Use of human hepatocyte-like cells derived from induced pluripotent stem cells as a model for hepatocytes in hepatitis C virus infection

Abstract

Host tropism of hepatitis C virus (HCV) is limited to human and chimpanzee. HCV infection has never been fully understood because there are few conventional models for HCV infection. Human induced pluripotent stem cell-derived hepatocyte-like (iPS-Hep) cells have been expected to use for drug discovery to predict therapeutic activities and side effects of compounds during the drug discovery process. However, the suitability of iPS-Hep cells as an experimental model for HCV research is not known. Here, we investigated the entry and genomic replication of HCV in iPS-Hep cells by using HCV pseudotype virus (HCVpv) and HCV subgenomic replicons, respectively. We showed that iPS-Hep cells, but not iPS cells, were susceptible to infection with HCVpv. The iPS-Hep cells expressed HCV receptors, including CD81, scavenger receptor class B type I (SR-BI), claudin-1, and occludin; in contrast, the iPS cells showed no expression of SR-BI or claudin-1. HCV RNA genome replication occurred in the iPS-Hep cells. Anti-CD81 antibody, an inhibitor of HCV entry, and interferon, an inhibitor of HCV genomic replication, dose-dependently attenuated HCVpv entry and HCV subgenomic replication in iPS-Hep cells, respectively. These findings suggest that iPS-Hep cells are an appropriate model for HCV infection.

Introduction

Hepatitis C virus (HCV), a hepatotropic member of the Flaviviridae family, is the leading cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma. Approximately 130–200 million people are estimated to be infected with HCV worldwide. Each year, 3–4 million people are newly infected with HCV [1]. Thus, overcoming HCV is a critical issue for the World Health Organization.

HCV contains a positive strand ∼9.6 kb RNA encoding a single polyprotein (∼3000 aa), which is cleaved by host and viral proteases to form structural proteins (core, E1, E2, and p7) and non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) [1]. These virus proteins might be potent targets for anti-HCV drugs. However, combination therapy with interferon and ribavirin, which often causes severe side-effects leading to treatment termination, has been the only therapeutic choice [2]. Very recently, new direct antiviral agents have been approved or are under clinical trials; these agents include NS3 protease inhibitors, NS5A inhibitors, and NS5B polymerase inhibitors [2], [3], [4]. However, the emergence of drug resistance is a serious problem associated with the use of direct antiviral agents [5].

Host targets are alternative targets for the development of anti-HCV drugs. A liver-specific microRNA (miRNA), miR-122, facilitates the replication of the HCV RNA genome in cultured liver cells [6]. Administration of a chemically modified oligonucleotide complementary to miR-122 results in long-lasting suppression of HCV with no appearance of resistant HCV in chimpanzees [7]. Epidermal growth factor receptor (EGF-R) and ephrin factor A2 (EphA2) are host cofactors for HCV entry [8]. Inhibitors of EGF-R and EphA2 attenuated HCV entry, and prevented the appearance of viral escape variants [8]. These findings strongly indicate that identification of host factors associated with infection of human liver by HCV is a potent strategy for anti-HCV drug development. Because the host tropism of HCV is limited to human and chimpanzee [9], there is no convenient model for the evaluation of HCV infections. This has led to a delay in the development of anti-HCV agents targeting host factors.

Takahashi and Yamanaka developed human induced pluripotent stem (iPS) cells from human somatic cells [10]. The stem cells can be redifferentiated in vitro, leading to new models for drug discovery, including iPS-based models for drug discovery, toxicity assessment, and disease modeling [11], [12].

Recently, several groups reported that iPS cells can be successfully differentiated into hepatocyte-like (iPS-Hep) cells that show many functions associated with mature hepatocytes [13], [14], [15], [16], [17], [18], [19]. However, whether iPS-Hep cells are suitable as a model for HCV infection has not been fully determined. Here, we investigated HCV entry and genomic replication in iPS-Hep cells by using HCV pseudotype virus (HCVpv) and HCV subgenomic replicons, respectively.