title: A robust model of natural hepatitis C infection using hepatocyte-like cells derived from human induced pluripotent stem cells as a long-term host.
abstract: BACKGROUND
  Hepatitis C virus (HCV) could induce chronic liver diseases and hepatocellular carcinoma in human. The use of primary human hepatocyte as a viral host is restrained with the scarcity of tissue supply. A culture model restricted to HCV genotype 2a (JFH-1) has been established using Huh7-derived hepatocyte. Other genotypes including the wild-type virus could not propagate in Huh7, Huh7.5 and Huh7.5.1 cells.
  
  
  METHODS
  Functional hepatocyte-like cells (HLCs) were developed from normal human iPS cells as a host for HCV infection. Mature HLCs were identified for selective hepatocyte markers, CYP450s, HCV associated receptors and HCV essential host factors. HLCs were either transfected with JFH-1 HCV RNA or infected with HCV particles derived from patient serum. The enhancing effect of α-tocopherol and the inhibitory effects of INF-α, ribavirin and sofosbuvir to HCV infection were studied. The HCV viral load and HCV RNA were assayed for the infection efficiency.
  
  
  RESULTS
  The fully-developed HLCs expressed phase I, II, and III drug-metabolizing enzymes, HCV associated receptors (claudin-1, occludin, CD81, ApoE, ApoB, LDL-R) and HCV essential host factors (miR-122 and SEC14L2) comparable to the primary human hepatocyte. SEC14L2, an α-tocopherol transfer protein, was expressed in HLCs, but not in Huh7 cell, had been implicated in effective HCVser infection. The HLCs permitted not only the replication of HCV RNA, but also the production of HCV particles (HCVcc) released to the culture media. HLCs drove higher propagation of HCVcc derived from JFH-1 than did the classical host Huh7 cells. HLCs infected with either JFH-1 or wild-type HCV expressed HCV core antigen, NS5A, NS5B, NS3 and HCV negative-stand RNA. HLCs allowed entire HCV life cycle derived from either JFH-1, HCVcc or wild-type HCV (genotype 1a, 1b, 3a, 3b, 6f and 6n). Further increasing the HCVser infection in HLCs was achieved by incubating cell with α-tocopherol. The supernatant from infected HLCs could infect both naïve HLC and Huh7 cell. Treating infected HLC with INF-α and ribavirin decreased HCV RNA in both the cellular fraction and the culture medium. The HLCs reacted to HCVcc or wild-type HCV infection by upregulating TNF-α, IL-28B and IL-29.
  
  
  CONCLUSIONS
  This robust cell culture model for serum-derived HCV using HLCs as host cells provides a remarkable system for investigating HCV life cycle, HCV-associated hepatocellular carcinoma development and the screening for new anti HCV drugs.
authors.affiliation: Department of Biochemistry, Faculty of Pharmacy, Mahidol University, Bangkok, 10400, Thailand.
authors.forename: Khanit
authors.initials: K
authors.lastname: Sa-Ngiamsuntorn
authors.affiliation: Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Wanglang Road, Bangkoknoi, Bangkok, 10700, Thailand. adisak.won@mahidol.ac.th.
authors.forename: Adisak
authors.initials: A
authors.lastname: Wongkajornsilp
authors.affiliation: Stem Cell Research Group, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, 73170, Thailand.
authors.forename: Phetcharat
authors.initials: P
authors.lastname: Phanthong
authors.affiliation: Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand.
authors.forename: Suparerk
authors.initials: S
authors.lastname: Borwornpinyo
authors.affiliation: Stem Cell Research Group, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, 73170, Thailand.
authors.forename: Narisorn
authors.initials: N
authors.lastname: Kitiyanant
authors.affiliation: Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand.
authors.forename: Wasun
authors.initials: W
authors.lastname: Chantratita
authors.affiliation: Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, 270 Rama VI Road, Ratchatewi, Bangkok, 10400, Thailand. suradej.hon@mahidol.ac.th.
authors.forename: Suradej
authors.initials: S
authors.lastname: Hongeng
mesh_terms: D000328:Adult; D002454:Cell Differentiation; D016174:Hepacivirus / Q000254:growth & development*; D006526:Hepatitis C / Q000473:pathology / Q000821:virology; D022781:Hepatocytes / Q000821:virology*; D054884:Host-Pathogen Interactions; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000821:virology*; D008954:Models, Biological; D014776:Virus Cultivation / Q000379:methods*
keywords: Cell culture model; Cytochrome P450; Differentiation; Drug metabolism; HCV; Hepatitis C virus; Hepatocyte; Hepatocyte-like cell; Induced pluripotent stem cell; JFH-1; iPSC