PROJECT SUMMARY Cardiomyocytes from human induced pluripotent stems cells (iPSC-CMs) possess the potential to study patient-specific heart disease in a dish. Their usefulness ranges from being important in drug development, cellular therapies, as well as understanding human cardiac development. Although we can generate beating cardiomyocytes in a dish, their representation of human adult subtypes (e.g., atrial, ventricular, and nodal) is still rudimentary. To understand the factors which regulate iPSC-CMs into various cardiomyocyte subtypes, we will utilize a single cell RNA sequencing approach to identify critical transcription factors involved in the transition of progenitor cardiomyocytes into an atrial-like (retinoic acid-treated cultures) or ventricular-like (default differentiation parameters) phenotype. Furthermore, we will genetically engineer our iPSCs to be deficient in or mark the expression of core subtype specific markers using CRISPR/Cas9 technology. In using this approach, we will assess what are the master transcription factors involved in cardiomyocyte subtype specification and whether a specific cardiomyocyte subpopulation formed in vitro is better at repairing the damaged heart. In using a miniswine myocardial infarction model, we will assess how well subpopulations integrate within the ischemic miniswine hearts as well as assess the functional recovery parameters that each cardiomyocyte subpopulations will contribute to repairing the damaged myocardium. Finally, by recovering the iPSC-CMs that have integrated within the heart, we will assess by single-cell sequencing how the transcriptome of these cells changes after being functional in vivo. We anticipate that these studies will shed light on how various subtypes can be differentiated in vitro which will further accelerate our use of iPSC-CMs for research and therapeutic applications.