Hypoimmunogenic hPSC-derived cardiac organoids for immune evasion and heart repair [bulkRNA-seq]

Human pluripotent stem cell (hPSC)-derived cardiac therapies hold great promise for heart 41 regeneration but face major translational barriers due to allogeneic immune rejection. Here, we 42 engineered hypoimmunogenic hPSCs using a two-step CRISPR-Cas9 strategy: (1) B2M 43 knockout, eliminating HLA class I surface expression, and (2) knock-in of HLA-E or HLA-G trimer 44 constructs in the AAVS1 safe harbor locus to confer robust immune evasion. Hypoimmunogenic 45 hPSCs maintained pluripotency, efficiently differentiated into cardiac cell types that resisted both 46 T and NK cell-mediated cytotoxicity in vitro, and self-assembled into engineered cardiac 47 organoids. Comprehensive analyses of the hypoimmunogenic cells and organoids revealed 48 preservation of transcriptomic, structural, and functional properties with minimal off-target effects 49 from gene editing. In vivo, hypoimmunogenic cardiac organoids restored contractile function in 50 infarcted rat hearts and demonstrated superior graft retention and immune evasion in humanized 51 mice compared to wild-type counterparts. These findings establish the therapeutic potential of 52 hypoimmunogenic hPSC-CMs in the cardiac organoid platform, laying the foundation for off-the shelf cardiac cell therapies to treat cardiovascular disease, the leading cause of death worldwide. Control (19-9-11) and hypoimmune (B2M KO/HLA-E KI) hiPSC lines were developed. mRNA from both hiPSC conditions was isolated for RNA-seq (n = 4 per condition). Following this, hiPSCs were differentiatied into four terminal cell types: hPSC-cardiomyocytes (iCMs), hPSC-epicardial cardiac fibroblasts (iECFs), hPSC-endothelial cells (iECs), and hPSC-pericytes (iPCs). mRNA was isolated from each terminal cell for both conditions for RNA-seq (n = 4 per condition). Cardiac organoids were fabricated from terminally differentiated cells for each condition, cultured for 5 days, where they were dissociated and mRNA was isolated for RNA-seq (n = 4 per condition).