Oxygen drives hepatocyte differentiation and phenotype stability in liver cell lines

Martien Van Wenum,Ruurdtje Hoekstra,Erik J Hendriks,Robert A F M Chamuleau,Thomas M Van Gulik,Perry D Moerland,Vincent A Van Der Mark,Aziza A A Adam,Ronald P Oude Elferink,Jung-Chin Chang,Manon E Wildenberg,Aldo Jongejan

doi:10.1007/s12079-018-0456-4

Martien Van Wenum, Ruurdtje Hoekstra + Show 10 more

Open Access

https://doi.org/10.1007/s12079-018-0456-4

Copy DOI

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 CEBPα, hepatic progenitor marker SOX9, and hypoxia inducible factor (HIF)1α. 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 HIF1α was strongly positive in hypoxic cultures, variable in normoxic cultures and predominantly negative in hyperoxic cultures. Conversely, albumin and CEBPα 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 HIF1α.

Highlights

There is a need for terminally differentiated hepatocytes that can be maintained in vitro
HepaRG cells were cultured under Normoxia (21% ambient O2), Hypoxia (5% ambient O2) or Hyperoxia (40% ambient O2)
In this study we show that oxygen has a significant effect on the differentiation state of HepaRG cells; hypoxia promotes stem cell characteristics with increased cell line stability, whereas hyperoxia induces hepatic differentiation

Summary

Introduction

There is a need for terminally differentiated hepatocytes that can be maintained in vitro. Data on the effects of atmospheric hyperoxia on cultured primary hepatocytes are contradicting, some reporting improvement (Kidambi et al 2009; Poyck et al 2008; Buck et al 2014) and other deterioration (Lillegard et al 2011), of hepatic functions. This may be explained by differences in experimental set-up leading to a difference in oxygen flux at equal starting concentrations, as well as the use of primary hepatocytes, which display biological variability and enter a condition of stress and dedifferentiation after harvesting, leading to significant batch-to-batch variation (Meyer et al 2013)

Methods

Results

Conclusion