Humanized liver mouse model with transplanted human hepatocytes from patients with ornithine transcarbamylase deficiency.

Ornithine transcarbamylase deficiency (OTCD) is a metabolic and genetic disease caused by dysfunction of the hepatocytic urea cycle. To develop new drugs or therapies for OTCD, it is ideal to use models that are more closely related to human metabolism and pathology. Primary human hepatocytes (HHs) isolated from two patients (a 6-month-old boy and a 5-year-old girl) and a healthy donor were transplanted into host mice (hemi-, hetero-OTCD mice, and control mice, respectively). HHs were isolated from these mice and used for serial transplantation into the next host mouse or for in vitro experiments. Histological, biochemical, and enzyme activity analyses were performed. Cultured HHs were treated with ammonium chloride or therapeutic drugs. Replacement rates exceeded 80% after serial transplantation in both OTCD mice. These highly humanized OTCD mice showed characteristics similar to OTCD patients that included increased blood ammonia levels and urine orotic acid levels enhanced by allopurinol. Hemi-OTCD mice showed defects in OTC expression and significantly low enzymatic activities, while hetero-OTCD mice showed residual OTC expression and activities. A reduction in ammonium metabolism was observed in cultured HHs from OTCD mice, and treatment with the therapeutic drug reduced the ammonia levels in the culture medium. In conclusion, we established in vivo OTC mouse models with hemi- and hetero-patient HHs. HHs isolated from the mice were useful as an in vitro model of OTCD. These OTC models could be a source of valuable patient-derived hepatocytes that would enable large scale and reproducible experiments using the same donor.

Estimating Drug Efficacy with a Diet-Induced NASH Model in Chimeric Mice with Humanized Livers.

Nonalcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) is the most common liver disorder in developed countries. Although many new therapeutics for NASH are present in the drug development pipeline, there are still no approved drugs. One of the reasons that makes NASH drug development challenging is the lack of appropriate animal NASH models that resolve issues arising from inter-species differences between humans and rodents. In the present study, we developed a choline-deficient, L-amino-acid-defined, high-fat-diet (CDAHFD)-induced human NASH model using human liver chimeric mice. We demonstrated human hepatocyte injury by an elevation of plasma human alanine aminotransferase 1 in mice fed CDAHFD. Histological analysis showed that CDAHFD feeding induced similar histological changes to human NASH patients, including ballooning, inflammation, apoptosis, regeneration of human hepatocytes, and pericellular and perisinusoidal fibrosis. The chimeric mice fed CDAHFD were treated with a peroxisome-proliferator-activated receptor α/δ agonist, Elafibranor. Elafibranor ameliorated steatosis, ballooning of hepatocytes, and preserved fibrosis progression. We developed a novel humanized NASH model that can elucidate pathophysiological mechanisms and predict therapeutic efficacy in human NASH. This model will be useful in exploring new drugs and biomarkers in the early stages of human NASH.

Detection of acute toxicity of aflatoxin B1 to human hepatocytes in vitro and in vivo using chimeric mice with humanized livers.

Aflatoxin B1 (AFB1), a mycotoxin, is acutely hepatotoxic to many animals including humans. However, there are marked interspecies differences in sensitivity to AFB1-induced toxicity depending on bioactivation by cytochrome P450s (CYPs). In the present study, we examined the applicability of chimeric mice with humanized livers and derived fresh human hepatocytes for in vivo and vitro studies on AFB1 cytotoxicity to human hepatocytes. Chimeric mice with highly humanized livers and SCID mice received daily injections of vehicle (corn oil), AFB1 (3 mg/kg), and carbon tetrachloride (50 mg/kg) for 2 days. Histological analysis revealed that AFB1 promoted hepatocyte vacuolation and inflammatory cell infiltration in the area containing human hepatocytes. A novel human alanine aminotransferase 1 specific enzyme-linked immunosorbent assay demonstrated the acute toxicity of AFB1 to human hepatocytes in the chimeric mouse livers. The sensitivity of cultured fresh human hepatocytes isolated from the humanized liver mice for AFB1 cytotoxicity was comparable to that of primary human hepatocytes. Long-term exposure to AFB1 (6 or 14 days) produced a more severe cytotoxicity. The half-maximal lethal concentration was 10 times lower in the 2-week treatment than after 2 days of exposure. Lastly, the significant reduction of AFB1 cytotoxicity by a pan-CYP inhibitor or transfection with CYP3A4 specific siRNA clearly suggested that bioactivation of AFB1 catalyzed by CYPs was essential for AFB1 cytotoxicity to the human hepatocytes in our mouse model. Collectively, our results implicate the humanized liver mice and derived fresh human hepatocytes are useful models for studies of AFB1 cytotoxicity to human hepatocytes.