349 General Discussion - hiPSC-CMs Disease Modelling and Future Perspectives 13 provides a framework for advanced basic and translational cell biology applications. From here, methods for defined culture, efficient differentiation, and the upscaling of hiPSC-CMs for the generation of large batches of cardiomyocytes are elucidated. As presented in Chapter 3, we identified the earliest and most potent mitogen to stimulate the canonical Wnt signal via the LEF/TCF pathway to delay hiPSC-CM maturation. Notably, we found that persistent CM proliferation required both LEF/TCF activity and AKT phosphorylation, which was independent of yes-associated protein (YAP) signaling. Importantly, in this study, we also investigated the contractility of previously expanded hiPSC-CMs in 3D microtissues and showed the uncompromised cellular functionality after expansion compared to non-expanded hiPSCCMs. The massive expansion of hiPSC-derived cardiomyocytes via Wnt/β-catenin signaling modulation holds great potential for the generation of large batches of cardiomyocytes (Chapter 3). Biobanking of expanding hiPSC-CMs, as performed in Chapter 4, now allows scientists and clinicians to generate an ‘’off-the-shelf’’ human cardiomyocyte source. Through these recent developments to generate and biobank billions of cardiomyocytes, hiPSC-CMs will be useful for drug screening, disease modeling, and regenerative approaches in cardiovascular medicine. In addition, we were one of the first to report on sarcomere distribution during cardiac mitosis, followed by cytokinesis, multinucleation, or self-duplication in massively expanding human cardiomyocytes (Chapter 5). Interestingly, we found that both mono- and binuclear hiPSC-CMs give rise to mononuclear daughter cells or binuclear progeny, which give novel insights into the potential proliferative strategy in adult CMs. However, the specific role of mononuclear and diploid cardiomyocytes during cardiac repair following myocardial infarction remains unclear3, although small case studies have shown evidence of postnatal human heart recovery post-injury in a newborn and 6 years old child suffering from myocardial dilation or infarction.4,5 More research is required to discover and combine new mechanisms to manipulate the cardiomyocyte cell cycle and cell-cell contact in adults to induce cardiomyocyte mitosis. Future research could focus on combining the overexpression of cell cycle promoters (cyclins and CDKs), cell cycle progression pathways (neuregulin/ ERBB2/ERBB4), cell cycle arrest pathways (hippo-YAP), and miRNA modulation.6 However, these processes should be tightly controlled to avoid tumorigenesis, endoreplication, or endomitosis. The verification of the massive expansion of human CMs in vivo is still pending, which is of significant interest to cardiovascular investigators to restore the adult myocardium. Cardiomyocyte maturation, are we close enough? Although scalability has improved for the majority of hiPSC-derived cell types, the immaturity of the differentiated cells, specifically hiPSC-CMs, limits their clinical potential.7 Many biochemical and biophysical strategies have been invented to induce a mature phenotype of hiPSC-CMs. For example, during prolonged culture time, hiPSC-CMs displayed more mature phenotypes in morphology (increased cell size), structure (sarcomeric organization),
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