In Vitro Ballooned Hepatocytes Can Be Produced By Primary Human Hepatocytes and Hepatic Stellate Cell Sheets

Abstract Background Despite the increasing prevalence of Nonalcoholic steatohepatitis (NASH) worldwide, there is no effective treatment available for this disease. “Ballooned hepatocyte” is a characteristic finding in NASH and is correlated with disease prognosis, but their mechanisms of action are poorly understood; furthermore, neither animal nor in vitro models of NASH have been able to adequately represent ballooned hepatocytes. Herein, we engineered cell sheets to develop a new in vitro model of ballooned hepatocytes. Methods Primary human hepatocytes (PHH) and Hepatic stellate cells (HSC) were co-cultured to produce cell sheets, which were cultured in glucose and lipid containing medium, following which histological and functional analyses were performed. Results Histological findings showed hepatocyte ballooning, accumulation of fat droplets, abnormal cytokeratin arrangement, and the presence of Mallory-Denk bodies and abnormal organelles. These findings are similar to those of ballooned hepatocytes in human NASH. Functional analysis showed elevated levels of TGFβ-1, SHH, and p62, but not TNF-α, IL-8. Conclusions Exposure of PHH/HSC sheets to a glucolipotoxicity environment induces ballooned hepatocyte without inflammation. Moreover, fibrosis is an important mechanism underlying ballooned hepatocytes and could be the basis for the development of a new in vitro NASH model with ballooned hepatocytes.

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Primary Human Hepatocytes as an In Vitro Toxicology System

Advances In Animal Alternatives For Safety And Efficacy Testing ◽

10.1201/9781439805817-47 ◽

1997 ◽

pp. 379-388

Keyword(s):

Human Hepatocytes ◽

In Vitro Toxicology ◽

Primary Human Hepatocytes

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Expression of the Metalloproteinase ADAM8 Is Upregulated in Liver Inflammation Models and Enhances Cytokine Release In Vitro

Mediators of Inflammation ◽

10.1155/2021/6665028 ◽

2021 ◽

Vol 2021 ◽

pp. 1-17

Author(s):

Tanzeela Awan ◽

Aaron Babendreyer ◽

Justyna Wozniak ◽

Abid Mahmood Alvi ◽

Viktor Sterzer ◽

...

Keyword(s):

Cell Lines ◽

Inflammatory Mediators ◽

Liver Tissue ◽

Stellate Cell ◽

Hepatoma Cell ◽

Cell Types ◽

Chronic Liver ◽

Liver Inflammation ◽

Tnf Α

Acute and chronic liver inflammation is driven by cytokine and chemokine release from various cell types in the liver. Here, we report that the induction of inflammatory mediators is associated with a yet undescribed upregulation of the metalloproteinase ADAM8 in different murine hepatitis models. We further show the importance of ADAM8 expression for the production of inflammatory mediators in cultured liver cells. As a model of acute inflammation, we investigated liver tissue from lipopolysaccharide- (LPS-) treated mice in which ADAM8 expression was markedly upregulated compared to control mice. In vitro, stimulation with LPS enhanced ADAM8 expression in murine and human endothelial and hepatoma cell lines as well as in primary murine hepatocytes. The enhanced ADAM8 expression was associated with an upregulation of TNF-α and IL-6 expression and release. Inhibition studies indicate that the cytokine response of hepatoma cells to LPS depends on the activity of ADAM8 and that signalling by TNF-α can contribute to these ADAM8-dependent effects. The role of ADAM8 was further confirmed with primary hepatocytes from ADAM8 knockout mice in which TNF-α and IL-6 induction and release were considerably attenuated. As a model of chronic liver injury, we studied liver tissue from mice undergoing high-fat diet-induced steatohepatitis and again observed upregulation of ADAM8 mRNA expression compared to healthy controls. In vitro, ADAM8 expression was upregulated in hepatoma, endothelial, and stellate cell lines by various mediators of steatohepatitis including fatty acid (linoleic-oleic acid), IL-1β, TNF-α, IFN-γ, and TGF-β. Upregulation of ADAM8 was associated with the induction and release of proinflammatory cytokines (TNF-α and IL-6) and chemokines (CX3CL1). Finally, knockdown of ADAM8 expression in all tested cell types attenuated the release of these mediators. Thus, ADAM8 is upregulated in acute and chronic liver inflammation and is able to promote inflammation by enhancing expression and release of inflammatory mediators.

