Engraftment of human hepatocytes in the livers of rats bearing bone marrow reconstructed with immunodeficient mouse bone marrow cells.

ABSTRACT

BACKGROUND: Previously, we created, a chimeric mouse (humanized mouse), a severe combined immunodeficiency (SCID) mouse whose liver was >90% repopulated with human (h)-hepatocytes, which are useful for the testing of drug metabolism and toxicity, as well as a hepatitis B virus and hepatitis C virus-susceptible animal model. However, their small body size and small total blood volume limited the utilization for analytical purposes, which led us to develop a method to create a chimeric rat bearing h-hepatocyte-repopulated liver. METHODS: F344 nude rats devoid of T cells were irradiated with X-rays and injected with bone marrow cells (BMCs) from SCID mice (m(SCID)). The rate of replacement with m(SCID)-BMCs was evaluated by two-color flow cytometry analysis of peripheral blood mononuclear cells (PBMCs). After m(SCID)-BMCs repopulated the host bone marrow (BM), the rats were treated with retrorsine, partially hepatectomized (PHx), and transplanted with 5 x 10(6) h-hepatocytes isolated from the chimeric mice. h-Albumin (h-Alb) concentrations in the host blood and the expression levels of protein and mRNA of hepatocyte differentiation markers in the h-hepatocytes were evaluated by ELISA, immunostaining, and reverse transcription-PCR, respectively. RESULTS: The m(SCID)-BMCs successfully repopulated the rats, the percentage of mouse cells reaching 94% among host (r(nudeF344)) PBMCs at 4 weeks after m-BMC transplantation. h-Hepatocytes isolated from the chimeric mice were transplanted to the liver of the m(SCID)-BMC-repopulated rats. The engrafted h-hepatocytes expressed h-Alb and h-cytochrome P450 (CYP) subtypes and survived showing normal phenotypes until at least 3 weeks post-h-hepatocytes transplantation (h-HPCT). However, the blood concentrations of h-Alb declined at 4 weeks post-HPCT, concomitant with the emergence of both r(nudeF344)- and m(SCID)-macrophages, suggesting the rejection of h-hepatocytes due to the activation of macrophages. CONCLUSION: We developed a novel method to create a rat that bears the liver engrafted with h-hepatocytes, utilizing a rat with the BM composed of m(SCID)-BMCs as a host. This h-hepatocyte-bearing rat will be a valuable model for studying the immunologic mechanisms involved in xenogeneic transplantation and for generating rats with higher rates of repopulation with h-hepatocytes.