Getting closer to the patient: Upgrade of hepatitis C virus infection in primary human hepatocytes
Authors: Mirjam B. Zeisel, Marine Turek, Thomas F. Baumert
Journal: Journal of Hepatology (2010)

COMMENTARY ON: Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures. Ploss A, Khetani SR, Jones CT, Syder AJ, Trehan K, Gaysinskaya VA, Mu K, Ritola K, Rice CM, Bhatia SN. Proc Natl Acad Sci U S A. 2010;107(7):3141–5. Copyright (2010). Abstract reprinted with permission from National Academy of Sciences, U.S.A. www.ncbi.nlm.nih.gov/pubmed/20133632 Abstract: Hepatitis C virus (HCV) remains a major public health problem, affecting approximately 130million people worldwide. HCV infection can lead to cirrhosis, hepatocellular carcinoma, and end-stage liver disease, as well as extrahepatic complications such as cryoglobulinemia and lymphoma. Preventative and therapeutic options are severely limited; there is no HCV vaccine available, and nonspecific, IFN-based treatments are frequently ineffective. Development of targeted antivirals has been hampered by the lack of robust HCV cell culture systems that reliably predict human responses. Here, we show the entire HCV life cycle recapitulated in micro-patterned co-cultures (MPCCs) of primary human hepatocytes and supportive stroma in a multiwell format. MPCCs form polarized cell layers expressing all known HCV entry factors and sustain viral replication for several weeks. When coupled with highly sensitive fluorescence- and luminescence-based reporter systems, MPCCs have potential as a high-throughput platform for the simultaneous assessment of in vitro efficacy and toxicity profiles of anti-HCV therapeutics. With an estimated 170 million infected individuals, hepatitis C virus (HCV) has a major impact on public health. The majority of HCV-infected individuals develop chronic hepatitis that may progress to liver cirrhosis and hepatocellular carcinoma. Although new antivirals are entering clinical trials, current treatment options are limited and a vaccine is not available. In recent years, tremendous progress has been made in deciphering HCV–host cell interactions. The establishment of novel and robust infectious model systems has allowed for a detailed study of the entire HCV life cycle in vitro [[1]Wakita T. Pietschmann T. Kato T. Date T. Miyamoto M. Zhao Z. et al.Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.Nat Med. 2005; 11: 791-796Crossref PubMed Scopus (2449) Google Scholar]. However, these model systems rely on unique HCV isolates (i.e. JHF1) and on specific cell lines (i.e. Huh7) for a reproducible and standardized analysis of HCV–host cell interactions. In vivo, the hepatocyte is the primary target cell of HCV. The liver’s architecture is elaborate and hepatocytes are characterized by a complex and highly-polarized morphology [[2]Wang L. Boyer J.L. The maintenance and generation of membrane polarity in hepatocytes.Hepatology. 2004; 39: 892-899Crossref PubMed Scopus (80) Google Scholar]. Human hepatoma cells differ from primary human hepatocytes (PHH) with respect to morphology, proliferation, gene expression, and innate immune responses but are widely used as a surrogate model for human hepatocytes because PHH are difficult to maintain in cell culture and infection with recombinant or serum-derived HCV is not robust. Indeed, PHH do not proliferate and tend to rapidly loose their phenotype and viability upon isolation. Therefore, innovative strategies that circumvent these drawbacks and improve PHH culture are needed. Furthermore, a PHH-based HCV infectious model system would be of great interest to determine the efficacy and toxicity of novel antivirals. Interestingly, transgenic immunodeficient mice with hepatocyte-lethal phenotype have been successfully transplanted with PHH and are susceptible to HCV infection [3Mercer D.F. Schiller D.E. Elliott J.F. Douglas D.N. Hao C. Rinfret A. et al.Hepatitis C virus replication in mice with chimeric human livers.Nat Med. 2001; 7: 927-933Crossref PubMed Scopus (764) Google Scholar, 4Bissig K.D. Wieland S.F. Tran P. Isogawa M. Le T.T. Chisari F.V. et al.Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment.J Clin Invest. 2010; 120: 924-930Crossref PubMed Scopus (287) Google Scholar]. In the transplanted mice, PHH may occupy the majority of the liver parenchyma and preserve their phenotype for several months, suggesting that the in vivo microenvironment stabilizes the PHH phenotype. In the past years, tissue engineering has greatly improved PHH culture conditions in order to preserve and modulate PHH phenotype. Micro-patterned co-culture (MPCC) of PHH with mouse fibroblasts (3T3-J2 cells) (Fig. 1) stabilizes the PHH phenotype (e.g. synthesis of albumin, urea, and cytochrome P450 activity) for several weeks and has been proposed as a model to study drug safety [[5]Khetani S.R. Bhatia S.N. Microscale culture of human liver cells for drug development.Nat Biotech. 