Project Summary: Cardiovascular disease is the cause of 1 out of every 4 deaths in the United States, with >800,000 people suffering from myocardial infarction each year. Human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) hold remarkable promise for treating infarcted hearts. To accelerate clinical translation of hPSC-CMs, our lab developed nanowired human cardiac organoids composed of hPSC-CMs, human cardiac fibroblasts, endothelial cells, stromal cells, and electrically conductive silicon nanowires (e- SiNWs). Our in vivo data showed that the organoids with/without e-SiNWs (1E6: 1x106 cells/rat) robustly engrafted in Ischemia/Reperfusion (I/R) injured athymic rat hearts, with an engraftment rate of ~30% 1-week post-implantation. The unwired organoids showed comparable functional recovery (~39% Fractional Shortening (FS) recovery) to previous studies that injected 10-fold greater dissociated hPSC-CMs/rat (1E7 cells/rat). Further, the nanowired organoids illustrated superior functional recovery (~69% FS recovery). Despite the progress, current cardiac organoids are prepared through spontaneous assembly of hPSC-CMs and primary cells, leading to undefined human leukocyte antigen (HLA) expressions and immune rejection after implantation. To alleviate allogenic rejection, key genes involved in immune activation/suppression (e.g., B2M necessary for HLA-A/B/C expression, HLA-E/CD47) have been knocked-out/in to hPSCs to derive hypoimmunogenic hPSCs. Notably, islets derived from hypoimmunogenic hPSCs have been shown to effectively evade host immune surveillance post-implantation and functionally recover diabetes in a humanized mouse model. Unlike hypoimmunogenic hPSC islets, there have been few studies on the derivation and validation of hypoimmunogenic hPSC cardiac cells to date. Further, effects of hypoimmunogenic gene editing on therapeutic efficacies of hPSC cardiac cells have not been assessed. In addition, the effects of e-SiNWs on the hypoimmunogenic cardiac cells has yet to be explored. The goal of the proposal is to test if hypoimmunogenic gene editing of hPSCs will affect therapeutic efficacies of nanowired hPSC isogenic cardiac organoids. The central hypotheses of the proposal are hypoimmunogenic gene editing does not significantly affect therapeutic efficacy of nanowired cardiac organoids. The innovations of the proposal include, for the first time, we will 1) determine the effects of hypoimmunogenic gene editing on structures/functions of hPSC cardiac organoids, and 2) illustrate the synergy between biomaterials and hypoimmune on regenerative medicine. Accordingly, we will pursue 3 Aims: 1) Determine the effects of hypoimmunogenic gene editing on hPSC cardiac cells, 2) Determine the effects of e-SiNWs on hypoimmunogenic hPSC isogenic cardiac organoids, and 3) Determine therapeutic efficacy and hypoimmunogenicity of the nanowired hPSC cardiac organoids optimized in Aim 2 in vivo. The proposed studies will lay down the foundation to develop readily available, “off-the-shelf” hPSC cardiac organoids to treat infarcted patients.

PUBLIC HEALTH RELEVANCE: Cardiovascular disease is the leading cause of death worldwide. The goal for the proposed research is to develop nanowired, hypoimmunogenic hPSC cardiac organoids to provide “off-the- shelf” therapeutics for heart repair.