352 Chapter 13 significantly improve calcium handling, metabolic function, and hypertrophy in day 17 2D hiPSC-CMs. Another study used single-cell RNA sequencing to compare hiPSC-CM monolayers versus 3D sheets, tissue strips, and organoid chambers, revealing increasing levels of maturity by increasing tissue complexity. Interestingly, myofibril structure was shown to be the first to mature, followed by electrophysiological function and oxidative metabolism.24 In summary, the limited maturation and predictivity in the current hiPSC-CM models pose the question if these methods can yield mature enough cells, in a realistic, large-scale, and financially appropriate culture time frame. Combining the current strategies such as long-term culturing, 3D tissue engineering, and metabolic supplementation with future improvements such as TFs/Cx43/KCNJ2 enhancement could take us to the final steps in getting as close as possible to our ‘holy grail’; an hiPSC derived adult-like CM. PART TWO: MODELING THE PHOSPHOLAMBAN R14DEL MUTATION USING PATIENT-SPECIFIC HIPSC-CMS In Part 1, we discussed the substantial technological innovations observed from cardiac development regarding the differentiation, proliferation, and maturation of hiPSC-CMs. Especially with the advanced hiPSC technology, the possibility arises to create essentially limitless numbers of hiPSC-derived CMs, harboring a diversity of patient genetics. Genetic cardiac hiPSC-CM models have been constructed using hiPSCs derived from patients suffering from ACM, DCM, HCM, non-compaction cardiomyopathy (NC), and lysosomal storage disorders which were thoroughly reviewed elsewhere.25–27 In this thesis, we study the deletion of arginine 14 (R14) in the phospholamban protein (PLN-R14del), causing the panoply of DCM and ACM manifestations in heart failure patients. By systematically reviewing the current evidence of all studies describing PLN-R14del mutation, we summarized the potential molecular mechanisms contributing to the disease pathophysiology (Chapter 7). We emphasized how the discovery of PLN-R14del fuelled insights into the basic biology of calcium handling, protein toxicity, metabolism, and fibrosis, and we reinforced the idea that PLN is a crucial dynamic regulator of SERCA2a (ATP2A2) that contributes to the speed and force of calcium-driven muscle contraction. Metabolic maturation and multi-omics analysis of hiPSC-CMs reveal the pathological features of the R14del cardiomyopathy in vitro. RNA sequencing (RNA-seq) is a genomic approach for the detection and quantitative analysis of messenger RNA molecules to study cellular gene expression. In Chapter 5, we used whole genome RNA-seq to study the gene expression patterns in expanding hiPSC-CMs, versus hiPSC-CMs that were metabolically matured for +77 days. As expected, genes associated with embryonic signaling pathways and mitosis were upregulated in proliferating hiPSC-CMs, whereas the expression of sarcomere genes was upregulated in matured CMs and human
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