ABSTRACT Pluripotent stem cells (PSCs) can be an unlimited source for generation of cardiovascular cells at different stages of 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 PSC-based cell therapy is limited by many technical challenges including the inability to deliver a pure population of cardiomyocytes (CMs) that can survive and functionally integrate into the host myocardium without adverse effects. Endogenous CMs, including fetal, have been reported to exhibit modest engraftment after transplantation with varying reports of post-transplantation maturation. Considering that the maturity of PSC-CMs is nearly equivalent to native embryonic CMs, it is not clear whether the outcomes of PSC-CM transplantation is similar to fetal CM transplantation with regards to integration and functional improvement. Ideally, the best approach to investigate the efficacy of PSC-derived and endogenous CMs for heart regeneration would include a head-to- head comparison between native human CMs at different stages of development (i.e. fetal, neonatal, and adult) vs. hESC- and hiPSC-derived CMs. However, this approach has many practical limitations. First, it is challenging to obtain freshly isolated human endogenous CMs for transplantation. Second, considering that differences between ESCs and iPSCs may stem from their source of origin, it is impossible to have hESCs and hiPSCs from the same individual or from individuals with similar genetic background. Therefore, it has not been possible to perform an adequate comparison of human endogenous and PSC-CMs using an identical genetic background. We hypothesize that a single transgenic mouse model (aMHCCre;Polr2aCAG-GCaMP5G,tdT) can be used as a source for isolation of endogenous CMs, as well as generation of mESC- and miPSC-derived CMs. This system enables: (i) tracking transplanted CMs by permanently labeling them TdTomato, and (ii) investigating their functional integration into the host via a sensitive calcium reporter (GCaMP5). In specific aim 1, we propose to use this mouse model to (i) derive mESC and miPSCs from which CMs are generated and (ii) isolate endogenous CMs at different stages of development (fetal, neonatal, and adult). We will investigate the electrophysiological properties and transcriptional profile of these cells at a single cell level. In specific aim 2, we will transplant each population (fetal, neonatal, adult, mESC-, and miPSC-CMs) in the infarct border of NSG mice after I/R injury. We will then investigate if the transplanted CMs lead to functional improvement and electromechanical coupling with the host tissue. Transcriptional profiling and pseudo-time analysis of each CM population before and after transplantation may reveal key factors that regulate the...