Long-term maintenance of human fetal hepatocytes and prolonged susceptibility to HBV infection by co-culture with non-parenchymal cells

Zhou, Ming; Zhao, Fei; Li, Jiafu; Cheng, Zhikui; Tian, Xin; Zhi, Xiaoguang; Huang, Yayun; Hu, Kanghong

doi:10.1016/j.jviromet.2013.10.010

Cited by 24 publications

(31 citation statements)

References 46 publications

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“…17 ), with none successful in microscale formats. Fetal human hepatocytes (FHHs) and human hepatocyte-like cells (HLCs) derived from induced pluripotent stem cells (iPSCs) have also been shown to be susceptible to HBV infection, but neither exhibits the fully mature adult PHH phenotype 14, 16, 41, 42 , limiting their utility for antiviral drug screening. Furthermore, while HLCs can be produced in unlimited quantities through directed differentiation of iPSCs, few FHHs can be isolated from a single donor, increasing inter-experimental variability.…”

Section: Discussionmentioning

confidence: 99%

“…For in vitro experiments, primary hepatocyte cultures are thus more desirable 10 . Previous work has indeed shown that primary human hepatocytes (PHHs) of both adult and fetal origin can be infected with HBV 11–16 . However, long-term infections of PHHs with HBV or other hepatotropic pathogens, such as hepatitis C virus (HCV) or parasites that cause malaria in humans, have been notoriously difficult due to their rapid dedifferentiation and loss of characteristic hepatic functions following isolation and plating.…”

Section: Introductionmentioning

confidence: 99%

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Long-term hepatitis B infection in a scalable hepatic co-culture system

et al. 2017

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Hepatitis B virus causes chronic infections in 250 million people worldwide. Chronic hepatitis B virus carriers are at risk of developing fibrosis, cirrhosis, and hepatocellular carcinoma. A prophylactic vaccine exists and currently available antivirals can suppress but rarely cure chronic infections. The study of hepatitis B virus and development of curative antivirals are hampered by a scarcity of models that mimic infection in a physiologically relevant, cellular context. Here, we show that cell-culture and patient-derived hepatitis B virus can establish persistent infection for over 30 days in a self-assembling, primary hepatocyte co-culture system. Importantly, infection can be established without antiviral immune suppression, and susceptibility is not donor dependent. The platform is scalable to microwell formats, and we provide proof-of-concept for its use in testing entry inhibitors and antiviral compounds.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Long-term hepatitis B infection in a scalable hepatic co-culture system

et al. 2017

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“…Western blot was performed as previously described (Zhou et al, 2014b). The primary antibodies used were rabbit anti-human hNTCP polyclonal antibody (Sigma: HPA042727) and mouse anti-human GAPDH monoclonal antibody (Proteintech TM , Chicago, USA).…”

Section: Western Blotmentioning

confidence: 99%

“…An immunofluorescence assay was performed as described previously (Zhou et al, 2014b). The primary antibodies were rabbit anti-HBcAg polyclonal antibody (Dako, Glostrup, Denmark), mouse anti-HBsAg monoclonal antibody (Cao L. et al, 2014), mouse anti-CD81 antibody (Santa Cruz, California, USA), and rabbit antihNTCP polyclonal antibody (Sigma: HPA042727).…”

Section: Immunofluorescence Assaymentioning

confidence: 99%

Productive HBV infection of well-differentiated, hNTCP-expressing human hepatoma-derived (Huh7) cells

et al. 2017

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Feasible and effective cell models for hepatitis B virus (HBV) infection are required for investigating the complete lifecycle of this virus, including the early steps of viral entry. Resistance to dimethyl sulfoxide/polyethylene glycol (DMSO/PEG), hNTCP expression, and a differentiated state are the limiting factors for successful HBV infection models. In the present study, we used a hepatoma cell line (Huh7D hNTCP ) to overcome these limiting factors so that it exhibits excellent susceptibility to HBV infection. To achieve this goal, different hepatoma cell lines were tested with 2.5% DMSO / 4% PEG8000, and one resistant cell line (Huh7D) was used to construct a stable hNTCP-expressing cell line (Huh7D hNTCP ) using a recombinant lentivirus system. Then, the morphological characteristics and differentiation molecular markers of Huh7D hNTCP cells with or without DMSO treatment were characterized. Finally, the susceptibility of Huh7D hNTCP cells to HBV infection was assessed. Our results showed that Huh7D cells were resistant to 2.5% DMSO / 4% PEG8000, whereas the others were not. Huh7D hNTCP cells were established to express a high level of hNTCP compared to liver extracts, and Huh7D hNTCP cells rapidly transformed into a non-dividing, well-differentiated polarized phenotype under DMSO treatment. Huh7D hNTCP cells fully supported the entire lifecycle of HBV infection. This cell culture system will be useful for the analysis of host-virus interactions, which should facilitate the discovery of antiviral drugs and vaccines.

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“…Further, spherical hepatocyte masses, initially cultured in the presence of preattached stellate cells, efficiently replicated HCV clones of gt1a, gt1b, and gt2a (150). Similarly, long-term cultures of primary human fetal hepatocytes were developed by plating a hepatic cell mixture at low density, allowing outgrowth of nonparenchymal cells and formation of hepatic islands that were subsequently cultured under DMSO-containing conditions to maintain characteristic hepatocytic features; these cultures were susceptible to HBV infection for up to 10 weeks in culture (151). In 3D engineered tissues, Bcl-2-transduced HUVECs have been used to modulate fetal hepatocyte phenotype and permit HCV replication (151a).…”

Section: Hepatotropic Virus Infection In Models With Emergent Propertiesmentioning

confidence: 99%

New Methods in Tissue Engineering: Improved Models for Viral Infection

et al. 2014

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New insights in the study of virus and host biology in the context of viral infection are made possible by the development of model systems that faithfully recapitulate the in vivo viral life cycle. Standard tissue culture models lack critical emergent properties driven by cellular organization and in vivo–like function, whereas animal models suffer from limited susceptibility to relevant human viruses and make it difficult to perform detailed molecular manipulation and analysis. Tissue engineering techniques may enable virologists to create infection models that combine the facile manipulation and readouts of tissue culture with the virus-relevant complexity of animal models. Here, we review the state of the art in tissue engineering and describe how tissue engineering techniques may alleviate some common shortcomings of existing models of viral infection, with a particular emphasis on hepatotropic viruses. We then discuss possible future applications of tissue engineering to virology, including current challenges and potential solutions.

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Long-term maintenance of human fetal hepatocytes and prolonged susceptibility to HBV infection by co-culture with non-parenchymal cells

Cited by 24 publications

References 46 publications

Long-term hepatitis B infection in a scalable hepatic co-culture system

Long-term hepatitis B infection in a scalable hepatic co-culture system

Productive HBV infection of well-differentiated, hNTCP-expressing human hepatoma-derived (Huh7) cells

New Methods in Tissue Engineering: Improved Models for Viral Infection

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