77 Massive Expansion of Functional Human iPSC-derived Cardiomyocytes 3 DISCUSSION A major goal of cardiac regenerative medicine is the restoration of functional cardiac tissue in damaged or defected hearts. Identification of a strategy to stimulate proliferation of preexisting CMs would allow for repairing failing hearts induced by CM loss and generating patient-specific, engineered heart tissue for regenerative therapy or drug screening applications. Here, we achieved massive expansion of functional hiPSC-CMs in vitro through concomitant GSK-3β inhibition and removal of cell-cell contact. While we and others have previously demonstrated the ability of canonical Wnt signaling activation or direct GSK-3β inhibition to transiently expand mouse and human PSC-CMs in vitro, the extent remained modest (<10-fold) (Buikema et al., 2013b; Mills et al., 2019; Sharma et al., 2018; Titmarsh et al., 2016; Uosaki et al., 2013). In this study, we show that direct cell-cell contact is a major inhibitor for continuous CM proliferation, and the modest expansion of hiPSC-CMs by GSK-3β inhibition alone can be overcome by removal of cell-cell contact following multiple passages, which can cumulatively result in a 250-fold expansion (Figure 1 and 2). Mechanistically, we demonstrate that GSK-3β inhibition suppresses CM maturation via LEF/TCF activity, while concomitant removal of cell-cell contact stimulates the cell cycle activation by AKT T308 phosphorylation (Figure 3 and 5). The combined CHIR treatment with low density hiPSC-CM culture allowed for continuous cell cycle activation and CM proliferation, independent from upregulation of YAP activity (Figure 1 and 4) These findings support the role of Wnt/ β-catenin signaling in suppressing hiPSC-CM maturation and uncover a role of cell-cell contact in inhibiting CM proliferation. As a proof-of-concept, we show the feasibility of using hiPSC-CMs expanded by the presented method for mass production of engineered heart tissues (Figure 6). We believe the ability to generate hiPSC-CMs at this scale will greatly facilitate in vitro disease modeling, high throughput drug screening and in vivo tissue engineering applications. In summary, we demonstrate massive expansion of human cardiomyocytes in vitro by simultaneously “removing brakes and pushing accelerators” (He and Zhou, 2017). Our finding here complements the recent studies demonstrating the generation of a large number of cardiac cells for regenerative applications with noted differences. First, this study reveals an important finding that mitotic cardiomyocytes quickly lose proliferation capacity via juxtacrine signaling (Figure 1), highlighting the importance of removal of cell-cell contact. Contact inhibition of cell proliferation is an essential regulatory process to control tissue growth during embryonic development and tissue homeostasis, and is known to be dysregulated in uncontrolled tumor growth (Kim et al., 2011). Historically for continuous cell growth upon reaching high cell confluence, reducing cell density and removing contact inhibition have been standard in vitro cell culture procedure, also known as passaging. However, passaging of hiPSC-CMs has not been possible because passaging alone leads to expanding only nonmyocytes (Figure 2F and 2H). We show a remarkable role of CHIR in delaying hiPSC-CM maturation and widening their mitotic window to enable continuous CM passaging and expansion (Figure 2 and 3). In addition, this study represents a unique
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