Dissection of cardiomyocyte differentiation process at the cellular level is indispensable in the research for cardiac development and regeneration. Previously, we have established an embryonic stem cell differentiation system that reproduces early vascular development from progenitor cells that express Flk1, a vascular endothelial growth factor receptor, by the combinatory application of 2-dimensional culture and flowcytometry. Here we show that cardiomyocytes can be successfully induced from a single Flk1+ cell on 2-dimensional culture, enabling the direct observation of differentiating cardiomyocytes and the prospective identification of cardiac progenitor potentials. Flk1+ cells could give rise to cardiomyocytes, as well as endothelial cells, from a single cell by the co-culture on OP9 stroma cells in a fusion-independent manner. Among the cell populations in intermediate stages from Flk1+ cells to cardiomyocytes, Flk1+/CXCR4+/vascular endothelial cadherin- cells were cardiac-specific progenitors at the single cell level. Noggin, a bone morphogenetic protein inhibitor, abolished cardiomyocyte differentiation by inhibiting the cardiac progenitor induction. However, wnt inhibitors Dkk-1 or Frizzled-8/Fc chimeric protein augmented, but wnt3a inhibited, cardiomyocyte differentiation. In vitro reproduction of cardiomyocyte differentiation process should be a potent tool for the cellular and molecular elucidation of cardiac development, which would provide various targets for cardiac regeneration.

Induced pluripotent stem cells (iPSCs) are novel stem cells derived from adult mouse and human tissues by reprogramming. Elucidation of mechanisms and exploration of efficient methods for their differentiation to functional cardiomyocytes are essential for developing cardiac cell models and future regenerative therapies. We previously established a novel mouse embryonic stem cell (ESC) and iPSC differentiation system in which cardiovascular cells can be systematically induced from Flk1+ common progenitor cells, and identified highly cardiogenic progenitors as Flk1+/CXCR4+/VE-cadherin− (FCV) cells. We have also reported that cyclosporin-A (CSA) drastically increases FCV progenitor and cardiomyocyte induction from mouse ESCs. Here, we combined these technologies and extended them to mouse and human iPSCs. Co-culture of purified mouse iPSC-derived Flk1+ cells with OP9 stroma cells induced cardiomyocyte differentiation whilst addition of CSA to Flk1+ cells dramatically increased both cardiomyocyte and FCV progenitor cell differentiation. Spontaneously beating colonies were obtained from human iPSCs by co-culture with END-2 visceral endoderm-like cells. Appearance of beating colonies from human iPSCs was increased approximately 4.3 times by addition of CSA at mesoderm stage. CSA-expanded human iPSC-derived cardiomyocytes showed various cardiac marker expressions, synchronized calcium transients, cardiomyocyte-like action potentials, pharmacological reactions, and ultra-structural features as cardiomyocytes. These results provide a technological basis to obtain functional cardiomyocytes from iPSCs.

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