HBV infection-induced liver cirrhosis development in dual-humanised mice with human bone mesenchymal stem cell transplantation

Abstract

Objective: Developing a small animal model that accurately delineates the natural history of hepatitis B virus (HBV) infection and immunopathophysiology is necessary to clarify the mechanisms of host-virus interactions and to identify intervention strategies for HBV-related liver diseases. This study aimed to develop an HBV-induced chronic hepatitis and cirrhosis mouse model through transplantation of human bone marrow mesenchymal stem cells (hBMSCs).

Design: Transplantation of hBMSCs into Fah^-/-Rag2^-/-IL-2Rγc^-/- SCID (FRGS) mice with fulminant hepatic failure (FHF) induced by hamster-anti-mouse CD95 antibody JO2 generated a liver and immune cell dual-humanised (hBMSC-FRGS) mouse. The generated hBMSC-FRGS mice were subjected to assessments of sustained viremia, specific immune and inflammatory responses and liver pathophysiological injury to characterise the progression of chronic hepatitis and cirrhosis after HBV infection.

Results: The implantation of hBMSCs rescued FHF mice, as demonstrated by robust proliferation and transdifferentiation of functional human hepatocytes and multiple immune cell lineages, including B cells, T cells, natural killer cells, dendritic cells and macrophages. After HBV infection, the hBMSC-FRGS mice developed sustained viremia and specific immune and inflammatory responses and showed progression to chronic hepatitis and liver cirrhosis at a frequency of 55% after 54 weeks.

Conclusion: This new humanised mouse model recapitulates the liver cirrhosis induced by human HBV infection, thus providing research opportunities for understanding viral immune pathophysiology and testing antiviral therapies in vivo.

Keywords: chronic hepatitis; hepatitis B; liver cirrhosis; stem cells.

Figure 1

Generation of human bone marrow mesenchymal stem cells-Fah^-/- Rag2^-/- IL-2Rγc^-/-SCID (hBMSC-FRGS) mice through the transplantation of hBMSCs into fulminant hepatic failure (FHF)-FRGS mice. (A) Schematic design of the rescue of FRGS mice with life-threatening FHF by hBMSC transplantation, the generation of hBMSC-derived human hepatocytes and immune cell lineages, and the modelling of the progression of HBV infection-induced viremia, immune and inflammatory responses, antibody synthesis, chronic hepatitis and liver cirrhosis. (B) Schematic design of the cell transplantation treatments administered to mice. During the perioperative period, FHF-FRGS mice with or without hBMSC transplantation received the same treatments, including 2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) on-off treatment, JO2 and busulfan injection. FHF-FRGS mice with hBMSC transplantation received a splenic injection of 1×10⁶ hBMSCs at day 0. The normal FRGS mice were administered 100% NTBC in their drinking water without any extra treatment. (C) Survival analysis of mice in the three groups during the perioperative period. The survival of hBMSC-FRGS and normal FRGS mice was observed for 60 weeks (n=30/group). (D) Temporal changes in eight typical biochemical markers of liver function (n=8/group). (E) H&E staining of liver tissue collected from FHF-FRGS with or without hBMSC transplantation and normal FRGS mice (bar=50 µm). The yellow arrow indicates the haemorrhage point, the red arrow indicates dead cells and the black arrow indicates residual cells. a) P<0.05; b) p<0.01; c) p<0.001. ALT, alanine transaminase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; cccDNA, covalently closed circular DNA; DC, dendritic cell; HBcAg, hepatitis B core antigen; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; NS, not significant; PT, prothrombin time; TB, total bilirubin; TBA, total bile acid; TC, total cholesterol; TP, total protein.

Figure 2

Characterisation of human bone marrow mesenchymal stem cell (hBMSC)-derived hepatocytes in hBMSC-Fah^-/- Rag2^-/- IL-2Rγc^-/-SCID (FRGS) mice. (A) Detection of the serum human albumin (hALB) level and percentage of human leucocyte antigen-positive (HLA⁺) cells in the total population of liver cells (n=8/group). (B) Percentage of hALB⁺, hNTCP⁺ and hCD45⁺ cells in the HLA⁺ cells from week 0 to 60 after hBMSC transplantation (n=8/group). (C) Correlation analysis between the serum hALB level and the percentage of hNTCP⁺ cells in the whole population of liver cells collected from hBMSC-FRGS mice (n=8/group). (D) Morphology of fluorescent-activated cell sorting (FACS)-sorted hALB⁺ hBMSC-hepatocytes (Heps) at week 12 after transplantation (bar=50 µm). (E) Detection of perfused hBMSC-Heps by immunofluorescence (IF) staining for hALB and hNTCP at week 12 after transplantation (bar=50 µm). (F) Quantitative reverse transcription PCR (qRT-PCR) analysis of the expression of 19 hepatic genes in the perfused hBMSC-Heps from week 12 to 60 after transplantation; primary human hepatocytes and hBMSCs were used as controls (n=6/group). (G) Immunohistochemistry (IHC) staining of liver tissues collected from hBMSC-FRGS mice at week 12 after transplantation for human hepatocyte markers, including hALB, hCK18, hAAT, hFAH and hNTCP (bar=100 µm). (H) IHC staining for hALB⁺ hBMSC-Heps in the whole liver lobe showed multiple distribution patterns, including periportal, non-periportal, scatter-like, island-like and cluster-like patterns (bar=1 mm). hFAH, human hepatocyte-specific fumarate dehydrogenase; hAAT, human hepatocyte-specific alpha-1-antitrypsin; hCK18, human hepatocyte-specific cytokeratin 18; hNTCP, human sodium-sodium taurocholate co-transporting polypeptide.

