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

Abstract

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.

Drug metabolic activity is a critical cell-intrinsic determinant for selection of hepatocytes during long-term culture

“…Human hepatocytes were isolated from surplus liver tissue of a 1-year-old girl with DILI (Human hepatocyte 2064), a 7-month-old girl with DILI (Human hepatocyte 2061), a 1-month-old girl with DILI (Human hepatocyte 2062), a 7-month-old boy with DILI (Hep(C)/Hep2054)and a 9-month-old girl with DILI (Human hepatocyte 2055). Human primary hepatocytes were isolated according to the collagenase perfusion method [25][26][27]. First, liver tissues were shredded and washed with HEPES buffer (pH 7.7; 140 mM NaCl/2.68 mM KCl/0.2 mM Na 2 HPO 4 /10 mM HEPES).…”

“…The liver tissues were obtained from the surplus liver of living donors from a 35-year-old woman (donor ID: 0988). Hepatocytes were isolated by the collagenase perfusion method (Enosawa, 2017;Enosawa et al, 2014;Sugahara et al, 2020). Collagenase type I (1 mg/mL, 035-17604, Fujifilm Wako Pure Chemicals, Osaka, Japan) in Hanks' solution was used to separate hepatocytes from resected liver tissue, and liver parenchymal cells were separated by low-speed centrifugation (50 g).…”

Characterization and Treatment Responsiveness of Genetically Engineered Ornithine Transcarbamylase-Deficient Pig

“…We also followed up the blood ammonia levels of the carrier female pigs to investigate the possibility of a late-onset decrease in OTC activity as shown in chimeric mice with humanized livers with OTCD patients [ 12 ]. A small number of carrier females showed a gradual increase in blood ammonia levels until about 1 month of age.…”

Ammonia-based enrichment and long-term propagation of zone I hepatocyte-like cells

“…Human cells were isolated from surplus liver tissue of a one-year-old girl with fulminant hepatitis (Hep2064). Primary cells were isolated according to the collagenase perfusion method 25 , 29 , 30 . iPSC-O cells were generated by introduction of Sendai virus carrying the 4 Yamanaka factors 31 .…”

“…Healthy human hepatocytes were isolated from surplus liver tissue of a 35-year-old healthy female according to the collagenase perfusion method 25 , 29 , 30 . First, liver tissues were shredded and washed with HEPES buffer (pH 7.7; 140 mM NaCl/2.68 mM KCl/0.2 mM Na 2 HPO 4 /10 mM HEPES).…”