REPORTS

Cite as: N. Cao et al., Science 10.1126/science.aaf1502 (2016).

Advances in reprogramming enable the switch of one cell fate to another with potential use in regenerative therapy. Cardiomyocyte (CM)-like cells can be reprogrammed from somatic fibroblasts by overexpression of cardiac genes in vitro (1–6) and in vivo (5, 7–10). However, efficiently trans-differentiating human non-cardiac cells into highly func-tional CMs remains a significant challenge (1, 4, 6). In contrast to conventional reprogramming by genetic meth-ods, a chemical reprogramming approach introduces small molecules that interact with and modulate endogenous fac-tors in the starting cell type (e.g., fibroblast) in the absence of target cell type–specific proteins. Small molecules have certain advantages: they are convenient to use, can be effi-ciently delivered into cells, provide greater temporal control, are nonimmunogenic and more cost-effective. Moreover, their effects can be fine-tuned by varying their concentra-tions and combinations. Here, we report identification of a combination of small molecules that enable 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 quantita-tive RT-PCR (qPCR), flow-cytometry, and immunofluores-

cence analyses (fig. S1). HFFs were virally transduced with an alpha myosin heavy chain (αMHC)-GFP reporter (11) to specifically label CMs. Based on the cell-activation and sig-naling-directed/CASD reprogramming paradigm (12, 13), we treated cells with compounds that induce or enhance cellu-lar 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 known to facilitate reprogramming (ta-ble 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 induc-tion medium (CIM) containing cardiogenic molecules in-cluding activin A, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor, and CHIR99021 (fig. S2A). Positive hits that enhanced cardiogenic gene expres-sion, as assayed by qPCR, after CIM treatment were identi-fied, and further tested in different combinations. Cells were then replated in human CM–conditioned medium, which may provide cardiac paracrine signals that mimic the in vivo environment for further maturation (14). After iterative

Conversion of human fibroblasts into functional cardiomyocytes by small molecules Nan Cao,1,2 Yu Huang,1 Jiashun Zheng,4,5 C. Ian Spencer,1 Yu Zhang,1,2 Ji-Dong Fu,6 Baoming Nie,1,2 Min Xie,1,2 Mingliang Zhang,1,2 Haixia Wang,1,2 Tianhua Ma,1,2 Tao Xu,1,2 Guilai Shi,1,2 Deepak Srivastava,1,3,4* Sheng Ding1,2*† 1Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA. 2Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA. 3Department of Pediatrics, University of California, San Francisco, San Francisco, CA 94158, USA. 4Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA. 5California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, CA 94158, USA. 6Department of Medicine, Heart and Vascular Research Center, Case Western Reserve University, Cleveland, OH 44106, USA.

*These authors contributed equally to this work.

†Corresponding author. Email: sheng.ding@gladstone.ucsf.edu

Reprogramming somatic fibroblasts into alternative lineages would provide a promising source of cells for regenerative therapy. However, transdifferentiating human cells to 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 (9C). The chemically induced cardiomyocyte-like cells (ciCMs) 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/enhancers to bind effectors of major cardiogenic signals. When transplanted into infarcted mouse hearts, 9C-treated fibroblasts were efficiently converted to ciCMs. This pharmacological approach for lineage-specific reprogramming may have many important therapeutic implications after further optimization to generate mature cardiac cells.

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rounds of testing, we found that a combination of total 15 compounds (15C) generated about 5 αMHC-GFP-positive clusters that beat spontaneously from 3x105 replated cells at day 30 (Fig. 1A), indicating reprogramming toward the car-diac fate.

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 significant-ly reduced by removal of CHIR99021, A83-01, BIX01294, AS8351, SC1, Y27632, or OAC2 (Fig. 1B). Together, these sev-en compounds (7C) were sufficient and necessary to effi-ciently 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 re-ceptors with 7C as the baseline. Two inhibitors of the plate-let-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). The 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 induc-tion efficiency (Fig. 1E and fig. S2B). Similarly, 9C repro-grammed human fetal lung fibroblasts (HLFs) into ciCMs with comparable efficiency (fig. S5).

