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  • The chronotropic responses of the hPSC CMs to various

    2018-11-08

    The chronotropic responses of the hPSC-CMs to various cardio-active drugs in our study are well in line with the published results (Mandel et al., 2012; Pillekamp et al., 2009; Pillekamp et al., 2012; Xu et al., 2002), in which the beating rates of CMs were increased in response to isoproterenol and phenylephrine, and decreased in response to carbamylcholine. While the pharmacological responsiveness of the cells was similar, the calcium cycling of hPSC-CMs from cultures on the two types of fibrous scaffolds were significantly prolonged. Lundy et al. found that late stage hPSC-CMs (differentiation day 80+) exhibited faster spontaneous Ca transient kinetics, i.e., significant reduction in time to peak and time to 50% decay, while the transient peak amplitude remained unchanged, comparing with early stage cells (differentiation day 20–40) (Lundy et al., 2013). Similarly, we found statistically unchanged peak amplitude in spontaneous Ca transients. However, the increases in 50% rise to peak time and peak to 50% decay time suggest unimproved maturation for cells on fibrous scaffolds, comparing with cells on TCPs. There are several challenges and considerations in promoting the maturation of hPSC-CMs in vitro. First, hPSC-CMs are dramatically different from human adult ventricular cardiomyocytes. For example, human adult ventricular cardiomyocytes in culture have a surface area of approximately 12,000μm2 (Li et al., 1996), which is at least 11 fold larger than hPSC-CMs examined rac inhibitor in the current study, and the sarcomere length of relaxed human rac inhibitor cell is approximately 2.2μm (Bird et al., 2003), which is about 1.5 fold of the value in hPSC-CMs observed in this study. In vivo, it can take 6–7years for human CMs to reach certain features of adult CMs (Peters et al., 1994), suggesting the necessity of expedited maturation for hPSC-CMs differentiated in vitro. Second, most studies to date including our present study used hPSC-CM population with heterogeneous maturation levels of immature embryonic or neonatal-like CMs, as reflected in different levels of maturation in sarcomeric organizations and electrophysiological properties (Veerman et al., 2015; Yang et al., 2014). Third, the hPSC-CMs generated so far are mixed populations of nodal-, atrial-, or ventricular-like cells (Veerman et al., 2015; Yang et al., 2014). Distinct profiles in their gene expression as well as the electrophysiological properties will undoubtedly lead inaccurate evaluation in CM maturation. Yet with subtype specification, we can precisely gauge and evaluate the maturation stage of hPSC-CMs, which in turn, will contribute to the homogeneity of the cell maturation. Forth, the molecular, structural, and functional phenotypes of CMs change constantly during development. Thus using stage-specific human CMs, like the fetal, neonatal, postnatal, or adult CMs as the references to gauge the maturation level, will provide a more accurate characterization (Ribeiro et al., 2015). Last but not the least, in native 3-D cardiac environment, the underlying ECM, the neighboring non-CMs, together with the surrounding biochemical, mechanical, and electrical signals act synergistically to modulate the maturation of CMs (Ma et al., 2014; Veerman et al., 2015; Yang et al., 2014). Combinatorial methods by applying 3-D culturing with electrical stimulation, mechanical stretch and co-culture with vascular cells, or microgroove alignment and biochemical treatment, have thus far achieved promising results (Nunes et al., 2013; Ribeiro et al., 2015; Tulloch et al., 2011). Therefore, the enhanced and expedited maturation of hPSC-CMs may be significantly advanced by addressing the present challenges in CM purification (subtype specification/differentiation), heterogeneity and variability in cell maturity, using the stage-relevant human CMs as the references, as well as the combinatorial use of multiple microenvironment factors.
    Conclusions The following are the supplementary data related to this article.