71 Massive Expansion of Functional Human iPSC-derived Cardiomyocytes 3 shown that overexpression of active YAP is sufficient to induce cardiomyocyte proliferation (Bassat et al., 2017; von Gise et al., 2012; Lin et al., 2015). We examined whether YAP is responsible for density-dependent proliferation of CHIR-treated hiPSC-CMs. Immunostaining of YAP revealed that ratio of nuclear to cytoplasmic YAP increases when hiPSC-CMs are sparsely plated (Figure 4A–B). Given the positive correlation between nuclear YAP translocation and cardiomyocyte proliferation (Figure 1K–N, ,4A4A–B), we next asked whether this increase in YAP nuclear translocation results from mechanosensing of stiffness. Since densely-packed hiPSC-CMs would sense their microenvironment to be softer than sparsely-plated hiPSC-CMs that are cultured directly over tissue culture plastic, we addressed whether substrate stiffness could alter the proliferation of hiPSC-CMs via YAP activity (Figure 4C). Interestingly, we found no difference in the fraction of Ki67+ CMs with changing substrate stiffness (Figure 4D–E), despite a significant increase in nuclear translocation of YAP when hiPSC-CMs were cultured on stiff hydrogel (60 kPa) or rigid plastic (Figure 4F–G). In line with this finding, reduced YAP nuclear translocation via treatment of verteporfin (Figure 4H–I), an inhibitor of YAP nuclear translocation, did not lead to a reduction in hiPSC-CM proliferation (Figure 4J–K). In summary, these data demonstrate an uncoupling of CHIR and cell-cell contact regulated hiPSC-CM proliferation from nuclear YAP activity. Mechanistic analysis of GSK-3β inhibition and low-density culture on hiPSC-CM proliferation reveals an interplay between canonical Wnt and AKT signaling To further elucidate the underlying molecular mechanisms, we first examined whether CHIR acts downstream of Wnt ligand binding to its receptor Frizzled and co-receptor low-density lipoprotein receptor 5/6 (LRP 5/6) (Nusse and Clevers, 2017). We found that a panel of Wnt ligands (i.e. canonical Wnt agonist Wnt3A and a previously described Wnt surrogate ligand scFv-DKK1c (Janda et al., 2017; Yan et al., 2017) in combination with R-Spondin (RSPO)) robustly mediated immature hiPSC-CM expansion comparable to CHIR-treatment, suggesting CHIR acts downstream of Wnt ligand activity (Figure S3A–G). Since GSK-3β is known to be involved in multiple cellular processes beyond canonical Wnt signaling modulation (Beurel et al., 2015) and we show here that cell contact is a potent inhibitor of hiPSC-CM proliferation, we sought to examine whether a β-catenin-independent mechanism is involved in hiPSC-CM proliferation. Interestingly, we found that while CHIRinduced activation of the TCF/LEF luciferase reporter was completely abolished in the presence of PNU74654, a specific TCF/LEF β-catenin signaling blocker (Trosset et al., 2006) (Figure 5A), the CHIR-mediated hiPSC-CM proliferation decreased only by ~50% (Figure 5B). This suggests that a β-catenin-independent mechanism contributes to roughly half of the proliferative activity observed with CHIR treatment. We confirmed, by real time quantitative PCR, the induction of Wnt signaling target gene expression (e.g. Axin2, LEF, and CCND2) following CHIR treatment in hiPSC-CMs and the ability of PNU74654 treatment to abolish this increase
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