Cardiogenesis involves sequential induction of meso-derm, cardiac progenitor cells (CPCs), and CMs (15). This developmental sequence, which was apparent in differentia-tion of human pluripotent stem cells (hPSCs) (16–18), 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 sequen-tially expressing mesoderm, CPC, and CM genes (figs. S7 and S8), and finally became beating ciCMs (fig. S6). Expression of CPC genes, particularly markers of second but not first heart field progenitors, was further confirmed by qPCR and immunofluorescence analysis in 9C-treated cells (figs. S8A and S9). Small contracting clusters of ciCMs began to ap-pear 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 repro-grammed human iCMs achieving a high degree of repro-gramming as characterized to beat spontaneously, display cardiac action potentials (APs), and express multiple cardiac

genes uniformly in vitro (10). In contrast, we found that more than 97% of the ciCMs spontaneously beat and uni-formly expressed multiple cardiac structural proteins, simi-lar to hPSC-CMs (fig. S10). The homogeneity of ciCMs was further confirmed by single-cell qPCR analyses, revealing little, if any, difference in the expression levels of cardiac genes between individual ciCMs (fig. S11). Collectively, these results confirm that ciCMs are highly reprogrammed and largely homogeneous.

Next, we further characterized ciCMs reprogrammed from HFFs (HFF-CMs) and HLFs (HLF-CMs). Immunofluo-rescence and transmission electron microscopy revealed that ciCMs exhibited well-organized sarcomere organization closely resembling hPSC-CMs (Fig. 2A and fig. S12). ciCMs also highly expressed genes involved in CM function, includ-ing atrial natriuretic factor, connexin 43, Cav3.2, HCN4, and Kir2.1 (Fig. 2A and fig. S13). Intracellular electrical record-ings from ciCMs at reprogramming day 45–50 revealed ro-bust APs that were synchronized 1:1 with rhythmic Ca2+ transients (Fig. 2B and fig. S14, A and B), similar to hPSC-CMs (19), suggesting ciCMs are highly reprogrammed and have a comparable maturity to hPSC-CMs. Most ciCMs ex-hibited ventricular-like APs 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 similar to hPSC-CMs.

We next compared the transcriptomes of ciCMs, their parental fibroblasts, hPSC-CMs, purified human fetal CMs (1), and primary human hearts (20) by microarray analyses. ciCMs, hPSC-CMs, and primary CM/hearts displayed tran-scriptional profiles that clearly differed from those of the fibroblasts (Fig. 3 and fig. S16, A and B). Genes most signifi-cantly 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).

We examined the expression of several maturation-related genes that are distinctly expressed during cardi-ogenesis (20). ciCMs closely resembled hPSC-CMs in a hier-archical 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), indi-cating ciCMs acquire maturity similar to hPSC-CMs. To de-termine whether ciCMs had an established CM-like chromatin state, we analyzed histone and DNA methylation status in the promoter regions of several fibroblast and car-diac genes and found that ciCMs gained key epigenetic fea-tures similar to hPSC-CMs (fig. S19). Furthermore, ciCMs

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were directly reprogrammed without going through a PSC-like state (fig. S20) and maintain genomic stability when compared 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 infarct-ed hearts of immunodeficient mice. HFFs not treated with 9C served as the negative control. Two weeks after trans-plantation, 9C-treated HFFs (identified by human-specific lamin A/C staining) robustly expressed CM markers, exhib-ited well-organized sarcomeres, and partially re-muscularized the infarcted area (Fig. 4). These results sug-gest that 9C-treated cells are compatible with the diseased-heart environment to further mature into CMs in vivo.

