385 General Summary 14 replating of hiPSC-CMs in culture flasks, the addition of CHIR to the cardiac culture medium efficiently induces hiPSC-CM proliferation up to 37% and subsequent passaging. Moreover, we estimated a price reduction of ~70% when compared to producing the same number of day 20 hiPSC-CMs following the standard Wnt-based differentiation protocol. Lastly, we describe the cryopreservation of expanded cardiomyocytes at passages 1-2 to allow the biobanking of large batches of hiPSC-CMs. We conclude that the removal of cell-cell contact in combination with GSK-3β inhibition is a cost-effective strategy for the massive expansion (> 250-fold) that will greatly facilitate in vitro disease modeling, large-scale drug screening, and in vivo tissue engineering applications. In Chapter 5, we provided insights into sarcomere disassembly during cardiac mitosis and created a tool for gene manipulation studies in hiPSC-CMs. We utilized time-lapse recordings to study the sequence of sarcomere distribution during mitosis, followed by cytokinesis, multinucleation, or self-duplication in massively expanding hiPSC-CMs. 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 binuclear cells going through the M phase formed a rare event (0.1–0.2%). We observed 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. Secondly, we found that CHIR administration in hiPSC-CMs increased the transfection efficiency to over 30%, which is in the range of conventional non-cardiomyocyte (HEK293) cells. We conclude that sarcomere disassembly is required during cardiomyocyte mitosis and that the increase in sarcomere disassembly allows efficient non-viral vector incorporation. In Chapter 6, we studied the hypoxic effect in hiPSC-CMs cultured in a maturation medium. The immaturity of hiPSC-CMs remains a roadblock for disease modeling. This study shows that only after metabolic maturation in low glucose, high oxidative substrate media, hiPSCCMs become susceptible to hypoxia-induced cellular damage. Secondly, we validated the cardioprotective effects of necroptosis inhibitor (nec-1) and confirmed that nec-1 prevented the hypoxia-induced decrease in mitochondrial respiration and cell death in the hiPSCCMs cultured in a maturation medium. We conclude that metabolic maturation renders the susceptibility of hiPSC-CMs to hypoxia further toward a clinically representative phenotype, thereby creating a valid and predictive human in vitro model of ischemic heart disease. PART II: MODELING THE PHOSPHOLAMBAN R14DEL MUTATION USING PATIENT-SPECIFIC HIPSC-CMS In Chapter 7, we systematically reviewed the effect of the deletion of arginine 14 (p.Arg14del) in the phospholamban protein (PLN-R14del) and summarized all the current studies that
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