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Implications of Efficient Hepatic Delivery by Tenofovir Alafenamide (GS-7340) for Hepatitis B Virus Therapy

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00128-15 ◽

2015 ◽

Vol 59 (6) ◽

pp. 3563-3569 ◽

Cited By ~ 72

Author(s):

Eisuke Murakami ◽

Ting Wang ◽

Yeojin Park ◽

Jia Hao ◽

Eve-Irene Lepist ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Human Hepatocytes ◽

Target Tissue ◽

Small Contribution ◽

Primary Human Hepatocytes ◽

B Virus ◽

Tenofovir Alafenamide ◽

Pharmacologically Active

ABSTRACTTenofovir alafenamide (TAF) is a prodrug of tenofovir (TFV) currently in clinical evaluation for treatment for HIV and hepatitis B virus (HBV) infections. Since the target tissue for HBV is the liver, the hepatic delivery and metabolism of TAF in primary human hepatocytesin vitroand in dogsin vivowere evaluated here. Incubation of primary human hepatocytes with TAF resulted in high levels of the pharmacologically active metabolite tenofovir diphosphate (TFV-DP), which persisted in the cell with a half-life of >24 h. In addition to passive permeability, studies of transfected cell lines suggest that the hepatic uptake of TAF is also facilitated by the organic anion-transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3, respectively). In order to inhibit HBV reverse transcriptase, TAF must be converted to the pharmacologically active form, TFV-DP. While cathepsin A is known to be the major enzyme hydrolyzing TAF in cells targeted by HIV, including lymphocytes and macrophages, TAF was primarily hydrolyzed by carboxylesterase 1 (CES1) in primary human hepatocytes, with cathepsin A making a small contribution. Following oral administration of TAF to dogs for 7 days, TAF was rapidly absorbed. The appearance of the major metabolite TFV in plasma was accompanied by a rapid decline in circulating TAF. Consistent with thein vitrodata, high and persistent levels of TFV-DP were observed in dog livers. Notably, higher liver TFV-DP levels were observed after administration of TAF than those given TDF. These results support the clinical testing of once-daily low-dose TAF for the treatment of HBV infection.

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In vitro infection of primary human hepatocytes by HCV-positive sera: insights on a highly relevant model

Gut ◽

10.1136/gutjnl-2013-304623 ◽

2013 ◽

Vol 63 (9) ◽

pp. 1490-1500 ◽

Cited By ~ 14

Author(s):

Claire Gondeau ◽

Philippe Briolotti ◽

Francia Razafy ◽

Cédric Duret ◽

Pierre-Alain Rubbo ◽

...

Keyword(s):

Human Hepatocytes ◽

Relevant Model ◽

Primary Human Hepatocytes

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Long-term functional maintenance of primary human hepatocytes in vitro

Science ◽

10.1126/science.aau7307 ◽

2019 ◽

Vol 364 (6438) ◽

pp. 399-402 ◽

Cited By ~ 36

Author(s):

Chengang Xiang ◽

Yuanyuan Du ◽

Gaofan Meng ◽

Liew Soon Yi ◽

Shicheng Sun ◽

...

Keyword(s):

Cell Biology ◽

Expression Profiles ◽

Antiviral Drug ◽

Gene Expression Profiles ◽

Global Gene Expression ◽

Human Hepatocytes ◽

Hbv Infection ◽

Primary Human Hepatocytes

The maintenance of terminally differentiated cells, especially hepatocytes, in vitro has proven challenging. Here we demonstrated the long-term in vitro maintenance of primary human hepatocytes (PHHs) by modulating cell signaling pathways with a combination of five chemicals (5C). 5C-cultured PHHs showed global gene expression profiles and hepatocyte-specific functions resembling those of freshly isolated counterparts. Furthermore, these cells efficiently recapitulated the entire course of hepatitis B virus (HBV) infection over 4 weeks with the production of infectious viral particles and formation of HBV covalently closed circular DNA. Our study demonstrates that, with a chemical approach, functional maintenance of PHHs supports long-term HBV infection in vitro, providing an efficient platform for investigating HBV cell biology and antiviral drug screening.

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