PROJECT SUMMARY Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) currently generated in culture suffer from a major limitation of cellular immaturity, unable to recapitulate the phenotypes of the adult cardiomyocytes. No single method to date has been accepted as the ‘gold standard’ in achieving cellular maturity of iPSC-CMs. To address this critical limitation of iPSC-CM technology, I will identify growth factors secreted from developing coronary vessel cells that regulate myocardial compaction during cardiac development. I will then test whether the identified factors can robustly enhance iPSC-CM maturation using various functional phenotyping techniques. Specifically, in Aim 1 (K99), I will perform single-cell RNA-seq on embryonic hearts to uncover the interplay between coronary vessel endothelial/endocardial cells and cardiomyocytes. Using the state-of-the-art bioinformatics pipelines for intercellular communication analysis, I will identify a set of angiocrine factors secreted from developing coronary vessels predicted to augment cardiomyocyte maturation. In Aim 2 (K99), I will test the direct effects of the identified factors on cardiomyocyte maturation, by exogenously treating the factors to explanted embryonic hearts ex vivo and to human iPSC-CMs in vitro. I will employ various functional phenotyping methods to assess the level of cellular maturity after the treatment of the factors. Lastly in Aim 3 (R00) I will use single-cell RNA-seq and ATAC-seq to unveil the transcriptomic and epigenetic information of the maturation process of iPSC-CMs at the single-cell level. The completion of this R00 aim will allow me to determine key transcription factors regulating iPSC-CM maturation, to identify subpopulations of cells resistant or conducive to maturation protocol, and to generate cellular trajectory of iPSC-CM differentiation and maturation. My K99 training will be guided by an exceptional team of mentors and advisory committee whom will provide support and mentorship in all phases of my transition to independence. During the K99 phase I will engage in two externships to further develop technical skills and conceptual knowledge in electrophysiology and single-cell epigenetics. With my existing expertise in single-cell sequencing and the proposed training opportunities, I foresee no major obstacles in completing the proposed experiments. The K99/R00 award will thus propel me to realize my longstanding career goal of becoming an independent investigator in cardiovascular research and to continue making impactful discoveries in our field.