Post-doctoral Fellow Centre for Translational Stem Cell Biology, Science Park, Hong Kong
Abstract: Xenotransplantation is promising in overcoming organ shortages. Currently, somatic cells are used for genetic modifications to generate the donor pig, yet they face technical challenges like limited gene editing capacity and subsequent phenotyping. Here, we report using porcine expanded potential stem cells (EPSCs) as a unique cellular source in xenotransplantation research. We simultaneously knocked out hyperacute rejection related genes GGTA1, CMAH and B4GALNT2, and inactivated GHR to reduce organ overgrowth. Subsequently we engineered a recombinase-mediated cassette exchange (RMCE) construct at the porcine ROSA26 locus for introducing human immune modulating genes. Then we readily targeted human CD47 cDNA via this cassette exchange method. The engineered EPSCs remained pluripotent and genetically stable. By differentiating them into highly immunogenic endothelial cells and directly immunophenotyping them, we demonstrated that the edited cells exhibited substantially lower complement-mediated lysis, decreased antibody binding, and reduced macrophage phagocytosis compared to the unedited cells. The efficient and robust gene editing in porcine EPSCs and the subsequent cellular immunophenotyping of the differentiated cells can potentially streamline the xenotransplantation study and facilitate generation of improved donor animals.
Funding Source: National Key Research and Development Program of China (nos. 2022YFA1105401); Health@InnoHK, Innovation Technology Commission; HKSAR, Hong Kong Research Council (Germany/Hong Kong travel grant G-HKU704/21)
