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Received: 6 January 2018 Accepted: 20 February 2018 Published: xx xx xxxx
Functional innate immunity restricts Hepatitis C Virus infection in induced pluripotent stem cell– derived hepatocytes Anja Schöbel, Kathrin Rösch & Eva Herker Knowledge of activation and interplay between the hepatitis C virus (HCV) and the hosts’ innate immunity is essential to understanding the establishment of chronic HCV infection. Human hepatoma cell lines, widely used as HCV cell culture system, display numerous metabolic alterations and a defective innate immunity, hindering the detailed study of virus-host interactions. Here, we analysed the suitability of induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iHLCs) as a physiologically relevant model to study HCV replication in vitro. Density gradients and triglyceride analysis revealed that iHLCs secreted very-low density lipoprotein (VLDL)-like lipoproteins, providing a putative platform for bona fide lipoviroparticles. iHLCs supported the full HCV life cycle, but in contrast to Huh7 and Huh7.5 cells, replication and viral RNA levels decreased continuously. Following HCV infection, interferon-stimulated gene (ISG)-expression significantly increased in iHLCs, whereas induction was almost absent in Huh7/7.5 cells. However, IFNα-stimulation equally induced ISGs in iHLCs and hepatoma cells. JAK-STAT pathway inhibition increased HCV replication in mature iHLCs, but not in Huh7 cells. Additionally, HCV replication levels where higher in STAT2-, but not STAT1knockdown iHLCs. Our findings support iHLCs as a suitable model for HCV-host interaction regarding a functional innate immunity and lipoprotein synthesis. Chronic hepatitis C virus (HCV) infection still remains a major public health problem worldwide, leading to severe secondary liver diseases such as cirrhosis or hepatocellular carcinoma. Current knowledge of molecular mechanisms in HCV-host interaction is often based on in vitro experiments using well-established hepatoma cell lines (Huh7 and its derivates). Despite their convenience, those cell lines frequently differ from the in vivo state and primary hepatocytes in important aspects regarding metabolic pathways, proliferation, and innate immune response1,2. For example, Huh7 cells show an impaired lipoprotein metabolism, as they do not produce bona fide very low-density lipoproteins (VLDLs) but apolipoprotein B (ApoB)-containing particles that resemble low-density lipoproteins (LDLs)3,4. The HCV life cycle is closely linked to the hepatic lipoprotein metabolism as viral particles associate with lipoproteins, most prominently ApoE, and lipids during maturation to form lipoviroparticles (LVPs)5. Accordingly, cell culture-derived HCV particles (HCVcc) produced in Huh7-derived cells show a higher buoyant density compared to in vivo- or primary hepatocyte–derived samples, correlating with a lower specific infectivity3,6. Intriguingly, production of infectious particles in Huh7-derived cells depends on ApoE but not ApoB expression7. Another drawback of using the hepatoma cell lines to study infectious processes is their diminished innate immunity8–10. In order to understand viral persistence, studying the interplay of HCV and the host cells in a physiologically intact model system is thus an important aspect. As access to primary human hepatocytes is limited and their long-term cultivation remains challenging, the creation of induced pluripotent stem cells (iPSCs) opened up possibilities for an alternative model for in vitro studies11,12. iPSCs provide a robust regenerating source for various cell types and, derived from different donors, enable the analysis of different genetic backgrounds as well as sex dependencies in various disease-related questions13. Successful differentiation into functional hepatocyte-like cells (iHLCs) has been described in several reports14–16.
Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany. Correspondence and requests for materials should be addressed to E.H. (email:
[email protected]) SCIEntIfIC REPorTS | (2018) 8:3893 | DOI:10.1038/s41598-018-22243-7
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Figure 1. Differentiation and characteristics of iHLCs. (a) Cell morphology and culture conditions at different stages of differentiation from iPSCs into iHLCs. Bright-field images were taken at the indicated time points (scale bar 100 µm). (b) Sequential expression and repression of transcription factors marking successful differentiation. Cells were immunostained with antibodies against Oct 3/4 (pluripotency marker), the endodermal transcription factor GATA-4, and the hepatic marker HNF4α at day 0, 5, and 10 of differentiation (scale bars 20 µm). Shown are representative fluorescence and DIC images. (c) mRNA expression levels of different hepatocyte-specific factors in iHLCs, Huh7.5 cells, and primary human hepatocytes (PHHs) were analysed via qRT-PCR. Expression levels are displayed as fold over iPSCs normalised to GAPDH and 18S rRNA (mean ± SEM, n = 3–4, *p
