Cellular Landscapes of Different Gene‐Edited Pig Hearts Associated With Cardiac Xenotransplantation

Gene-edited pigs are currently considered the most appropriate donors for cardiac xenotransplantation. Although gene editing has improved donor-recipient compatibility, the effects of gene editing on donor organs, and the comprehensive cellular landscape of different gene-edited pig hearts remain unclear. This study characterized the cardiomyocyte and non-cardiomyocyte cellular landscape of wild-type (WT), alpha 1-3 galactosyltransferase gene knockout (GTKO), and triple gene edited (3GE: GTKO·hCD46·hTBM) porcine hearts using single-nucleus RNA sequencing (snRNA-seq) and single-cell RNA sequencing (scRNA-seq). We identified seven major cell types, including cardiomyocyte, fibroblast, endothelial cell, smooth muscle cell, macrophage, T lymphocyte, and pericyte. Integrative analysis revealed significantly more profound transcriptomic alterations in hearts from 3GE pigs compared to those from GTKO pigs. Cellular heterogeneity within cardiomyocytes and non-cardiomyocytes was identified, and pathological staining validated key sequencing findings. Analyses of cell-to-cell interactions identified the core position of cardiomyocytes and fibroblasts in intercellular communications, alongside the characteristic cellular interactions within each genotype. Transcriptomic differences at the transcriptomic level between porcine and normal human hearts were also compared. Our data revealed key cell subsets potentially involved in immune rejection and cardiac hypertrophy in cardiac xenotransplantation. This approach also provides a foundation for applying transcriptomic analysis to donor pigs with increasingly complex genetic modifications, enabling unbiased documentation of transgene expression and assessment of the impact of multigene engineering on endogenous cardiac gene expression.