Single ventricle congenital heart defects, in which one ventricle fails to develop leading to mixing of oxygenated and deoxygenated blood, affect approximately 1 in 1,000 live births and have a 70% mortality rate. The Fontan operation is the standard surgical treatment, where venous blood is diverted directly to the pulmonary artery via synthetic or tissue-engineered vascular conduits (TEVCs). TEVCs are of great interest and promise due to their ability to remodel and grow with children. However, an unexpectedly high incidence of graft stenosis was reported in a prior TEVC clinical trial, leading to termination. It is likely that overt inflammatory responses caused by graft material degradation and pre-seeded bone marrow cells may have led to over-proliferation of repopulated host cells and graft stenosis. By replacing bone marrow cells with human induced pluripotent cell-derived endothelial cells (hiPSC-ECs) and substituting biodegradable synthetic grafts with native decellularized human umbilical arteries (dHUAs), we generated TEVCs via coating the lumen of dHUAs with hiPSC-ECs under physiological shear stress in a flow bioreactor. TEVCs prevented luminal stenosis and clotting after implantation as inferior vena cava (IVC) interposition grafts in nude rats, a validated model for studying grafts for Fontan procedures. To make TEVCs immunocompatible to any patient, we have generated hypoimmunogenic universal hiPSC-ECs via ablation of human leukocyte antigens (HLAs) using the CRISPR gene editing. The therapeutic efficacy and immunocompatibility of universal TEVCs will be investigated via IVC implantation in immune-humanized rats. Graft patency and blood flow will be monitored by ultrasound, and grafts will be harvested for histological analysis within 3 months post-implantation. Expanding on using universal, endothelialized vascular conduits, the PI’s group will also develop a contractile Fontan conduit as a generation 2 therapy to assist blood flow from IVC to the pulmonary artery, as none of the conduits now in use provide pumping activity, leading to insufficient tissue perfusion, heart failure, and pulmonary vascular disease. We have recently developed a tissue-engineered pulsatile conduit (TEPC) by deriving engineered heart tissues (EHTs) made by seeding hiPSC-derived cardiomyocytes into decellularized native heart matrices and then wrapping the EHTs around a dHUA. We will develop an immunocompatible TEPC by wrapping EHTs based on universal hiPSC-derived cardiomyocytes and cardiac fibroblasts around the above vascular conduits. TEPCs will be matured under electro-mechanical training conditions in bioreactors to achieve enhanced contractile output to make a strong Fontan conduit that assists pulmonary circulation. The PI will test the hypothesis that universal TEPCs are immunocompatible and maintain contractility in the IVC graft model in immune-humanized rats. Ultrasound will be employed to monitor the patency and pulsatility of TEPCs, and TE...