78 Chapter 3 strategy to generate a massive amount of CMs from differentiated CMs. Earlier studies have shown to produce cardiovascular progenitor cells using growth factor combinations during hiPSC differentiation or small molecules plus overexpression of reprogramming factor for direct lineage conversion into cardiovascular progenitors (Birket et al., 2015; Zhang et al., 2016). Given the mix population of the differentiated progeny of multipotent cardiovascular progenitors, this strategy to generate a massive amount of pure hiPSC-CMs provides a unique opportunity. For example, ~10 billion hiPSC-CMs (the total estimated number of CMs in an adult heart) with >95% purity can be prepared from two 6-wells plate of day 12 hiPSC-CMs. Mechanistically, none of previous studies that demonstrated the ability of GSK-3β inhibition and Wnt signaling to induce CM division (Buikema et al., 2013b; Heallen et al., 2011; Kerkela et al., 2008; Titmarsh et al., 2016; Tseng et al., 2006; Uosaki et al., 2013), has reported that canonical Wnt signaling activation via GSK-3β inhibition prevents CM maturation and extends the proliferative window of immature CMs (Figure 2 and 5). Furthermore, we report CHIR/low density-induced hiPSC-CM proliferation is independent from YAP activity (Figure 4), which was previously shown to be sufficient for promoting cardiomyocyte proliferation mediated by β-catenin (Heallen et al., 2011; Mills et al., 2017). Since β-catenin was a required mediator of Hippo signaling to induce cardiomyocyte proliferation and cardiomegaly (Heallen et al., 2011), CHIR treatment may have overwritten the effect of YAP modulation, thereby revealing a YAP-independent effect of contact inhibition. Further study is being actively conducted to further elucidate the underlying molecular mechanisms. One concern associated with massively expanding CMs is the possibility of their oncogenic transformation. We demonstrate that after the withdrawal of CHIR, hiPSC-CMs can mature normally and display a similar degree of sarcomere organization, electrophysiological response, and force generation as control hiPSC-CMs that were withheld from GSK-3β inhibition (Figure 3 and Figure 6). Gene expression analysis showed an increase in most sarcomere and ionchannel gene expression upon withdrawal of GSK-3β inhibition (Figure 3). Furthermore, we observed that proliferating hiPSC-CMs are mostly mononucleated (Figure 2 and 3) and that after withdrawal of CHIR, the number of binucleated CMs increased while the fraction of cell-cycleentered hiPSC-CMs decreased (Figure S1H–I). Our single-cell RNA sequencing data also supports the maintenance of the immature state of hiPSC-CMs by Wnt signaling pathway activation as a mean to extend their proliferative window, leading to a moderate level of increase in the expression of cell cycle proteins compared with untreated hiPSC-CMs (Figure 3 and 5 and S2). Taken together, this confirms that GSK-3β inhibition in immature hiPSC-CMs serves to increase proliferation by arresting these cells from their normal maturation process and not by driving these beating CMs into an alternative fate that is more proliferative, as with overexpression of cell cycle genes, which should minimize the potential for oncogenic transformation. Finally, one of the most promising therapeutic strategies to treat ischemic heart failure is to generate new CMs in an injured myocardium. Our in vivo studies indicate CHIR treatment during late gestation results in significantly more mitotic CMs, while this effect was not found
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