UC San Diego

Cardiac tissue engineering has the potential to develop regenerative therapies for damaged myocardium, but due to the complex structure and composition of the tissue in both healthy and diseased states, a clinical solution has not been realized. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are self- renewing, pluripotent cell sources that can potentially generate an unlimited number of cardiomyocytes (CMs). However, the clinical application of these cells is hampered by the limited knowledge of 1) the long-term ability of transplanted cells to maintain a therapeutic effect and 2) the eventual fate of these still very immature cells. Identifying the biochemical and physical factors that govern cardiac maturation and understanding how they influence this process will be imperative to propel this field forward. This dissertation describes the influence of endothelial cells on major aspects of hiPSC- derived CM physiology and their functional maturation. To date, neither repopulation of lost myocardial mass nor functional recovery of cardiac infarct regions has been demonstrated after transplantation of human pluripotent stem cell (hPSC)-derived CMs. Strikingly, even after long- term incubation, transplanted CMs remain morphologically and physiologically immature. To this end, we postulate the importance of endothelial influences on CM maturation based on their intimate association from the formation of the early heart tube through adulthood. In the subsequent chapters, we demonstrate the maturative influences of co- culture hESC-derived CMs with human umbilical vein endothelial cells (HUVECs) on the electrophysiology and gene expression profiles towards a more mature phenotype. Moreover, we investigate the specific effect of endothelial paracrine factors on Ca²⁺-handling machinery by evaluating changes in kinetic parameters and pharmacological sensitivity. Ultimately, we demonstrate that treatment of hiPSC-derived CMs with endothelial conditioned media increases 3Hz pacing ability, Ca²⁺ transient kinetics, and sensitivity to pharmacological agents that affect the functionality of Ca²⁺ reuptake and initiation of the Ca²⁺ transient. We argue that the functional maturation of hPSC-derived CMs and elucidating the mechanisms that govern these processes will significantly contribute to the therapeutic efficacy and safety of cell-based therapies for myocardial repair