Figure 3

Characterisation of human bone marrow mesenchymal stem cell (hBMSC)-derived multiple immune cell lineages in hBMSC-Fah^-/- Rag2^-/- IL-2Rγc^-/- SCID (FRGS) mice. (A) Total number of hCD45⁺ cells in the peripheral blood, spleen, liver, bone marrow (BM), thymus and lymph nodes in hBMSC-FRGS mice (blue circle) and control FRGS mice (black circle) at week 12 after transplantation (n=8/group). (B) Detection of human cytokine expression in hCD45⁺ cells isolated from the livers of hBMSC-FRGS mice and subjected to in vitro stimulation with phytohemagglutinin (PHA) (green column) or phorbol 12-myristate 13-acetate (PMA)/ionomycin (violet column) for 24 hours (n=6/group). hCD45⁺ cells without stimulation (black column) were used as controls. (C) Detection of hBMSC-derived human immune cells in the livers of hBMSC-FRGS mice at 12 weeks after transplantation by immunohistochemistry (IHC) for B cells (hCD19⁺), T cells (hCD4⁺ and hCD8⁺), natural killer (NK) cells (hNKp46⁺), macrophages (hCD86⁺ and hCD163⁺) and dendritic cells (DCs) (hCD11c⁺ and hCD123⁺) (bar=100 µm). (D) Gating scheme and representative fluorescent-activated cell sorting (FACS) contour plots of hBMSC-derived multiple human immune cell lineages in the livers of hBMSC-FRGS mice. (E) Reconstitution and maintenance of hCD45⁺ cells, hCD19⁺ B cells, hCD3⁺ T cells and hCD3^-hNKp46⁺ NK cells in the peripheral blood, spleen and livers of hBMSC-FRGS mice from week 3 to 60 after transplantation (10 different donors, n=3). Relative proportions of (F) hCD4⁺ helper T (T_H) cells and hCD8⁺ cytotoxic T (T_C) cells within the population of hCD45⁺hCD3⁺ T cells in the spleen and liver of hBMSC-FRGS mice (n=6/group); (G) hCD86⁺ M1 and hCD163⁺ M2 subset within the population of hCD45⁺hCD3^-hCD14⁺hCD68⁺ macrophages in the spleen and liver of hBMSC-FRGS mice (n=6/group) and (H) hCD11c⁺ myeloid and hCD123⁺ plasmacytoid subsets within the population of hCD45⁺hCD3^-hCD14^-HLA-DR⁺ DCs in the spleen and liver of hBMSC-FRGS mice (n=6/group). a) P<0.05; b) p<0.01; c) p<0.001). U.D., undetectable.

Figure 4

Establishment of sustained HBV infection in human bone marrow mesenchymal stem cell-Fah^-/- Rag2^-/- IL-2Rγc^-/- SCID (hBMSC-FRGS) mice. (A) Serum human albumin (hALB) (grey columns), HBV DNA (black lines), (B) hepatitis B surface antigen (HBsAg) (grey columns) and hepatitis B e antigen (HBeAg) (black lines) levels in hBMSC-FRGS mice inoculated with HBV (genotype C) from 0 to 56 weeks postinfection (w.p.i.) (n=8/group). (C) Immunohistochemistry (IHC) staining for hALB⁺, HBsAg⁺ and HBcAg⁺ cells of liver tissues collected from HBV-infected and uninfected hBMSC-FRGS mice and FRGS mice at 16 w.p.i. (bar=200 µm). (D) Statistical analysis of IHC images to determine the proportion of HBsAg⁺ cells in the population of hALB⁺ cells from 0 to 56 w.p.i. (n=8/group). (E) Fluorescence in situ hybridisation (FISH) analysis for HBV DNA in the population of hALB⁺ cells collected from HBV-infected hBMSC-FRGS mice and uninfected controls at 4 w.p.i. (bar=20 µm). Detection of intrahepatic HBV covalently closed circular DNA (cccDNA) levels in HBV-infected hBMSC-FRGS mice from 0 to 56 w.p.i. by (F) Southern blot and (G) quantitative reverse transcription PCR (qRT-PCR) (n=8/group). (H) Analysis of the correlation between the serum HBV DNA and serum HBsAg levels and the correlation between the serum HBV DNA and intracellular HBV cccDNA levels (n=10). PEIU, Paul Ehrlich Institute units.

