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Human induced pluripotent stem cell-derived hepatic cell lines as a new model for host interaction with hepatitis B virus.

ABSTRACT: Hepatitis B virus (HBV) is not eradicated by current antiviral therapies due to persistence of HBV covalently closed circular DNA (cccDNA) in host cells, and thus development of novel culture models for productive HBV infection is urgently needed, which will allow the study of HBV cccDNA eradication. To meet this need, we developed culture models of HBV infection using human induced pluripotent stem cell-derived hepatocyte lineages, including immature proliferating hepatic progenitor-like cell lines (iPS-HPCs) and differentiated hepatocyte-like cells (iPS-Heps). These cells were susceptible to HBV infection, produced HBV particles, and maintained innate immune responses. The infection efficiency of HBV in iPS-HPCs predominantly depended on the expression levels of sodium taurocholate cotransporting polypeptide (NTCP), and was low relative to iPS-Heps: however, long-term culture of iPS-Heps was difficult. To provide a model for HBV persistence, iPS-HPCs overexpressing NTCP were established. The long-term persistence of HBV cccDNA was detected in iPS-HPCs overexpressing NTCP, and depended on the inhibition of the Janus-kinase signaling pathway. In conclusion, this study provides evidence that iPS-derived hepatic cell lines can be utilized for novel HBV culture models with genetic variation to investigate the interactions between HBV and host cells and the development of anti-HBV strategies.

SUBMITTER: Kaneko S

PROVIDER: S-EPMC4937433 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Human induced pluripotent stem cell-derived hepatic cell lines as a new model for host interaction with hepatitis B virus.

Kaneko Shun S Kakinuma Sei S Asahina Yasuhiro Y Kamiya Akihide A Miyoshi Masato M Tsunoda Tomoyuki T Nitta Sayuri S Asano Yu Y Nagata Hiroko H Otani Satoshi S Kawai-Kitahata Fukiko F Murakawa Miyako M Itsui Yasuhiro Y Nakagawa Mina M Azuma Seishin S Nakauchi Hiromitsu H Nishitsuji Hironori H Ujino Saneyuki S Shimotohno Kunitada K Iwamoto Masashi M Watashi Koichi K Wakita Takaji T Watanabe Mamoru M

Scientific reports 20160708

Hepatitis B virus (HBV) is not eradicated by current antiviral therapies due to persistence of HBV covalently closed circular DNA (cccDNA) in host cells, and thus development of novel culture models for productive HBV infection is urgently needed, which will allow the study of HBV cccDNA eradication. To meet this need, we developed culture models of HBV infection using human induced pluripotent stem cell-derived hepatocyte lineages, including immature proliferating hepatic progenitor-like cell l ...[more]

PMID: 27386799

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Project description:BackgroundThe use of a personalized liver organoid derived from human-induced pluripotent stem cells (HuiPSCs) is advancing the use of in vitro disease models for the design of specific, effective therapies for individuals. Collecting patient peripheral blood cells for HuiPSC generation is preferable because it is less invasive; however, the capability of blood cell-derived HuiPSCs for hepatic differentiation and liver organoid formation remains uncertain. Moreover, the currently available methods for liver organoid formation require a multistep process of cell differentiation or a combination of hepatic endodermal, endothelial and mesenchymal cells, which is a major hurdle for the application of personalized liver organoids in high-throughput testing of drug toxicity and safety. To demonstrate the capability of blood cell-derived HuiPSCs for liver organoid formation without support from endothelial and mesenchymal cells.MethodsThe peripheral blood-derived HuiPSCs first differentiated into hepatic endoderm (HE) in two-dimensional (2D) culture on Matrigel-coated plates under hypoxia for 10 days. The HE was then collected and cultured in 3D culture using 50% Matrigel under ambient oxygen. The maturation of hepatocytes was further induced by adding hepatocyte growth medium containing HGF and oncostatin M on top of the 3D culture and incubating the culture for an additional 12-17 days. The function of the liver organoids was assessed using expression analysis of hepatocyte-specific gene and proteins. Albumin (ALB) synthesis, glycogen and lipid storage, and metabolism of indocyanine were evaluated. The spatial distribution of albumin was examined using immunofluorescence and confocal microscopy.ResultsCD34+ hematopoietic cell-derived HuiPSCs were capable of differentiating into definitive endoderm expressing SOX17 and FOXA2, hepatic endoderm expressing FOXA2, hepatoblasts expressing AFP and hepatocytes expressing ALB. On day 25 of the 2D culture, cells expressed SOX17, FOXA2, AFP and ALB, indicating the presence of cellular heterogeneity. In contrast, the hepatic endoderm spontaneously formed a spherical, hollow structure in a 3D culture of 50% Matrigel, whereas hepatoblasts and hepatocytes could not form. Microscopic observation showed a single layer of polygonal-shaped cells arranged in a 3D structure. The hepatic endoderm-derived organoid synthesis ALB at a higher level than the 2D culture but did not express definitive endoderm-specific SOX17, indicating the greater maturity of the hepatocytes in the liver organoids. Confocal microscopic images and quantitative ELISA confirmed albumin synthesis in the cytoplasm of the liver organoid and its secretion. Overall, 3D culture of the hepatic endoderm is a relatively fast, simple, and less laborious way to generate liver organoids from HuiPSCs that is more physiologically relevant than 2D culture.

Project description:Progression of fibrosis and the development of cirrhosis are responsible for the liver related morbidity and mortality associated with chronic liver diseases. There is currently a great unmet need for effective anti-fibrotic strategies. Stem cells play a central role in wound healing responses to restore liver homeostasis following injury. Here we tested the hypothesis that extracellular vesicles (EVs) isolated from induced pluripotent stem cells (iPSC) modulate hepatic stellate cell (HSCs) activation and may have anti-fibrotic effects. Human iPSCs were generated by reprogramming primary skin fibroblasts. EVs were isolated by differential centrifugation, quantified by flow cytometry (FACS) and characterized by dynamic light scattering (DLS) and electron microscopy (TEM). Primary human HSCs were activated with TGFβ (10 ng/mL) and exposed to iPSC-EVs. Efficacy of iPSC-EVs was tested on HSC in vitro and in two murine models of liver injury (CCl4 and bile duct ligation). Characterization of iPSC-derived EVs by flow cytometry identified a large population of EVs released by iPSC, primarily with a diameter of 300 nm and that could be visualized by TEM as round, cup-shaped objects. Fluorescent tracing assays detected iPSC-EVs in HSC cytosol after a short incubation and EV uptake by HSCs resulted in both decrease of pro-fibrogenic markers αSMA, CollagenIα1, Fibronectin and TIMP-1 and HSC pro-fibrogenic responses such as chemotaxis and proliferation. Genomics analyses of iPSC-EV miRNA cargo revealed 22 highly expressed miRNAs, among which miR-92a-3p resulted the most abundant. Transcriptome analysis identified 60 genes down-modulated and 235 up-regulated in TGF-β-primed HSC in presence or absence of iPSC-EVs. Intravenous injection of iPSC-EVs in CCl4 and bile duct ligation-induced liver fibrosis resulted in anti-fibrotic effects at protein and gene levels. Results of this study identify iPSC-EVs as a novel anti-fibrotic approach that may reduce or reverse liver fibrosis in patients with chronic liver disease.

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Human induced pluripotent stem cell-derived hepatic cell lines as a new model for host interaction with hepatitis B virus.

Publications

Human induced pluripotent stem cell-derived hepatic cell lines as a new model for host interaction with hepatitis B virus.

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