Johan M Waern

Abstract: Macrophages play an important role in the rejection of xenogeneic cells and therefore represent a major obstacle to generating chimeric mice with human xenografts that are useful tools for basic and preclinical medical research. The inhibitory SIRPα receptor is a negative regulator of macrophage phagocytic activity and interacts in a species-specific fashion with its ligand CD47. Furthermore, SIRPα polymorphism amongst laboratory mouse strains significantly affects the extent of human CD47-mediated toleration of human xenotransplants. Aiming to minimize macrophage activity and thus optimize human cell engraftment in immunodeficient mice, we lentivirally transduced murine CD47 (Cd47) into human liver cells. Human HepG2 liver cells expressing Cd47 were less frequently contacted and phagocytosed by murine RAW264.7 macrophages in vitro than their Cd47-negative counterparts. For the generation of human-mouse chimeric livers in immunodeficient BALB-ΔRAG/γ(c) -uPA mice, freshly thawed cryopreserved human hepatocytes were transduced with a lentiviral expression vector for Cd47 using a refined in vitro transduction protocol immediately before transplantation. In vivo, Cd47-positive human primary hepatocytes were selectively retained following engraftment in immunodeficient mice, leading to at least a doubling of liver repopulation efficiencies. CONCLUSION: We conclude that ectopic expression of murine Cd47 in human hepatocytes selectively favors engraftment upon transplantation into mice, a finding that should have a profound impact on the generation of robust humanized small animal models. Moreover, dominance of ectopically expressed murine Cd47 over endogenous human CD47 should also widen the spectrum of immunodeficient mouse strains suitable for humanization. (HEPATOLOGY 2012.).

Abstract: Humanised mouse models have emerged to become a cost-efficient and important tool in medical research to investigate the effects of innovative medical applications outside the human body. However, models still need improvement to explore highly complex interactions, which are in demand of high-level threshold of humanisation to be conducted in a representative manner. In respect to liver research, immunodeficient mice repopulated with human hepatocytes have already proven useful for the study of hepatitis virus life cycles and new antiviral approaches. In this project, a BALB/c Alb-uPAtg(+/+)Rag2(-/-)IL-2Rγc null mouse model was used for transplantation experiments. This mouse model is characterised by severe liver damage, requiring hepatocyte transplantation within two weeks after birth for survival. Although most of the immune system is dysfunctional, macrophages remain intact and pose a threat to transplanted xenogeneic hepatocytes. Macrophages play an important role in the rejection of xenogeneic cells. Some ligands are known to downregulate macrophage activity upon contact. One of the candidates is the integrin associated protein (IAP) CD47, which has been shown to inhibit macrophage phagocytosis by species-specific interaction with signal regulatory protein α (SIRPα). This study mainly focuses on the benefits of human hepatic cells expressing murine CD47 (mCD47) when encountering murine macrophages in vitro and upon transplantation into mice. Vsv-g pseudotyped lentiviral vectors encoding for mCD47 were generated to transduce human HepG2 cells. Expression of mCD47 in HepG2 cells was confirmed by Western blot and cells were incubated with RAW264.7 murine macrophages to monitor the downregulating effect of mCD47. These experiments clearly demonstrated that mCD47- HepG2 cells were more frequently contacted and phagocytosed by murine macrophages than mCD47+ HepG2 cells. Encouraged by these results, transplantation experiments were carried out to monitor engraftment of cryopreserved human hepatocytes expressing mCD47. Mice were sacrificed 8 weeks after transplantation and liver sections were analysed for expression of mCD47 and human serum albumin. The percentage of mCD47 expressing human hepatocyte clusters had increased by 101 % in comparison to the percentage of mCD47 expressing cells at the time of transplantation, indicating that human hepatocytes expressing mCD47 showed improved survival in the mice liver. The results were able to show that expression of mCD47 in hepatic cells protects from attacks by murine macrophages in vitro. Furthermore, mCD47+ human hepatocytes demonstrated higher engraftment rates when transplanted into immunodeficient mice. This may be an important finding for pre-clinical studies of novel medical approaches, as they are in need of robust humanised mouse models with high repopulation rates.