2008; 26: 120-126Crossref PubMed Scopus (1023) Google Scholar]. This model appears robust as stabilization of liver functions was observed with both fresh and cryopreserved PHH from different donors. Of note, like in monoculture, PHH do not seem to proliferate under MPCC culture conditions. On the other hand, virologists have been continuously trying to improve the HCV models and most recently two highly sensitive new reporter models were described [[6]Jones C.T. Catanese M.T. Law L.M. Khetani S.R. Syder A.J. Ploss A. et al.Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter system.Nat Biotech. 2010; 28: 167-171Crossref PubMed Scopus (213) Google Scholar]: an HCVcc reporter virus expressing secreted Gaussia luciferase (Jc1FLAG2(p7-nsGluc2A) and a fluorescent protein (EGFP or RFP) reporter containing an HCV protease cleavage site (IPS-1). In contrast to current model systems, these new reporter systems offer the advantage of determining HCV infection in living cells without the need for cell lysis (Fig. 1). Combining these two advancements, the Rice laboratory established a new model system to study persistent HCV infection in PHH [[7]Ploss A. Khetani S.R. Jones C.T. Syder A.J. Trehan K. Gaysinskaya V.A. et al.Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures.Proc Natl Acad Sci USA. 2010; 107: 3141-3145Crossref PubMed Scopus (179) Google Scholar]. Interestingly, MPCC PHH appear to polarize [[7]Ploss A. Khetani S.R. Jones C.T. Syder A.J. Trehan K. Gaysinskaya V.A. et al.Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures.Proc Natl Acad Sci USA. 2010; 107: 3141-3145Crossref PubMed Scopus (179) Google Scholar] and HCV host entry factor distribution seems to be comparable to liver hepatocytes [[8]Reynolds G.M. Harris H.J. Jennings A. Hu K. Grove J. Lalor P.F. et al.Hepatitis C virus receptor expression in normal and diseased liver tissue.Hepatology. 2008; 47: 418-427Crossref PubMed Scopus (87) Google Scholar]. Thus, MPCC PHH appear to reflect hepatocyte morphology in the liver more closely than PHH monoculture or hepatoma cells. Noteworthy, MPCC PHH were able to replicate HCV for 2 weeks as shown by luciferase secretion that was reduced in the presence of HCV polymerase and protease inhibitor [[7]Ploss A. Khetani S.R. Jones C.T. Syder A.J. Trehan K. Gaysinskaya V.A. et al.Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures.Proc Natl Acad Sci USA. 2010; 107: 3141-3145Crossref PubMed Scopus (179) Google Scholar]. In addition, expression of the fluorescent reporter RFP-NLS-IPS allowed for the assessment of replication of cell culture and serum-derived HCV at a single cell level. Importantly, in line with previous results obtained in hepatoma cells, antibodies directed against CD81 and HCV E2 inhibited HCV infection. Of note, MPCC PHH are able to support the entire HCV lifecycle as supernatants from infected MPCC PHH were infectious in hepatoma cells [[7]Ploss A. Khetani S.R. Jones C.T. Syder A.J. Trehan K. Gaysinskaya V.A. et al.Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures.Proc Natl Acad Sci USA. 2010; 107: 3141-3145Crossref PubMed Scopus (179) Google Scholar]. Although HCV infection and viral spread occurred only in a small fraction of cells, MPCC PHH appear to be a valuable and robust model system to study HCV infection in primary cells. The possibility to use fluorescent reporter proteins in MPCC PHH broadens the scope of this model system as it will allow for studying HCV infection of a large panel of genotypes and isolates without engineering reporter viruses. Moreover, as HCV associates with lipoproteins in vivo and this impacts viral entry and neutralization [9Popescu C.I. Dubuisson J. Role of lipid metabolism in hepatitis C virus assembly and entry.Biol Cell. 2009; 102: 63-74Crossref PubMed Scopus (91) Google Scholar, 10Zeisel M.B. Cosset F.L. Baumert T.F. Host neutralizing responses and pathogenesis of hepatitis C virus infection.Hepatology. 2008; 48: 299-307Crossref PubMed Scopus (44) Google Scholar], it will be of interest to compare infectious virions released from MPCC and hepatoma cells with respect to biochemical properties, host factor usage, infectivity, and neutralization. Furthermore, this model also offers a unique opportunity to assess the efficacy of novel antivirals but also to determine drug interactions and toxicity in HCV-infected cells [[7]Ploss A. Khetani S.R. Jones C.T. Syder A.J. Trehan K. Gaysinskaya V.A. et al.Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures.Proc Natl Acad Sci USA. 2010; 107: 3141-3145Crossref PubMed Scopus (179) Google Scholar]. In conclusion, this original model system based on the natural host cell of HCV infection clearly provides an important step forward for the understanding of virus–host interactions in the HCV-infected liver. The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript The authors acknowledge financial support of their work by the European Union (ERC-2008-AdG-233130-HEPCENT and INTERREG-IV-2009-FEDER-Hepato-Regio-Net), ANRS (2007/306 and 2008/354), the Région Alsace (2007/09), the Else Kröner-Fresenius Foundation (EKFS P17//07//A83/06), the Ligue Contre le Cancer (CA 06/12), Inserm, University of Strasbourg, and the Strasbourg University Hospitals, France.