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Oxygen drives hepatocyte differentiation and phenotype stability in liver cell lines.

ABSTRACT: The in vitro generation of terminally differentiated hepatocytes is an unmet need. We investigated the contribution of oxygen concentration to differentiation in human liver cell lines HepaRG and C3A. HepaRG cells were cultured under hypoxia (5%O2), normoxia (21%O2) or hyperoxia (40%O2). Cultures were analysed for hepatic functions, gene transcript levels, and protein expression of albumin, hepatic transcription factor CEBP?, hepatic progenitor marker SOX9, and hypoxia inducible factor (HIF)1?. C3A cells were analysed after exposure to normoxia or hyperoxia. In hyperoxic HepaRG cultures, urea cycle activity, bile acid synthesis, CytochromeP450 3A4 (CYP3A4) activity and ammonia elimination were 165-266% increased. These effects were reproduced in C3A cells. Whole transcriptome analysis of HepaRG cells revealed that 240 (of 23.223) probes were differentially expressed under hyperoxia, with an overrepresentation of genes involved in hepatic differentiation, metabolism and extracellular signalling. Under hypoxia, CYP3A4 activity and ammonia elimination were inhibited almost completely and 5/5 tested hepatic genes and 2/3 tested hepatic transcription factor genes were downregulated. Protein expression of SOX9 and HIF1? was strongly positive in hypoxic cultures, variable in normoxic cultures and predominantly negative in hyperoxic cultures. Conversely, albumin and CEBP? expression were highest in hyperoxic cultures. HepaRG cells that were serially passaged under hypoxia maintained their capacity to differentiate under normoxia, in contrast to cells passaged under normoxia. Hyperoxia increases hepatocyte differentiation in HepaRG and C3A cells. In contrast, hypoxia maintains stem cell characteristics and inhibits hepatic differentiation of HepaRG cells, possibly through the activity of HIF1?.

SUBMITTER: van Wenum M

PROVIDER: S-EPMC6039343 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Oxygen drives hepatocyte differentiation and phenotype stability in liver cell lines.

van Wenum Martien M Adam Aziza A A AAA van der Mark Vincent A VA Chang Jung-Chin JC Wildenberg Manon E ME Hendriks Erik J EJ Jongejan Aldo A Moerland Perry D PD van Gulik Thomas M TM Oude Elferink Ronald P RP Chamuleau Robert A F M RAFM Hoekstra Ruurdtje R

Journal of cell communication and signaling 20180204 3

The in vitro generation of terminally differentiated hepatocytes is an unmet need. We investigated the contribution of oxygen concentration to differentiation in human liver cell lines HepaRG and C3A. HepaRG cells were cultured under hypoxia (5%O2), normoxia (21%O2) or hyperoxia (40%O2). Cultures were analysed for hepatic functions, gene transcript levels, and protein expression of albumin, hepatic transcription factor CEBPα, hepatic progenitor marker SOX9, and h ...[more]

PMID: 29399736

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Project description:Mesenchymal stem cells (MSC) and induced pluripotent stem cells (iPSC) have been reported to be able to differentiate to hepatocyte in vitro with varying degree of hepatocyte maturation. A simple method to decellularize liver scaffold has been established by the Department of Histology, Faculty of Medicine, Universitas Indonesia, in SCTE IMERI lab.15 This study aims to evaluate hepatocyte differentiation from iPSCs compared to MSCs derived in our decellularized liver scaffold. The research stages started with iPSC culture, decellularization, seeding cell culture into the scaffold, and differentiation into hepatocytes for 21 days. Hepatocyte differentiation from iPSCs and MSCs in the scaffolds was characterized using hematoxylin-eosin, Masson Trichrome, and immunohistochemistry staining to determine the fraction of the differentiation area. RNA samples were isolated on days 7 and 21. Expression of albumin, CYP450, and CK-19 genes were analyzed using the qRT-PCR method. Electron microscopy images were obtained by SEM. Immunofluorescence examination was done using HNF4-α and CEBPA markers. The results of this study in hepatocyte-differentiated iPSCs compared with hepatocyte-differentiated MSCs in decellularized liver scaffold showed lower adhesion capacity, single-cell-formation and adhered less abundant, decreased trends of albumin, and lower CYP450 expression. Several factors contribute to this result: lower initial seeding number, which causes only a few iPSCs to attach to certain parts of decellularized liver scaffold, and manual syringe injection for recellularization, which abruptly and unevenly creates pattern of single-cell-formation by hepatocyte-differentiated iPSC in the scaffold. Hepatocyte-differentiated MSCs have the advantage of higher adhesion capacity to collagen fiber decellularized liver scaffold. This leads to positive result: increase trends of albumin and higher CYP450 expression. Hepatocyte maturation is shown by diminishing CK-19, which is more prominent in hepatocyte-differentiated iPSCs in decellularized liver scaffold. Confirmation of mature hepatocyte-differentiated iPSCs in decellularized liver scaffold maturation is positive for HNF4-a and CEBPA. The conclusion of this study is hepatocyte-differentiated iPSCs in decellularized liver scaffold is mature with lower cell-ECM adhesion, spatial cell distribution, albumin, and CYP450 expression than hepatocyte-differentiated MSCs in decellularized liver scaffold.

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Oxygen drives hepatocyte differentiation and phenotype stability in liver cell lines.

Publications

Oxygen drives hepatocyte differentiation and phenotype stability in liver cell lines.

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