Human hepatocytes were transplanted into urokinase-type plasminogen activator-transgenic SCID mice (uPA/SCID mice), which are immunodeficient and undergo liver failure. The transplanted cells were characterized in terms of their in vivo growth potential and functions. The human hepatocytes progressively repopulated the murine host liver. However, the recipients died when the replacement index (RI) of the human hepatocytes exceeded 50%. The hosts (chimeric mice) survived at RI >50% when treated with a drug that has anti-human complement factor activity, and these mice developed livers with RI values as high as 96%. In total, 36 chimeric mice were generated, and the rate of successful engraftment was as high as 92%. The yield of chimeric mice with RI >70% was 32%. The human hepatocytes in the murine host liver expressed mRNAs for a variety of human cytochrome P450 (hCYP) subtypes, in a manner that was similar to the donor liver. The mRNAs for hCYP3A4 and hCYP1A1/2 were induced in the liver in a CYP type-specific manner when the mice were treated with rifampicin and 3-methylcholanthrene, respectively. These results indicate that human hepatocytes that propagate in mice retain their normal pharmacological responses. We conclude that the chimeric mouse developed in the present study is a useful model for assessing the functions and pharmacological responses of human hepatocytes.

Severe combined immunodeficiency (SCID) mice, the most widely used animal model of DNA-PKcs (Prkdc) deficiency, have contributed enormously to our understanding of immunodeficiency, lymphocyte development, and DNA-repair mechanisms, and they are ideal hosts for allogeneic and xenogeneic tissue transplantation. Here, we use zinc-finger nucleases to generate rats that lack either the Prkdc gene (SCID) or the Prkdc and Il2rg genes (referred to as F344-scid gamma [FSG] rats). SCID rats show several phenotypic differences from SCID mice, including growth retardation, premature senescence, and a more severe immunodeficiency without "leaky" phenotypes. Double-knockout FSG rats show an even more immunocompromised phenotype, such as the abolishment of natural killer cells. Finally, xenotransplantation of human induced pluripotent stem cells, ovarian cancer cells, and hepatocytes shows that SCID and FSG rats can act as hosts for xenogeneic tissue grafts and stem cell transplantation and may be useful for preclinical testing of new drugs.

Clinical studies have shown a close association between nonalcoholic fatty liver disease and adult-onset GH deficiency, but the relevant molecular mechanisms are still unclear. No mouse model has been suitable to study the etiological relationship of human nonalcoholic fatty liver disease and human adult-onset GH deficiency under conditions similar to the human liver in vivo. We generated human (h-)hepatocyte chimeric mice with livers that were predominantly repopulated with h-hepatocytes in a h-GH-deficient state. The chimeric mouse liver was mostly repopulated with h-hepatocytes about 50 d after transplantation and spontaneously became fatty in the h-hepatocyte regions after about 70 d. Infusion of the chimeric mouse with h-GH drastically decreased steatosis, showing the direct cause of h-GH deficiency in the generation of hepatic steatosis. Using microarray profiles aided by real-time quantitative RT-PCR, comparison between h-hepatocytes from h-GH-untreated and -treated mice identified 14 GH-up-regulated and four GH-down-regulated genes, including IGF-I, SOCS2, NNMT, IGFLS, P4AH1, SLC16A1, SRD5A1, FADS1, and AKR1B10, respectively. These GH-up- and -down-regulated genes were expressed in the chimeric mouse liver at lower and higher levels than in human livers, respectively. Treatment of the chimeric mice with h-GH ameliorated their altered expression. h-Hepatocytes were separated from chimeric mouse livers for testing in vitro effects of h-GH or h-IGF-I on gene expression, and results showed that GH directly regulated the expression of IGF-I, SOCS2, NNMT, IGFALS, P4AH1, FADS1, and AKR1B10. In conclusion, the chimeric mouse is a novel h-GH-deficient animal model for studying in vivo h-GH-dependent human liver dysfunctions.

We performed in vivo and in vitro studies to determine the induction of human cytochrome P450 (CYP) using chimeric mice with humanized liver (PXB-mice®) and human hepatocytes isolated from the PXB-mice (PXB-cells), which were derived from the same donor. For the in vivo study, PXB-mice were injected with 3-methylcholanthrene (3-MC, 2 or 20 mg/kg) or rifampicin (0.1 or 10 mg/kg) for four days. For the in vitro study, PXB-cells were incubated with 3-MC (10, 50, or 250 ng/mL) or with rifampicin (5 or 25 μg/mL). The CYP1A1 and 1A2, and CYP3A4 mRNA expression levels increased significantly in the PXB-mouse livers with 20 mg/kg of 3-MC (Cmax, 12.2 ng/mL), and 10 mg/kg rifampicin (Cmax, 6.9 μg/mL), respectively. The CYP1A1 mRNA expression level increased significantly in PXB-cells with 250 ng/mL of 3-MC, indicating lower sensitivity than in vivo. The CYP1A2 and CYP3A4 mRNA expression levels increased significantly with 50 ng/mL of 3-MC, and 5 μg/mL of rifampicin, respectively, which indicated that the sensitivities were similar between in vivo and in vitro studies. In conclusion, PXB-mice and PXB-cells provide a robust model as an intermediate between in vivo and in vitro human metabolic enzyme induction studies.

We investigated effects of human (h) GH on the proliferation of h-hepatocytes that had been engrafted in the liver of albumin enhancer/promoter driven-urokinase plasminogen activator transgenic/severe combined immunodeficiency disease (uPA/SCID) mice (chimeric mice). The h-hepatocytes therein were considered to be deficient in GH, because hGH receptor (hGHR) is unresponsive to mouse GH. Actually, hIGF-1 was undetectable in chimeric mouse sera. The uPA/SCID mice were transplanted with h-hepatocytes from a 6-year (6Y)-old donor, and were injected with recombinant hGH (rhGH). rhGH stimulated the repopulation speed of h-hepatocytes; and up-regulated hIGF-1, human signal transducers and activators of transcription (hSTAT) 3, and cell cycle regulatory genes such as human forkhead box M1, human cell division cycle 25A, and human cyclin D1. To confirm the reproducibility of these effects of rhGH, similar experiments were run using h-hepatocytes from a 46-year (46Y)-old donor. rhGH similarly enhanced their repopulation speed and up-regulated the expression of the abovetested genes, especially hIGF-1 and hSTAT1. The extent of the enhancement by rhGH was much less than that in 6Y-hepatocyte-chimeric mice most probably due to the difference in GHR expression levels between the two donors. In conclusion, this study clearly demonstrated that rhGH stimulates the proliferation of h-hepatocytes in vivo.