Introduction: Porcine-to-human xenotransplantation offers a promising solution to the global organ shortage. While previous studies relied on somatic cells for genetic engineering, their limited lifespan and low editing efficiency hinder complex modifications. Porcine expanded potential stem cells (EPSCs), derived from preimplantation embryos via modulation of key developmental pathways, overcome these limitations. With unlimited proliferation, high editing efficiency, and the capacity to differentiate into all three germ layers and extra-embryonic lineages, EPSCs provide an ideal platform to generate humanized pigs. Here, we leverage these advantages to engineer immune-compatible porcine cells for clinical xenotransplantation.
Methods: We performed quadruple knockout (QKO) editing of porcine EPSCs, targeting GGTA1, CMAH, B4GALNT2 (to eliminate xenoantigens), and GHR (to prevent organ overgrowth). Human CD47 was inserted at the ROSA26 safe harbor locus via Cre-mediated recombination. Differentiated endothelial cells (ECs) from QKO-hCD47-EPSCs were assessed for immune compatibility.
Results: QKO-hCD47-EPSCs maintained genomic stability and differentiation potential. Derived ECs exhibited significantly reduced human antibody binding, complement-mediated lysis, and macrophage phagocytosis compared to wildtype controls.
Conclusion: This robust porcine EPSC-based gene editing system permits sophisticated genetic engineering. Meanwhile, the porcine EPSC-derived endothelial cells provide an in vitro platform for the fast functional assessment afterwards. These research efforts would benefit the eventual generation of ideal xenotransplantation donors in the future.