We hypothesized that 9C promotes an epigenetic state characterized by open chromatin, which renders cells re-sponsive to extrinsic cardiogenic signals. Indeed, we ob-served 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 de-condense closed chromatin regions in HFFs, possibly creating a more euchromatic structure at loci important for cardiogenesis. To assess this mechanism further, we ana-lyzed 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 D6 and D11. We observed a dynamic loss of most H3K27me3 and specific gain of H3K4me3 among a subset of genomic loci during reprogramming (Fig. 5B). These genes were fre-quently related to developmental processes and cell differ-entiation (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 downregulated (Fig. 5C and fig. S24). Consistently, 9C ena-bled the binding of β-catenin and Smad1 (effectors of major cardiogenic signal Wnt and BMP, respectively) to core pro-moter/enhancers of key genes important for heart develop-ment (Fig. 5, D and E). Thus, 9C appeared to convert the passive chromatin state of fibroblasts into a more euchro-matic state, gaining chromatin accessibility on core cardi-ogenesis gene loci, and thereby facilitating cardiac reprogramming (Fig. 5F).

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 epi-genetic enzymes, such as the JmjC-domain-containing his-tone 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 via mod-ulation 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 knockdown of KDM5B, or using a KDM5B inhibitor, PBIT could “pheno-copy” AS8351 in generating ciCMs (fig. S25), suggesting that it might be a target of AS8351. KDM5B catalyzes the de-methylation of tri-, di-, and mono-methylation states of H3K4 and facilitates heterochromatin formation (24). Con-sistent with the overall effect of 9C on re-opening 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.

The present study shows that reprogrammed and func-tional lineage-specific cells can be generated from human fibroblasts by defined small molecules/growth factors. This study not only achieves significantly higher quality human iCMs than previously reported, but also provides a chemical approach free of foreign genetic material that may be adapted toward generating multiple cell types. ciCMs are functionally comparable to PSC-CMs, albeit generating fully-mature CMs remains a critical challenge for cardiac regen-erative therapies. An important feature of ciCM induction is that, in response to signals mimicking the paracrine envi-ronment in the in vivo heart, 9C-treated cells are induced to become CMs. This finding may lay a foundation for ultimate in situ repair of the heart by targeting endogenous cardiac fibroblasts with small molecules. However, many critical challenges (e.g., reprogramming efficiency and tissue-specific delivery of multiple drugs in an efficient and con-trollable manner) need to be resolved before this strategy can be considered for in vivo therapeutic applications. Addi-tional studies are needed to determine whether unintended genomic changes occur in ciCM subpopulations, as well as increase the maturity of ciCMs. Furthermore, we need a bet-ter understanding of the underlying mechanisms for this reprogramming.

REFERENCES AND NOTES 1. J. D. Fu, N. R. Stone, L. Liu, C. I. Spencer, L. Qian, Y. Hayashi, P. Delgado-Olguin, S.

Ding, B. G. Bruneau, D. Srivastava, Direct reprogramming of human fibroblasts toward a cardiomyocyte-like state. Stem Cell Rev. 1, 235–247 (2013). Medline

2. M. Ieda, J. D. Fu, P. Delgado-Olguin, V. Vedantham, Y. Hayashi, B. G. Bruneau, D. Srivastava, Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell 142, 375–386 (2010). Medline doi:10.1016/j.cell.2010.07.002

3. T. M. Jayawardena, B. Egemnazarov, E. A. Finch, L. Zhang, J. A. Payne, K. Pandya, Z. Zhang, P. Rosenberg, M. Mirotsou, V. J. Dzau, MicroRNA-mediated in vitro and in vivo direct reprogramming of cardiac fibroblasts to cardiomyocytes. Circ. Res. 110, 1465–1473 (2012). Medline doi:10.1161/CIRCRESAHA.112.269035

4. Y. J. Nam, K. Song, X. Luo, E. Daniel, K. Lambeth, K. West, J. A. Hill, J. M. DiMaio, L. A. Baker, R. Bassel-Duby, E. N. Olson, Reprogramming of human fibroblasts toward a cardiac fate. Proc. Natl. Acad. Sci. U.S.A. 110, 5588–5593 (2013). Medline doi:10.1073/pnas.1301019110

