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 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.Electronic supplementary materialThe online version of this article (10.1007/s12079-018-0456-4) contains supplementary material, which is available to authorized users.

A 3D Cell Culture Organ-on-a-Chip Platform With a Breathable Hemoglobin Analogue Augments and Extends Primary Human Hepatocyte Functions in vitro

“…Hyperoxia (40% atmospheric oxygen) significantly elevated common markers of liver function and CYP3A4 activity, while hypoxia (5% atmospheric oxygen) kept the cells in a proliferative state and inhibited differentiation. Similar effects were observed with HepG2 cells (van Wenum et al, 2018). Therefore, a plausible explanation for the increase in respiratory metabolism in differentiated HepaRG cells, and HepG2 and BT-474 cell lines cultured in PerfusionPal is a metabolic switch toward OXPHOS that yields 15-fold higher net ATP than glycolysis.…”

“…Total cellular respiration in cell lines (HepG2 and BT-474) and in a terminally differentiated hepatocyte line (HepaRG) confirmed that 3D cultures were metabolically more active when Blood Substitute and perfusion were introduced, and in the case of the HepG2 cell line, when Blood Substitute was present even without perfusion ( Figure 5A). Previous studies have shown that oxygen contributes to differentiation and phenotypic stability in HepaRG cells (van Wenum et al, 2018). Hyperoxia (40% atmospheric oxygen) significantly elevated common markers of liver function and CYP3A4 activity, while hypoxia (5% atmospheric oxygen) kept the cells in a proliferative state and inhibited differentiation.…”

A practice-changing culture method relying on shaking substantially increases mitochondrial energy metabolism and functionality of human liver cell lines

“…Importantly, unlike DMF by simple shaking applied in our study, in all above mentioned DMF platforms, an internal oxygenation system was crucial for the viability and functionality of the hepatocytes in agreement with the assumption that improved oxygenation under shaking, stimulates mitochondrial biogenesis and thereby hepatic differentiation. Consistent with this, we recently established that oxygenation with 40%O 2 instead of 20%O 2 increased mitochondrial biogenesis, and hepatic differentiation of HepaRG cells Static-monolayers, however not to the level of DMF-culturing [ 11 , 39 ], suggesting a shared underlying mechanism, relying, at least in part, on improved oxygenation, however this requires further investigations.…”

Functional polarization of human hepatoma HepaRG cells in response to forskolin

“…Immunocytochemistry analyses were performed as previously described 11 . Briefly, wild-type, F5 or PXR-KO HepaRG cells were plated on 8-well chambered glass Lab-TEK™ systems (Thermo Fisher Scientific).…”

“…Alternatives to the use of DMSO for obtaining polarized differentiated HepaRG cells are therefore interesting to identify and characterize. In this context, culturing HepaRG cells in a three-dimensional environment or under hyperoxic conditions may help to generate polarized HepaRG cells displaying substantial hepatic functions without, or with reduced concentrations, of DMSO 10 , 11 . Similarly, overexpression of CAR, a nuclear receptor controlling various drug metabolism genes and acting as a key regulator for the hepatic differentiation and maturation of human embryonic stem cells (hESCs) 12 , has been recently shown to enhance the differentiation of HepaRG cells, in the absence of DMSO, creating thus a physiologically relevant environment for studies on hepatic drug metabolism 13 .…”