Engrafted human stem cell-derived hepatocytes establish an infectious HCV murine model

Abstract

The demonstrated ability to differentiate both human embryonic stem cells (hESCs) and patient-derived induced pluripotent stem cells (hiPSCs) into hepatocyte-like cells (HLCs) holds great promise for both regenerative medicine and liver disease research. Here, we determined that, despite an immature phenotype, differentiated HLCs are permissive to hepatitis C virus (HCV) infection and mount an interferon response to HCV infection in vitro. HLCs differentiated from hESCs and hiPSCs could be engrafted in the liver parenchyma of immune-deficient transgenic mice carrying the urokinase-type plasminogen activator gene driven by the major urinary protein promoter. The HLCs were maintained for more than 3 months in the livers of chimeric mice, in which they underwent further maturation and proliferation. These engrafted and expanded human HLCs were permissive to in vivo infection with HCV-positive sera and supported long-term infection of multiple HCV genotypes. Our study demonstrates efficient engraftment and in vivo HCV infection of human stem cell-derived hepatocytes and provides a model to study chronic HCV infection in patient-derived hepatocytes, action of antiviral therapies, and the biology of HCV infection.

Figure 7. HCV infection of engrafted HLCs.

Two weeks pe, chimeric mice were inoculated with HCV⁺ plasma of genotype 1a, 1b, or 3a. (A and B) HCV RNA quantified by RTqPCR, 1, 2, and 3 months after inoculation, in the sera of mice inoculated with (A) diluted chimpanzee sera containing 30 (black) or 1,000 (blue) CID₅₀ HCV of genotype 1a (strain HC-TN) or with (B) chimpanzee sera of HCV of genotype 1b (strain CG1B) (blue) or genotype 3a (strain S52) (black) (100 CID₅₀). Control noninoculated engrafted mice are depicted in red. Individual lines represent individual mice. (C) HCV core antigen assessed by ELISA in the sera of the mice 3 months after inoculation. Horizontal bars indicate the mean. Non inf, noninfected. (D) Correlation between HCV RNA titers and HCV core antigen concentrations in the sera of mice 3 months pe.

Figure 6. In situ maturation of engrafted HLCs.

(A and B) Coimmunostaining for hALB, hAAT, and hAFP on liver sections of engrafted mice 100 days pe, visualized by confocal microscopy. Asterisks indicate central veins. Original magnification, ×10; scale bar: 400 μm. Images at ×20 magnification are shown in Supplemental Figure 6. (C) IFA for hALB, hAFP, and hAAT 14 days pe. Scale bar: 200 μm. (D) ELISA for hAFP in sera of engrafted mice during the first 2 weeks pe (black lines) and control nonengrafted mice (red line). (E and F) Coimmunostaining for hALB and human and mouse CYP450 isoforms, confirming expression of mature hepatic markers in engrafted HLCs. Scale bars: 200 μm. Asterisks indicate central veins.

Figure 5. Maintenance of HLCs in the liver parenchyma of engrafted mice.

(A) ELISA for hALB 40, 70, and 100 days pe. For individual hALB ELISAs, see Supplemental Figure 5A. Horizontal bars indicate the mean. (B) Liver sections of MUP-uPA/SCID/Bg mice, 100 days pe, analyzed by H&E staining, showing white cells believed to be human engrafted cells. Scale bar: 200 μm. (C) Three representative IHC stainings for Hep Par-1 in mice showing high, average, and low concentrations of hALB 100 days pe. Scale bar: 1 mm. (D) IHC with antibodies anti–human hepatocyte-specific antigen (Hep Par-1) and CK18 and control without primary antibody. Scale bar: 200 μm; 1,000 μm (insets). (E) Immunostaining for hALB visualized using an inverted fluorescence microscope on sections of mouse liver 100 days pe. Scale bar: 1,000 μm. (F) Visualization of the difference in cell density and size between human and mouse cells. Scale bar: 200 μm. Asterisks indicate central veins.

Figure 4. Engraftment of HLCs in the liver parenchyma of engrafted MUP-uPA/SCID/Bg mice.

