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Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been widely used for the assessment of drug proarrhythmic potential through multielectrode array (MEA). HiPSC-CM cultures beat spontaneously with a wide range of frequencies, however, which could affect drug-induced changes in repolarization. Pacing hiPSC-CMs at a physiological heart rate more closely resembles the state of in vivo ventricular myocytes and permits the standardization of test conditions to improve consistency. In this study, we systematically investigated the time window of stable ion currents in high-purity hiPSC-derived ventricular cardiomyocytes (hiPSC-vCMs) and confirmed that these cells could be used to correctly predict the proarrhythmic risk of Comprehensive In Vitro Proarrhythmia Assay (CiPA) reference compounds. To evaluate drug proarrhythmic potentials at a physiological beating rate, we used a MEA to electrically pace hiPSC-vCMs, and we recorded regular field potential waveforms in hiPSC-vCMs treated with DMSO and 10 CiPA reference drugs. Prolongation of field potential duration was detected in cells after exposure to high- and intermediate-risk drugs; in addition, drug-induced arrhythmia-like events were observed. The results of this study provide a simple and feasible method to investigate drug proarrhythmic potentials in hiPSC-CMs at a physiological beating rate.

Most existing culture media for cardiac differentiation of human pluripotent stem cells (hPSCs) contain significant amounts of albumin. For clinical transplantation applications of hPSC-derived cardiomyocytes (hPSC-CMs), culturing cells in an albumin containing environment raises the concern of pathogen contamination and immunogenicity to the recipient patients. In addition, batch-to-batch variation of albumin may cause the inconsistent of hPSC cardiac differentiation. Here, we demonstrated that antioxidants l-ascorbic acid, trolox, N-acetyl-l-cysteine (NAC) and sodium pyruvate could functionally substitute albumin in the culture medium, and formulated an albumin-free, chemical-defined medium (S12 medium). We showed that S12 medium could support efficient hPSC cardiac differentiation with significantly improved reproducibility, and maintained long-term culture of hPSC-CMs. Furthermore, under chemical-defined and albumin-free conditions, human-induced pluripotent stem cells (hiPSCs) were established, and differentiated into highly homogenous atrial and ventricular myocytes in a scalable fashion with normal electrophysiological properties. Finally, we characterized the activity of three typical cardiac ion channels of those cells, and demonstrated that hPSC-derived ventricular cardiomyocytes (hPSC-vCMs) were suitable for drug cardiac safety evaluation. In summary, this simplified, chemical-defined and albumin-free culture medium supports efficient generation and maintaining of hPSC-CMs and facilitates both research and clinical applications of these cells.

The epicardium promotes neovascularization and cardiomyocyte regeneration by generating vascular smooth muscle cells (SMCs) and producing regenerative factors after adult heart infarction. It is therefore a potential cell resource for repair of the injured heart. However, the epicardium also participates in fibrosis and scarring of the injured heart, complicating its use in regenerative medicine. In this study, we report coexpression of TBX18 and WT1 in the majority of epicardial cells during mouse embryonic epicardial development. Furthermore, we describe a convenient chemically defined, immunogen-free, small molecule-based method for generating TBX18+/WT1+ epicardial-like cell populations with 80% homogeneity from human pluripotent stem cells by modulation of the WNT and retinoic acid signaling pathways. These epicardial-like cells exhibited characteristic epicardial cell morphology following passaging and differentiation into functional SMCs or cardiac fibroblast-like cells. Our findings add to existing understanding of human epicardial development and provide an efficient and stable method for generating both human epicardial-like cells and SMCs.