ABSTRACT Human embryonic stem cells (hESCs) can be an unlimited source for generation of cardiovascular cells at different stages of their development. The ultimate goal of cardiac stem cell therapy is to deliver therapeutically relevant cells to damaged hearts that can repopulate the injury area, electromechanically couple with the host myocardium, and provide functional benefit. However, the clinical application of hESC-based cell therapy is limited by many technical challenges including the inability to deliver a pure population of cardiomyocytes that can functionally integrate into the host myocardium without the risk of arrhythmias. Additionally, lack of appropriate large animal models with clinically relevant injury prototype has limited our understanding of the fate of hESC-derived cells after transplantation. Attempts to date for delivery of hESC-derived cardiac cells has relied on differentiation protocols that result in beating cells in a dish, which are considered cardiomyocytes for transplantation. However, their impurity, which may include cardiomyocyte subtypes such as pacemaker cells, may lead to serious safety issues, such as arrhythmias. Therefore, isolation of a pure population left ventricular (LV) cardiomyocytes that closely resemble the endogenous cardiomyocytes is an important goal of cell-based therapy. Recently, we isolated and characterized first- and second-heart filed cardiomyocytes, as well as pacemaker cells from differentiating hESCs. Additionally, with our computational biologist collaborator, we reported how cardiovascular progenitors make fate decisions during development. And finally, our group established the first large animal facility at UCLA where we have performed hESC-derived transplantation in porcine infarct hearts. We believe the expertise and experience of our group along with the available resources will contribute to the success of the proposed project. We hypothesize that hESC-derived LV cardiomyocytes can engraft into the host myocardium and provide functional benefit. In specific aim 1, we plan to isolate LV cardiomyocytes from differentiating hESCs by modifying Activin/Nodal and retinoic acid signaling pathway. We will fully characterize these cells based on their gene and protein expression, as well as electrophysiology at a single cell level. In specific aim 2, by analyzing single cell gene expression profile of hESC-derived and endogenous cardiomyocytes at different stages of development, we plan to explore the molecular signatures that mediate differentiation and maturation in vitro. And finally, in specific aim 3, we propose to transplant hESC-derived LV cardiomyocytes in a clinically relevant model of a porcine myocardial infarction. We will perform invasive and non-invasive tests to determine structural and functional integration of the transplanted cells into the host myocardium. We believe that the success of this project will enhance our understanding of hESC differentiation and maturation to chamber-specific cardiomyocytes, provide insight into their fate after transplantation, and set the platform for future cell-based therapies to treat heart disease.