113 Sarcomere Disassembly and Transfection E iciency in Proliferating Human iPSC-Derived Cardiomyocyt 5 RESULTS Sarcomere Organization of hiPSC-CMs during Cell Cycle Phases To visualize sarcomere organization during the cell cycle phases (G0, G1/S/G2 and M-phase), we differentiated hiPSC lines (Stanford Cardiovascular Institute 273 (SCVI-273 or SCVI-111) into CMs and performed imaging for sarcomere proteins and cell cycle marker expression (Ki67). Day 11 hiPSC-CMs were dissociated, replated and treated with glycogen synthase-3 beta (GSK3β) inhibitor to activate canonical Wnt signaling to maintain the proliferative capacity of early hiPSCCMs.3,24 (Figure 1A). To confirm purity after passage 1 (P1), hiPSC-CMs were stained for cardiac troponin T (cTnT) and cell counting indicated the presence of >95% pure CMs (Figure 1B, 1C). High resolution confocal imaging of hiPSC-CMs, stained for Ki67 and cTnT, demonstrated that CMs in G0 phase and G1/S/G2 phase had the highest degree of organized sarcomeres and were spontaneously beating, while cells in M phase, especially during metaphase, anaphase and telophase, disassembled their sarcomeres, had a rolled-up morphology and were not lively contracting (Figure 1D, 1E and Supplementary Figure 1). Interestingly, we observed that both mononuclear and binuclear hiPSC-CMs were found in G0 phase. Automated quantification of the amount of troponin T lines confirmed that quiescent CMs (G0) expressed more lines when compared to Ki67 positive CMs (p < 0.05). These results indicate that sarcomere breakdown, by reduced Troponin T lines, is present in hiPSC-CMs that undergo cytokinesis. Figure 1. hiPSC-CMs sarcomere disassembly during mitosis. (A) Bright-field images of expanding hiPSC-CMs at passage 1 (P1). (B) Immunofluorescence for cardiac troponin T (cTnT), Ki67 and DAPI in hiPSC-CMs expanded with 3 µM CHIR99021 for 2 days. (C) Quantification of cTnT positive cells represented as a percentage of total cells (n = 3). (D) Representative immunofluorescence wide-field images of cTnT, Ki67 and DAPI during the different cell cycle phases of hiPSC-CMs. Note: during metaphase, anaphase and telophase the hiPSC-CM have a rolled-up morphology while contraction is absent. Scale bar indicates 10 µm. (E) Quantification of cTnT density analyzed by image J (n = 50). Bar plot represents mean cTnT intensity ± SEM. Significance assessed by unpaired t-test and defined by ** p < 0.01, and *** p < 0.001.
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