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  • equol br Experimental Procedures br Author Contributions br

    2018-10-24


    Experimental Procedures
    Author Contributions
    Acknowledgments
    Introduction The reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) has revolutionized the fields of biology and medicine, providing new avenues for research and regenerative medicine applications. IPSCs are typically generated from somatic cells by overexpression of a set of pluripotency-specific transcription factors (Takahashi et al., 2007; Takahashi and Yamanaka, 2006; Yu et al., 2007). For many years, the fibroblast has been the main source from which to derive human iPSCs (hIPSCs), and numerous publications have described the functional properties of cardiomyocytes (CMs) differentiated from fibroblast-derived iPSCs (Itzhaki et al., 2011, 2012; Lahti et al., 2012; Liang et al., 2013; Malan et al., 2011; Matsa et al., 2011; Moretti et al., 2010; Navarrete et al., 2013; Yazawa et al., 2011). Disadvantages of fibroblasts, however, as a somatic cell source include the need for a minor surgical procedure (skin biopsy) and the requirement for several weeks of expansion prior to reprogramming (Lowry et al., 2008). To overcome these hurdles, Staerk and colleagues developed an approach to generate hiPSCs from peripheral blood T and myeloid cells that allows for transduction shortly after harvesting (Staerk et al., 2010). Here, we report the electrophysiological and pharmacological characterization of CMs differentiated from human peripheral blood mononuclear cell (PBMC)-derived iPSCs, using the patch-clamp technique, calcium transient (CaT) measurements, and multielectrode array (MEA) recordings. MEA-based assays using hiPSC-derived CMs are an attractive platform for the preclinical evaluation of potential QT-prolonging drug effects, a major obstacle in drug development, and the prevention of adverse outcomes. Most hiPSCCM studies report the field potential duration as a measure of repolarization, although this measure has no direct correlate to the transmembrane equol duration (APD). The activation-recovery interval (ARI) is a direct correlate of APD and has been extensively validated in animal models and human studies (Chinushi et al., 2001; Fuller et al., 2000a; Haws and Lux, 1990; Yue et al., 2004). In this study, we report the ARI as a robust, spatiotemporal measure of repolarization particularly when applied to the effects of pharmacological treatment of hiPSC CM in culture. Such proof-of-principal experiments provide a platform to study the (patho)physiological interactions between the initiating factors and the substrates underlying human tachyarrhythmias and to test the in vitro efficacy of Food and Drug Administration-approved therapeutic interventions.
    Results
    Discussion The ability to generate patient-specific CMs from somatic cells is revolutionizing the study of cardiovascular diseases and holds tremendous promise for drug discovery, toxicology screening, and personalized medicine approaches. Although a skin biopsy to obtain dermal fibroblasts is considered minimally invasive, a blood draw is a far simpler and more acceptable approach to obtain somatic cells for reprogramming, especially in the pediatric population. Although the majority of reprogramming studies have focused on fibroblasts as the somatic cell source, several studies have recently demonstrated the feasibility of reprogramming PBMCs. IPSCs derived from PBMCs have been successfully differentiated into mesenchymal stems cells, hepatocytes, and CMs (Brown et al., 2010; Churko et al., 2013; Sommer et al., 2012; Staerk et al., 2010). However, to date, there has been no comprehensive characterization of the electrophysiological properties of CMs differentiated from human PBMC-derived iPSCs. Here, we describe the electrophysiological and pharmacological characterization of CMs differentiated from human PBMC-derived iPSCs. CMs differentiated from PBMC-derived iPSCs demonstrated the typical AP features and morphologies consistent with nodal-, atrial-, and ventricular-like phenotypes. The specific electrophysiological features, including MDP, upstroke velocity, and APD10, APD50, and APD90, for the three cell types were generally similar to values reported for CMs differentiated from fibroblast-derived iPSCs and ESCs (Itzhaki et al., 2011; Lahti et al., 2012; Liang et al., 2013; Navarrete et al., 2013; Yazawa et al., 2011). We were surprised at the similarity in AP parameters given the distinct source of somatic cells and differences in the experimental conditions. These observations suggest that despite the reported differences in epigenetic memory between different somatic cell sources, the electrophysiological behavior of the differentiated human CMs remains remarkably similar.