5. K. Song, Y. J. Nam, X. Luo, X. Qi, W. Tan, G. N. Huang, A. Acharya, C. L. Smith, M. D. Tallquist, E. G. Neilson, J. A. Hill, R. Bassel-Duby, E. N. Olson, Heart repair by

First release: 28 April 2016 www.sciencemag.org (Page numbers not final at time of first release) 3

on June 18, 2020

http://science.sciencemag.org/

Dow

nloaded from

reprogramming non-myocytes with cardiac transcription factors. Nature 485, 599–604 (2012). Medline doi:10.1038/nature11139

6. R. Wada, N. Muraoka, K. Inagawa, H. Yamakawa, K. Miyamoto, T. Sadahiro, T. Umei, R. Kaneda, T. Suzuki, K. Kamiya, S. Tohyama, S. Yuasa, K. Kokaji, R. Aeba, R. Yozu, H. Yamagishi, T. Kitamura, K. Fukuda, M. Ieda, Induction of human cardiomyocyte-like cells from fibroblasts by defined factors. Proc. Natl. Acad. Sci. U.S.A. 110, 12667–12672 (2013). Medline doi:10.1073/pnas.1304053110

7. L. Qian, Y. Huang, C. I. Spencer, A. Foley, V. Vedantham, L. Liu, S. J. Conway, J. D. Fu, D. Srivastava, In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature 485, 593–598 (2012). Medline doi:10.1038/nature11044

8. K. Inagawa, K. Miyamoto, H. Yamakawa, N. Muraoka, T. Sadahiro, T. Umei, R. Wada, Y. Katsumata, R. Kaneda, K. Nakade, C. Kurihara, Y. Obata, K. Miyake, K. Fukuda, M. Ieda, Induction of cardiomyocyte-like cells in infarct hearts by gene transfer of Gata4, Mef2c, and Tbx5. Circ. Res. 111, 1147–1156 (2012). Medline doi:10.1161/CIRCRESAHA.112.271148

9. T. M. Jayawardena, E. A. Finch, L. Zhang, H. Zhang, C. P. Hodgkinson, R. E. Pratt, P. B. Rosenberg, M. Mirotsou, V. J. Dzau, MicroRNA induced cardiac reprogramming in vivo: Evidence for mature cardiac myocytes and improved cardiac function. Circ. Res. 116, 418–424 (2015). Medline doi:10.1161/CIRCRESAHA.116.304510

10. D. Srivastava, P. Yu, Recent advances in direct cardiac reprogramming. Curr. Opin. Genet. Dev. 34, 77–81 (2015). Medline doi:10.1016/j.gde.2015.09.004

11. H. Kita-Matsuo, M. Barcova, N. Prigozhina, N. Salomonis, K. Wei, J. G. Jacot, B. Nelson, S. Spiering, R. Haverslag, C. Kim, M. Talantova, R. Bajpai, D. Calzolari, A. Terskikh, A. D. McCulloch, J. H. Price, B. R. Conklin, H. S. Chen, M. Mercola, Lentiviral vectors and protocols for creation of stable hESC lines for fluorescent tracking and drug resistance selection of cardiomyocytes. PLOS ONE 4, e5046 (2009). Medline doi:10.1371/journal.pone.0005046

12. J. A. Efe, S. Hilcove, J. Kim, H. Zhou, K. Ouyang, G. Wang, J. Chen, S. Ding, Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. Nat. Cell Biol. 13, 215–222 (2011). Medline doi:10.1038/ncb2164

13. H. Wang, N. Cao, C. I. Spencer, B. Nie, T. Ma, T. Xu, Y. Zhang, X. Wang, D. Srivastava, S. Ding, Small molecules enable cardiac reprogramming of mouse fibroblasts with a single factor, Oct4. Cell Reports 6, 951–960 (2014). Medline doi:10.1016/j.celrep.2014.01.038

