384 Chapter 14 GENERAL SUMMARY The general aim of this thesis was to investigate the potentials of stem cell-derived cardiomyocytes to answer the question ’’Can we use stem cell-derived cardiomyocytes to find a cure for genetic cardiomyopathies?’’. This aim was achieved using two main parts. In part I, we examined the developmental cues leading to efficient differentiation, expansion, and maturation to study cardiomyocyte division, gene manipulation, and ischemic disease modeling of stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro. In part II, disease modeling and therapeutic strategies for the genetic cardiomyopathy PLN-R14del are described. In Chapter 1, we gave a general introduction to the clinical significance of a genetic variation in cardiomyopathies. We highlighted the knowledge derived from cardiogenesis and reproducible methods for the efficient generation of hiPSC-CMs to study the molecular mechanisms of genetic cardiomyopathy processes and contemporary challenges encountered in drug research. PART I: FROM HEART DEVELOPMENT TOWARDS OPTIMAL CARDIAC IN VITRO MODELS In Chapter 2, we summarize the signals that control human heart size and describe pivotal roles for Wnt and Hippo signaling during embryonic and fetal heart growth. Next, we compared the pathways regulating heart growth in vivo with the molecular targets that promote in vitro cardiomyocyte proliferation. Lastly, we provide a complete overview of Wntsignalling-based cardiomyocyte differentiation and cardiomyocyte expansion strategies. With this, we highlight the interaction of signaling pathways in heart development and discuss how this knowledge has been translated into current technologies for cardiomyocyte production. In Chapter 3, we achieved massive expansion of functional hiPSC-CMs in vitro through concomitant GSK-3β inhibition and removal of cell-cell contact. Mechanistically, we demonstrate that GSK-3β inhibition suppresses CM maturation via LEF/TCF activity while stimulating the cell cycle activation by AKT T308 phosphorylation, independent from upregulation of YAP activity. As a proof-of-concept, we demonstrated uncompromised cellular morphology and functionality in engineered heart tissues generated with previously expanded hiPSC-CMs. Lastly, we assessed the late gestational and postnatal CM proliferation in vivo and found that CHIR treatment is effective during late gestation but is not sufficient for cardiac regeneration in the postnatal and adult phases of the heart. We conclude that expanded cardiomyocytes have uncompromised cellular functionality making them suitable for multiple translational/regenerative applications. In Chapter 4, we offer a detailed protocol that allows massive expansion (up to a 250-fold increase of CM number within 3-5 weeks) from multiple hiPSC lines. After low cell-density
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