114 Chapter 5 Figure 2. Time-lapse analysis of cytokinesis and binucleation in hiPSC-CMs. (A) Mononuclear (Mono > Mono) or binuclear (Bi > Mono) hiPSC-CMs undergoing cytokinesis after chromosomal segregation (rolled-up morphology). Time is indicated in hours (h). Scale bar indicates 20 µm. (B) Relative number of mitotic mononuclear (Mono > Mono) or binuclear (Bi > Mono) hiPSC-CMs undergoing cytokinesis. Average consists of 4 replicates including, in total, 105 mitotic cells of 771 counted cells for the SCVI-273 hiPSC line. Error bars indicate standard error (C) Bright-field time-lapse images showing binucleation and chromosomal segregation of pre-existent mononuclear (Mono > Bi) or binuclear (Bi > Bi) hiPSC-CMs. Time is indicated in hours (h). Scale bar indicates 20 µm. (D) Relative number of mitotic mononuclear (Mono > Bi) or binuclear (Bi > Bi) hiPSC-CMs undergoing binucleation. Average consists of 4 replicates including, in total, 105 mitotic cells of 771 counted cells for the SCVI-273 hiPSC line. Error bars indicate standard error. (E) Schematic diagram visualizing cell cycle in relation to status of the sarcomeres. Live Tracing of Mitotic Figures in hiPSC-CMs To assess longitudinal characterization of CMs in M-phase (segregation of chromosomes) and their progeny, we recorded time-lapse videos of proliferating SCVI-273 hiPSC-CMs and analyzed the fraction of cells that went through the M-phase, indicated by a rolled-up morphology during the anaphase.13 We identified that both mononuclear (~13% of total) and binuclear cells (~1.5% of total) can enter the M-phase to segregate their chromosomes to give rise to two daughter cells (Figure 2A, 2B). Interestingly, binuclear CMs also (re)entered the M phase instead of directly providing one nucleus to each daughter cell (Figure 2A, arrows in lower panel).
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