(A) hALB quantification in the sera of engrafted mice within 2 weeks after intrasplenic injection of 4 million hiPSC-derived HLCs. (B) ELISA for hALB at day 10 pe did not reveal a significant difference between hESC- and hiPSC-derived HLCs. Horizontal bars indicate the mean. (C) Visualization of engrafted HLCs 14 days pe by IHC for Hep Par-1. cv, central vein; pa, portal area. Scale bars: 1 mm. (D) IFA for hALB and glutamine synthetase confirms engraftment around central veins. Scale bars: 200 μm. (E) Staining for Ki67 in the nuclei of cells positive for hALB confirms proliferation of engrafted HLCs (arrowheads). Scale bar: 200 μm (top); 50 μm (bottom). (F) Staining for hAAT and mouse ALB in distinct cells confirms the absence of cell fusion between human HLCs and mouse hepatocytes. Scale bars: 100 μm. Asterisks indicate central veins.

Figure 3. In vitro HCV infection of HLCs.

(A) Quantification of intracellular and extracellular HCV RNA by RTqPCR, and quantification of HCV core antigen in the supernatant by ELISA, after infection with JFH1-HCVcc. Inhibition of JFH1-HCV replication by 100 mM 2′-C-methylcytidine (2MC) is indicated in red (red arrows indicate when the 2MC was added). (B) Inoculation of Huh7.5.1 with supernatant of JFH1-HCVcc–infected HLCs, followed by immunofluorescence detection of HCV core protein, indicating production of infectious virus. Scale bar: 100 μm. (C and D) HLCs were transduced to express the IPS-NLS-RFP vector. One day later, cells were inoculated with (C) JFH1-HCVcc– or (D) HCV-positive clinical isolates of different genotypes. Two days pi, cells were observed for relocalization events, confirming infection of the cells by HCVcc and authentic HCV particles from patients’ sera. Scale bars: 50 μm. (E) Time-dependent visualization of HDFR, suggesting cell-to-cell transmission of HCV infection. Scale bars: 50 μm. (F) Induction of an antiviral innate immunity in response to JFH1-HCVcc infection of HLCs, as assessed by RTqPCR. *P < 0.05. Data are expressed as mean ± SEM.

Figure 2. Cellular factors associated with HCV infection.

(A and B) Expression of the 4 HCV entry factors during hepatic differentiation (day 0 [D0]) by (A) RTqPCR and (B) IFA. *P < 0.05. Scale bars: 50 μm. (C and D) Expression of apolipoproteins APOB and APOE during hepatic differentiation by (C) RTqPCR and (D) ELISA. For comparison to PHHs, see Supplemental Figure 3, C and D.

Figure 1. Hepatic differentiation of human pluripotent stem cells.

(A–D) Protocol, phase-contrast microscopy, immunofluorescent assay, and FACS analysis of the cells at different stages. (A) hESCs and hiPSCs. (B) Stem cells were treated for 3 days with 100 ng/ml activin A and basic fibroblast growth factor (bFGF) to generate definitive endoderm. (C) Hepatic specification was induced by maintaining the cells in presence of 100 ng/ml hepatocyte growth factor (HGF) and 0.1% DMSO for 8 days. (D) Finally, hepatic maturation was achieved by treating the hepatoblasts for 3 days with 10^–7 M dexamethasone. Differentiated HLCs were maintained for up to 2 weeks in culture in the presence of dexamethasone (DEX), hydrocortisone (HC), and insulin. Arrowheads indicate binucleate cells. Scale bars: 200 μm. (E) Expression of differentiation markers assessed by RTqPCR along the differentiation process (day 0: stem cells, day 3: definitive endoderm, day 11: hepatoblasts, days 14 and 18: differentiated hepatocytes). Results are expressed as relative expression. (F) Secretion of hepatic proteins AFP and albumin assessed by ELISA. Data represent mean ± SEM. (G–K) Functional characterization of HLCs at day 14 of differentiation. (G) Lipoprotein uptake assessed by incubation with Alexa 488–conjugated LDL. (H) Lipid storage demonstrated by Oil Red O staining of the lipid droplets. (I) Glycogen storage demonstrated by periodic acid–Schiff staining. (J) Cells were examined for uptake of indocyanine green. (K) Six hours later, internalized indocyanine green was released. Scale bars: 100 μm.