Abstract: Severe immunodeficient rats have served as a better model for transplantation, however, this tool cannot provide an appropriate xenogeneic regenerative microenvironment. The primary limitation is interspecies immune rejection, particularly is the clean of human cells by macrophages if with human donors. Macrophages recognize and phagocytose foreign cells via surface receptors, leading to the elimination of invasive allogeneic cells. Therefore, it is essential to avoiding from the recognition by host macrophage to prevent macrophage-mediated immune attack on human cells during xenotransplantation. To address the issue, we knocked in the human signal regulatory protein alpha (hSIRPA) gene sequence into wild-type rats. CD47 is the key molecule in the "Don't Eat Me" signaling pathway, which interacts with the SIRPĪ± receptor on the surface of macrophages, inhibiting their phagocytic function and thereby preventing the clearance of human-derived cells. With PCR assay, it is confirmed that the stable inheritance and expression of the hSIRPA gene in both homozygous and heterozygous rats and with flow cytometry, it is validated that macrophage-mediated phagocytosis of human red blood cells, whose surface express hCD47 in vitro and in vivo. The results showed that the wild-type had the highest phagocytic rate comparing with the heterozygotes and the homozygotes. Furthermore, it was crossbred homozygous hSIRPA rats with severe immunodeficient rats, established a novel rat model that is suitable for human-rat xenotransplantation and to generate humanized liver called FRG-pluS. This model effectively addresses the influence of macrophages and overcomes interspecies transplantation barriers, creating a microenvironment that is friendly to human-derived cells. The FRG-pluS rat model exhibits thymic hypoplasia, lacks T, B, and NK cells, and is compatible with human cells. During in vivo transplantation, it demonstrated an affinity for human cells, establishing a preconditioning protocol for human liver cell transplantation in large animal models. This significantly enhances the potential for organ transplantation, drug screening, and disease modeling.