14. G. Blin, D. Nury, S. Stefanovic, T. Neri, O. Guillevic, B. Brinon, V. Bellamy, C. Rücker-Martin, P. Barbry, A. Bel, P. Bruneval, C. Cowan, J. Pouly, S. Mitalipov, E. Gouadon, P. Binder, A. Hagège, M. Desnos, J. F. Renaud, P. Menasché, M. Pucéat, A purified population of multipotent cardiovascular progenitors derived from primate pluripotent stem cells engrafts in postmyocardial infarcted nonhuman primates. J. Clin. Invest. 120, 1125–1139 (2010). Medline doi:10.1172/JCI40120

15. P. W. Burridge, G. Keller, J. D. Gold, J. C. Wu, Production of de novo cardiomyocytes: Human pluripotent stem cell differentiation and direct reprogramming. Cell Stem Cell 10, 16–28 (2012). Medline doi:10.1016/j.stem.2011.12.013

16. M. J. Birket, M. C. Ribeiro, A. O. Verkerk, D. Ward, A. R. Leitoguinho, S. C. den Hartogh, V. V. Orlova, H. D. Devalla, V. Schwach, M. Bellin, R. Passier, C. L. Mummery, Expansion and patterning of cardiovascular progenitors derived from human pluripotent stem cells. Nat. Biotechnol. 33, 970–979 (2015). Medline doi:10.1038/nbt.3271

17. L. Bu, X. Jiang, S. Martin-Puig, L. Caron, S. Zhu, Y. Shao, D. J. Roberts, P. L. Huang, I. J. Domian, K. R. Chien, Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages. Nature 460, 113–117 (2009). Medline doi:10.1038/nature08191

18. S. J. Kattman, A. D. Witty, M. Gagliardi, N. C. Dubois, M. Niapour, A. Hotta, J. Ellis, G. Keller, Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 8, 228–240 (2011). Medline doi:10.1016/j.stem.2010.12.008

19. J. Zhang, G. F. Wilson, A. G. Soerens, C. H. Koonce, J. Yu, S. P. Palecek, J. A. Thomson, T. J. Kamp, Functional cardiomyocytes derived from human induced pluripotent stem cells. Circ. Res. 104, e30–e41 (2009). Medline doi:10.1161/CIRCRESAHA.108.192237

20. J. Synnergren, C. Améen, A. Jansson, P. Sartipy, Global transcriptional profiling reveals similarities and differences between human stem cell-derived cardiomyocyte clusters and heart tissue. Physiol. Genomics 44, 245–258 (2012). Medline doi:10.1152/physiolgenomics.00118.2011

21. J. Zhang, M. Klos, G. F. Wilson, A. M. Herman, X. Lian, K. K. Raval, M. R. Barron, L. Hou, A. G. Soerens, J. Yu, S. P. Palecek, G. E. Lyons, J. A. Thomson, T. J. Herron, J. Jalife, T. J. Kamp, Extracellular matrix promotes highly efficient cardiac differentiation of human pluripotent stem cells: The matrix sandwich method. Circ. Res. 111, 1125–1136 (2012). Medline doi:10.1161/CIRCRESAHA.112.273144

22. G. K. Azad, V. Singh, U. Golla, R. S. Tomar, Depletion of cellular iron by curcumin leads to alteration in histone acetylation and degradation of Sml1p in Saccharomyces cerevisiae. PLOS ONE 8, e59003 (2013). Medline doi:10.1371/journal.pone.0059003

23. I. P. Pogribny, V. P. Tryndyak, M. Pogribna, S. Shpyleva, G. Surratt, G. Gamboa da Costa, F. A. Beland, Modulation of intracellular iron metabolism by iron chelation affects chromatin remodeling proteins and corresponding epigenetic modifications in breast cancer cells and increases their sensitivity to chemotherapeutic agents. Int. J. Oncol. 42, 1822–1832 (2013). Medline

24. N. Mosammaparast, Y. Shi, Reversal of histone methylation: Biochemical and molecular mechanisms of histone demethylases. Annu. Rev. Biochem. 79, 155–179 (2010). Medline doi:10.1146/annurev.biochem.78.070907.103946

