Institute for Regenerative Medicine (CIRM). J.-D.F. is supported by the American Heart Association. S.D. and D.S. are supported by the CIRM, the National Heart, Lung, and Blood Institute, the National Institutes of Health, and the Roddenberry Foundation. D.S. is supported by the Younger Family Foundation and the Whittier Foundation. Data associated with this manuscript are available in the Gene Expression Omnibus (accession numbers GSE55820 and GSE78096). N.C. and S.D. are inventors on patent applications filed by the Gladstone Institute of Cardiovascular Disease that are related to the chemical reprogramming method reported in this paper.
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