Engrafted human stem cell–derived hepatocytes establish an infectious HCV murine model
Arnaud Carpentier, … , Stephen M. Feinstone, T. Jake Liang
Arnaud Carpentier, … , Stephen M. Feinstone, T. Jake Liang
Published October 8, 2014
Citation Information: J Clin Invest. 2014;124(11):4953-4964. https://doi.org/10.1172/JCI75456.
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Technical Advance Hepatology

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.

Authors

Arnaud Carpentier, Abeba Tesfaye, Virginia Chu, Ila Nimgaonkar, Fang Zhang, Seung Bum Lee, Snorri S. Thorgeirsson, Stephen M. Feinstone, T. Jake Liang

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Figure 1

Hepatic differentiation of human pluripotent stem cells.

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Hepatic differentiation of human pluripotent stem cells. (A–D) Protocol,...
(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.