Robust expansion of human hepatocytes in Fah-/-/Rag2-/-/Il2rg-/- mice

Abstract

Mice that could be highly repopulated with human hepatocytes would have many potential uses in drug development and research applications. The best available model of liver humanization, the uroplasminogen-activator transgenic model, has major practical limitations. To provide a broadly useful hepatic xenorepopulation system, we generated severely immunodeficient, fumarylacetoacetate hydrolase (Fah)-deficient mice. After pretreatment with a urokinase-expressing adenovirus, these animals could be highly engrafted (up to 90%) with human hepatocytes from multiple sources, including liver biopsies. Furthermore, human cells could be serially transplanted from primary donors and repopulate the liver for at least four sequential rounds. The expanded cells displayed typical human drug metabolism. This system provides a robust platform to produce high-quality human hepatocytes for tissue culture. It may also be useful for testing the toxicity of drug metabolites and for evaluating pathogens dependent on human liver cells for replication.

Figure 1

Engraftment and repopulation of Fah^−/−/Rag2^−/−/Il2rg^−/− mice with human hepatocytes. (a) A triple-mutant mouse (no. 469, red line) maintained its weight 6 weeks after transplantation whereas Il2rg gene heterozygote littermates (no. 470, no. 471) lost weight continuously after NTNC withdrawal. NTBC was given in weeks 1 and 4 (blue area), and was withdrawn at other times (white area). (b) Human Alu sequence PCR on genomic DNA from recipient livers. Only triple mutant mice were positive. (c-e) FAH enzyme assay with equal protein concentrations. Substrate concentration was measured at 330 nm, declined in wild-type mouse liver (c) but did not change with Fah^−/− mouse liver (d); a humanized mouse liver showed high enzyme activity (e). (f) FAH immunostaining (brown; black arrow) in a repopulated liver showed more than 80% of hepatocytes positive for FAH. Red arrow demarks FAH-negative cells. (g) H&E staining of the same liver section shows the less eosinophilic human hepatocytes (black arrow). Scale bars, 100 μm (f,g).

Figure 2

Histology and immunohistochemistry of chimeric mice. (a) FAH-positive human hepatocytes were integrated in mouse liver tissue and did not disturb recipient liver microstructure. (b) Highly repopulated chimeric livers also retained normal structure. (c,d) H&E staining readily distinguishes human hepatocyte clusters, which are less eosinophilic and hence appear paler than surrounding mouse cells. (e,f) Serial sections were stained for FAH (e) and HepPar (f). (g) Congruent staining is seen. Kidney section of highly repopulated mouse shows no tubular or glomerular destruction even 4 months after transplantation. (h) FAH-positive human hepatocytes in the spleen. Scale bars, 100 μm.

Figure 3

Human gene and protein expression in chimeric mice. (a) RT-PCR of chimeric liver. The human ALB, FAH, TAT, TF, TTR and UGT1A1 genes were expressed in chimeric mice livers (no. 697 and no. 785). Human hepatocytes and mouse hepatocytes were used as positive and negative control, respectively. (b,c) Blood human albumin concentration of human cell recipients was assayed by enzyme-linked immunosorbent assay (ELISA). The threshold concentration of our system is around 0.005 μg/ml. Primary recipients (b); secondary recipients (c). The degree of difference among secondary recipients was much smaller than primary recipients. Logarithmic plotting shows the doubling time of albumin concentration was around 1 week. (d–g) Human liver–specific gene expression levels (displayed on the y-axis) determined by quantitative RT-PCR and normalized to human cyclophilin (1). Adult, average of nine adult human cell donors; Fetal, five human fetal livers; FRG, average of three highly humanized (>10%) FRG mouse livers. (d=,e) All samples show high levels of albumin (d), but alpha-fetoprotein (AFP) is expressed highly only in fetal liver (e). (f) CytochromeP450 CYP3A4 (f) is highly expressed in adult liver and FRG mice, but not in fetal liver. (g) Similarly, CYP1A2 levels in FRG mice mimic those of adult liver.

Figure 4

Serial transplantation of human hepatocytes. (a) Serial transplantation scheme starting with the primary cells (brown). Red boxes indicate repopulated serial recipients, white boxes are nonengrafted mice. Only 1/4 of the primary recipients was repopulated but all 6 secondary recipients were engrafted. (b) Alu sequence PCR of serially transplanted recipient livers. (c–e). More than 70% of cultured hepatocytes from a tertiary mouse were positive for FAH. Phase contrast (c); Fah immuno stain (red) (d); merge (e). (f–h) FAH immunohistochemistry (brown stain) of serially transplanted mice liver. Primary (f), secondary (g) and tertiary (h) recipient livers were repopulated by human hepatocytes.

Figure 5

Absence of cell fusion. (a–c) Anti-mouse albumin (green) and anti-FAH (red) double staining showed most hepatocytes from chimeric liver were mouse albumin or FAH single positive. (d–f) Anti-human albumin (green) and anti-FAH (red) double staining showed most hepatocytes were human albumin and FAH double positive. Original magnification ×100. (g–l) Flow cytometric analysis ofhepatocytes from chimeric mouse liver. FITC-conjugated anti-HLA A,B,C and PE-conjugated anti-H-2Kb were used. Control human (g) and mouse (i) hepatocytes against HLA-A,B,C. Control human (h) and mouse (j) hepatocytes against H-2Kb. Hepatocytes in two highly chimeric mice were singly positive for either HLA or H-2Kb (k,l).

Figure 6

Drug metabolism. Cultured hepatocytes from three mice were analyzed. M790 had 10%; M697, 30%; and M785, 60% human repopulation. (a,b) Induction of drug metabolism enzymes in response to beta-naphthoflavone (BNF), phenobarbital (PB) and rifampicin (Rif). Control, noninduced cultures. EROD (CYP1A1 dependent) (a). Conversion of testosterone to 6-beta-hydroxyltestosterone (CYP3A4 mediated) (b). (c) mRNA levels of human-specific genes relevant to drug metabolism, transport and conjugation were determined by quantitative RT-PCR. The ratios of human drug metabolism genes are typical of adult human hepatocytes.