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

BackgroundHepatitis 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.MethodsFunctional 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.ResultsThe 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0519-1) contains supplementary material, which is available to authorized users.

Microarray Analysis Reveals a Potential Role of lncRNA Expression in 3,4-Benzopyrene/Angiotensin II-Activated Macrophage in Abdominal Aortic Aneurysm

“…SEC14L2, an α -tocopherol transfer protein (Ttpa), was expressed in hepatocyte-like cells (HLCs), starting from human mesenchymal stem cells (hMSC) through induced pluripotent stem (iPS) cell reprogramming. Sa-Ngiamsuntorn et al indicated that, together with the expression of SEC14L2 and the addition of α -tocopherol, the expressions of inflammatory cytokines were upregulated during the infection that mimicked the inflammatory process [ 36 ].…”

Current progress in hepatic tissue regeneration by tissue engineering

“…The basis of this protocol was the spontaneous formation of cell aggregates (embryoid bodies) from pluripotent cells which has the capacity to give rise to all three germ layers [16]. To expand the hepatocytes, the most well-organized method is a monolayer culture of distinct cells in a step-wise manner exposure to specific cocktails of recombinant growth factors and cytokines, including Activin A, Wnt3a, HGF, OSM, FGF4, VEGF, EGF and BMP4 [184][185][186][187][188][189][190][191]. These approaches were designed to simulate the embryonic stages of hepatocyte lineage.…”

Engineered Livers for Infectious Diseases

“…Several groups have demonstrated that induced pluripotent stem cells–derived HLCs (iHLCs) express HCV and HBV entry receptors, and support productive viral infection with HCV or HBV particles, 41 , 47 , 48 , 49 Viral load in iHLCs is higher than in hepatoma cell lines, 47 and antiviral innate immune response is readily detectable on HCV or HBV infection, with increased expression of various type I interferon-stimulated genes similar to that observed in primary human hepatocytes in MPCCs. 39 , 41 Furthermore, infected iHLCs can be used for drug screening purposes because they respond to anti-HCV and -HBV drugs.…”

“… 39 , 41 Furthermore, infected iHLCs can be used for drug screening purposes because they respond to anti-HCV and -HBV drugs. 39 , 41 , 47 …”