25. X. Lian, C. Hsiao, G. Wilson, K. Zhu, L. B. Hazeltine, S. M. Azarin, K. K. Raval, J. Zhang, T. J. Kamp, S. P. Palecek, Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc. Natl. Acad. Sci. U.S.A. 109, E1848–E1857 (2012). Medline doi:10.1073/pnas.1200250109

26. H. D. Devalla, V. Schwach, J. W. Ford, J. T. Milnes, S. El-Haou, C. Jackson, K. Gkatzis, D. A. Elliott, S. M. Chuva de Sousa Lopes, C. L. Mummery, A. O. Verkerk, R. Passier, Atrial-like cardiomyocytes from human pluripotent stem cells are a robust preclinical model for assessing atrial-selective pharmacology. EMBO Mol. Med. 7, 394–410 (2015). Medline doi:10.15252/emmm.201404757

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 Gene Expression Omnibus (GSE55820 and GSE78096). N.C. and S.D. are inventors on patent applications filed by the Gladstone Institute of Cardiovascular Disease related to the chemical reprogramming method reported in this paper.

SUPPLEMENTARY MATERIALS www.sciencemag.org/cgi/content/full/science.aaf1502/DC1 Materials and Methods Figs. S1 to S25 Tables S1 to S6 References (25, 26) Movies S1 and S2 23 December 2015; accepted 15 April 2016 Published online 28 April 2016 10.1126/science.aaf1502

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Fig. 1. Chemically induced CMs from HFFs. (A) A representative contracting cluster induced by 15C at reprogramming day 30. Scale bars, 100 μm. (B to E) Number of contracting clusters (B to D) and flow-cytometry analyses of cTNT (E) in indicated conditions (n = 3). *P < 0.05 vs. 15C (B), 7C (C and D), and 9C (E). #P < 0.05 vs. +SU16F/JNJ.

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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 APs and Ca2+ transients. Em, membrane potential. Dotted lines indicate 0 mV. F/F0, fluorescence relative to the baseline.

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Fig. 3. ciCMs acquire transcriptional signatures of normal CMs. (A) Hierarchical clustering analysis of genes that differently expressed in fibroblasts and fetal CMs, across all tested cell types. GO, gene ontology. (B) Cell/tissue classification heatmap of all tested cell types revealed by CellNet analyses. Higher classification scores indicate a higher probability that a query sample resembles the training sample.

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Fig. 4. 9C-treated fibroblasts convert into CMs in vivo. Immunofluorescence analyses of heart sections after transplantation of control or 9C-treated HFFs. Insets show boxed areas at higher magnification. Scale bars, 100 μm.

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Fig. 5. 9C-treated fibroblasts have globally more open chromatin structure and increased 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. (B) Tracing of gene status in D6 and D11, relative to HFFs. Percentage within each category represents the ratio of genes show dynamic changes. Digits represent number of genes in each bar. (C) Levels of H3K4me3, H3K27ac, and H3K27me3 enrichment in genes in the GO term of “heart development.” Black/blue band inside the box represents the median/mean value, respectively. (D and E) ChIP analyses of β-catenin (D) or Smad1 (E) binding on promoters/enhancers of cardiogenic genes (n = 3). (F) A model for 9C-induced cardiac reprogramming. *P < 0.05; n.s, P > 0.05.

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Conversion of human fibroblasts into functional cardiomyocytes by small molecules

Tianhua Ma, Tao Xu, Guilai Shi, Deepak Srivastava and Sheng DingNan Cao, Yu Huang, Jiashun Zheng, C. Ian Spencer, Yu Zhang, Ji-Dong Fu, Baoming Nie, Min Xie, Mingliang Zhang, Haixia Wang,

published online April 28, 2016

ARTICLE TOOLS http://science.sciencemag.org/content/early/2016/04/27/science.aaf1502

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REFERENCES

http://science.sciencemag.org/content/early/2016/04/27/science.aaf1502#BIBLThis article cites 26 articles, 9 of which you can access for free

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