115 Sarcomere Disassembly and Transfection E iciency in Proliferating Human iPSC-Derived Cardiomyocyt 5 Moreover, we observed that both mononuclear (~0.5% of total) and binuclear (~0.1% of total) hiPSC-CMs entered the M-phase to become or remain binuclear (Figure 2C, 2D). We repeated these experiments in SCVI-111 hiPSC-CMs treated with CHIR99021 and found that all four sequences were present with similar distributions, the binuclear-to-binuclear transition being the rarest event (Supplementary Figure 2). Given our study (Figure 1) on sarcomere density during M-phase it appears that for each form of nuclear or cytoplasmic division of hiPSC-CMs, cells underwent chromosomal segregation accompanied by sarcomere breakdown (Figure 2E). Gene Expression Analysis of Long-Term Proliferating, Matured Non-Proliferative hiPSC-CMs and Adult Human Heart Tissue To provide more background on the cell cycle status of immature hiPSC-CMs, which were longterm cultured with GSK3β inhibitor CHIR99021 and sequential serially passaged (up to P4), we performed gene expression profiling for each serial passage (P1 (day ~18), P2 (day ~24) and P3 (day ~35)) and compared this to long-term metabolic matured hiPSC-CMs of day 77, 84 and 97. Confirmative immunohistochemistry for α-actinin and Ki67 demonstrated that proliferation rates (Ki67 positivity) were decreased when reaching a higher passaging number in expanding CMs.25 (Figure 3A). In contrast, in long-term metabolic matured hiPSC-CMs, we observed almost complete absence of proliferative cells.25 (Figure 3A). Moreover, higher organization of sarcomeres was visible in mature versus immature hiPSC-CMs (Figure 3A, lower left panel). Whole genome RNA sequencing revealed that the expression of sarcomere genes in mature CMs and human heart tissue, such as TNNI3 and TNNT2, were inversely correlated to genes associated with proliferation, such as TOP2A, MKI67 and CCND2 (Figure 3B). The opposite phenomenon was observed in the gene expression of proliferative CMs from P1, P2 and P3. Furthermore, genes associated with embryonic signaling pathways, including Wnt and Hippo, were upregulated in proliferating hiPSC-CMs when compared to quiescent CMs (Figure 3B). Analysis of M-phase, G2 phase, S phase and G1 phase-related genes showed an increase in the long-term Wnt-treated hiPSC-CMs from P1 to P3 versus the metabolic maturated CMs from day 77, 84 and 97 and adult human hearts (Figure 3C). Altogether, these data showed that in hiPSC-CMs, the cell cycle gene expression is inversely correlated to sarcomere gene expression and maturity of CMs.
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