The direct conversion, or transdifferentiation, of non-cardiac cells into cardiomyocytes by forced expression of transcription factors and microRNAs provides promising approaches for cardiac regeneration. However, genetic manipulations raise safety concerns and are thus not desirable in most clinical applications. The discovery of full chemically induced pluripotent stem cells suggest the possibility of replacing transcription factors with chemical cocktails. Here, we report the generation of automatically beating cardiomyocyte-like cells from mouse fibroblasts using only chemical cocktails. These chemical-induced cardiomyocyte-like cells (CiCMs) express cardiomyocyte-specific markers, exhibit sarcomeric organization, and possess typical cardiac calcium flux and electrophysiological features. Genetic lineage tracing confirms the fibroblast origin of these CiCMs. Further studies show the generation of CiCMs passes through a cardiac progenitor stage instead of a pluripotent stage. Bypassing the use of viral-derived factors, this proof of concept study lays a foundation for in vivo cardiac transdifferentiation with pharmacological agents and possibly safer treatment of heart failure.

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Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have great potential in applications such as regenerative medicine, cardiac disease modeling, and in vitro drug evaluation. However, hPSC-CMs are immature, which limits their applications. During development, the maturation of CMs is accompanied by a decline in their proliferative capacity. This phenomenon suggests that regulating the cell cycle may facilitate the maturation of hPSC-CMs. Aurora kinases are essential kinases that regulate the cell cycle, the role of which is not well studied in hPSC-CM maturation. Here, we demonstrate that CYC116, an inhibitor of Aurora kinases, significantly promotes the maturation of CMs derived from both human embryonic stem cells (H1 and H9) and iPSCs (induced PSCs) (UC013), resulting in increased expression of genes related to cardiomyocyte function, better organization of the sarcomere, increased sarcomere length, increased number of mitochondria, and enhanced physiological function of the cells. In addition, a number of other Aurora kinase inhibitors have also been found to promote the maturation of hPSC-CMs. Our data suggest that blocking aurora kinase activity and regulating cell cycle progression may promote the maturation of hPSC-CMs.

The revolutionizing somatic cell reprogramming/transdifferentiation technologies provide a new path for cell replacement therapies and drug screening. The original method for reprogramming involves the delivery of exogenous transcription factors, leading to the safety concerns about the possible genome integration. Many efforts have been taken to avoid genetic alteration in somatic cell reprogramming or transdifferentiation by using non-integrating gene delivery approaches, cell membrane permeable proteins, and small molecule compounds.Our group is focused on identifying small molecules that can facilitate somatic cell reprogramming or transdifferentiation. Our previous studies have identified a number of small molecules (or condition), including LiCl, sunitinib, hyperosmosis, and etc., that can promote transcription factor-mediated reprogramming. Recently, we identified that the commonly used biological reagent, bromodeoxyuridine (BrdU), is able to enhance Yamanaka factor-mediated reprogramming. More interestingly, BrdU can replace Oct4, the most critical factor in iPSC generation. Further studies demonstrate that BrdU enables full-chemical induction of mouse iPSCs (CiPSCs) with several chemical cocktails, and the minimal combination being BrdU, CHIR99021, Repsox, and Forskolin. The CiPSCs resemble embryonic stem cells in terms of their gene expression, epigenetic status, in vivo differentiation and chimera generation. We also discovered the generation of automatically beating cardiomyocyte-like cells from mouse fibroblasts using only chemical cocktails. These chemical-induced cardiomyocyte-like cells (CiCMs) express cardiomyocyte-specific markers, exhibit sarcomeric organization, and possess typical cardiac calcium flux and electrophysiological features. Genetic lineage tracing confirms the fibroblast origin of these CiCMs. Further studies show the generation of CiCMs passes through a cardiac progenitor stage instead of a pluripotent stage. Bypassing the use of viral-derived factors, this proof of concept study lays a foundation for in vivo cardiac transdifferentiation with pharmacological agents and possibly safer treatment of heart failure.