119 Sarcomere Disassembly and Transfection E iciency in Proliferating Human iPSC-Derived Cardiomyocyt 5 DISCUSSION The cardiomyocyte compartment of the human heart is largely non-mitotic and with an annual cell turnover lower than 1%, this explains the lack of true regenerative capacity.28 In the neonatal period, CMs still proliferate and the heart possesses a capacity to regenerate upon injury, while in the adult myocardium the rate of CM self-renewal becomes too low to compensate for potential cell loss by, e.g., myocardial infarction. In mammals, cardiac growth by hyperplasia changes to hypertrophy early after birth, coinciding with increasing DNA content of the CMs via multinucleation and nuclear polyploidization.11 Previous studies have shown a correlation between the amount of diploid CMs and substantial regenerative capacity, raising the hypothesis that multinucleation and/or polyploidy forms a block for CM proliferation and heart regeneration.9,10,25,29,30 On the contrary, there is evidence for selfduplication of multinucleated CMs, suggesting a more complex situation of ploidy in relation to cardiac repair.31,32 For this study, we utilized time-lapse recordings to study the sequence of sarcomere distribution during mitosis, followed by cytokinesis, multinucleation or selfduplication in massively expanding hiPSC-CMs. In Figure 1 we demonstrate that during mitosis of hiPSC-CMs, sarcomere breakdown is predominantly activated during the metaphase, anaphase and telophase and cells transiently stop contracting during cytokinesis. After cytokinesis and the following G0 or G1/S/G2 phases, the sarcomeres are restored and spontaneous beating is reinitiated. Moreover, we observe that in hiPSC-CMs the sequence of mitosis is followed by cytokinesis and self-duplication versus multinucleation (Figure 2). Remarkably, for all of these sequences, we noticed that chromosomal segregation (M-phase), accompanied by sarcomere disassembly were both present during mitosis (Figure 1 and Figure 2). Mechanistically, we show that Wnt and Hippo genes are highly expressed in the high-proliferative juvenile hiPSC-CMs versus nonproliferative metabolic matured CMs (Figure 3). In addition, we demonstrate in Figure 4 that high-proliferative hiPSC-CMs in G1/S/G2 or M phase can incorporate non-viral vectors at a much higher degree than low-proliferative or non-proliferative hiPSC-CMs. Our data also indicates that the earliest expression of such vectors is present in CMs that just have gone through M phase/chromosomal segregation (Figure 5). Collectively, our study provides information on sarcomere density during mitosis and sequential cytokinesis, self-duplication or multinucleation. Our live-tracing studies (Figure 2 and Supplementary Figure 2) indicated that cell cycle activity of hiPSC-CMs was varying among different cell lines. We observed that cytokinesis occurred in 13–40%, self-duplication in 1.5–2.5% and multinucleation in 0.6–1.7% of events. Multinucleation as a result of a binuclear cells going through the M phase formed a rare event (0.1–0.2%). The observation that binucleated cells can self-duplicate is in line with previous studies in mice that have found that regenerative capacity also originates from pre-existing and multinucleated CMs.31,32 The occurrence of binucleation, however, is 5–10 times lower than self-renewal in our hiPSC-CM system, in which we concurrently activate Wnt signaling and remove cell–cell contacts (Figure 2B, 2D and Figure 5B). Therefore, this
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