Figure 5

Chronic HBV infection induced immune and inflammatory responses and chronic hepatitis in the human bone marrow mesenchymal stem cell-Fah^-/- Rag2^-/- IL-2Rγc^-/- SCID (hBMSC-FRGS) mice. (A) Amount of hCD45⁺ cells (top left) and percentages of hCD45⁺hCD3^-hNKp46⁺natural killer (NK) cells (top right), hCD45⁺hCD3^-hCD68⁺ macrophages (bottom left) and hCD45⁺hCD3⁺ T cells (bottom right) in the livers of uninfected controls and HBV-infected hBMSC-FRGS mice from 0 to 48 weeks postinfection (w.p.i.) (n=8/group). (B) Liver sections from uninfected controls (top) and HBV-infected hBMSC-FRGS mice (bottom) at 24 w.p.i. were co-stained for hepatitis B surface antigen (HBsAg) (red) and hCD68 (green). 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei are shown in blue (bar=100 µm). (C) Plasma sample from uninfected controls and HBV-infected hBMSC-FRGS mice were analysed by ELISA for various human cytokines (n=8/group). (D) Quantitative reverse transcription PCR (qRT-PCR) analysis for the expression of human cytokine genes in HLA⁺ liver cells collected from uninfected controls and HBV-infected hBMSC-FRGS mice (n=4/group). (E) ELISA for the concentrations of hIgM and hIgG in sera of uninfected controls and HBV-infected hBMSC-FRGS mice (n=8/group). (F) ELISA for the concentrations of HBsAg antibody (HBsAb), HBeAg antibody (HBeAb) and HBcAg antibody (HBcAb) in sera of uninfected controls and HBV-infected hBMSC-FRGS mice (n=8/group). (G) H&E staining of liver tissues collected from uninfected controls and HBV-infected hBMSC-FRGS mice. The yellow arrow indicates the haemorrhage point, and the black arrow indicates the recruitment and infiltration of immune cells (bar=50 µm). a) P<0.05; b) p<0.01; c) p<0.001). NS, not significant.

Figure 6

Progression of chronic HBV infection induced liver cirrhosis. (A) Concentration of the liver cirrhosis markers hyaluronic acid (HA) and gamma glutamate transpeptidase (GGT) in serum of uninfected controls (blue line) and HBV-infected human bone marrow mesenchymal stem cell-Fah^-/- Rag2^-/- IL-2Rγc^-/- SCID (hBMSC-FRGS) mice (red line) (n=8/group). (B) quantitative reverse transcription PCR (qRT-PCR) analysis of the expression of four human cirrhosis-related genes in liver tissues collected from uninfected controls (white column) and HBV-infected hBMSC-FRGS mice (red column) (n=8/group). (C) Morphology of liver cirrhosis and (D) statistical analysis of related morbidity in uninfected controls (white column) and HBV-infected hBMSC-FRGS mice (red column) during a 54-week HBV infection course (n=20/group). (E) M&T staining of liver tissues collected from uninfected controls and HBV-infected hBMSC-FRGS mice during the progression of HBV-induced liver cirrhosis (bar=500 µm). (F) Immunohistochemistry (IHC) staining for human leucocyte antigen (HLA) and pathological staining (H&E, M&T, SR/FG and VG) of serial sections collected from uninfected controls and HBV-infected hBMSC-FRGS mice with typical liver cirrhosis (bar=500 µm). a) P<0.05; b) p<0.01; c) p<0.001. NS, not significant; U.D., undetectabl; w.p.i., weeks postinfection.

Figure 7

Progression of chronic HBV infection induced hepatitis and liver cirrhosis in dual humanised human bone marrow mesenchymal stem cell-Fah^-/- Rag2^-/- IL-2Rγc^-/- SCID (hBMSC-FRGS) mice. A novel dual-humanised mouse model with efficient chimerism of human liver cells and human immune cell lineages was developed using a single transplantation of hBMSCs, which were sensitive to chronic HBV infection and generated sustained human immune and inflammatory responses that led to liver cirrhosis. FHF, fulminant hepatic failure.