Transcriptional and Epigenetic Consequences of DMSO Treatment on HepaRG Cells

Abstract

Dimethyl sulfoxide (DMSO) is used to sustain or favor hepatocyte differentiation in vitro. Thus, DMSO is used in the differentiation protocol of the HepaRG cells that present the closest drug-metabolizing enzyme activities to primary human hepatocytes in culture. The aim of our study is to clarify its influence on liver-specific gene expression. For that purpose, we performed a large-scale analysis (gene expression and histone modification) to determine the global role of DMSO exposure during the differentiation process of the HepaRG cells. The addition of DMSO drives the upregulation of genes mainly regulated by PXR and PPARα whereas genes not affected by this addition are regulated by HNF1α, HNF4α, and PPARα. DMSO-differentiated-HepaRG cells show a differential expression for genes regulated by histone acetylation, while differentiated-HepaRG cells without DMSO show gene signatures associated with histone deacetylases. In addition, we observed an interplay between cytoskeleton organization and EMC remodeling with hepatocyte maturation.

Liposome-Mediated Gene Transfer in Differentiated HepaRG™ Cells: Expression of Liver Specific Functions and Application to the Cytochrome P450 2D6 Expression

“…There is a large body of evidence demonstrating that DMSO promotes the expression of liver specific functions in primary hepatocytes [ 40 ] and in HepaRG™ cells [ 12 , 15 , 17 , 41 ]. Interestingly, we have recently shown that DMSO inhibited proliferation of differentiated HepaRG™ cells and that removal of DMSO from the culture medium triggered a moderate but regular proliferation of differentiated HepaRG™ cells correlating with a partial decrease in the expression levels of liver specific functions [ 41 ]. These data demonstrated that differentiated HepaRG™ keep their potential of proliferation even when maintained confluent without detachment of the culture dish leading to a significant cell expansion over several days after DMSO removal [ 41 ].…”

“…Interestingly, we have recently shown that DMSO inhibited proliferation of differentiated HepaRG™ cells and that removal of DMSO from the culture medium triggered a moderate but regular proliferation of differentiated HepaRG™ cells correlating with a partial decrease in the expression levels of liver specific functions [ 41 ]. These data demonstrated that differentiated HepaRG™ keep their potential of proliferation even when maintained confluent without detachment of the culture dish leading to a significant cell expansion over several days after DMSO removal [ 41 ]. We thought that the potential of differentiated HepaRG™ cells to proliferate could be used to improve the gene delivery.…”

“…In order to determine whether a strong proliferation of differentiated HepaRG™ cells can be obtained by mitogenic stimulation, we prepared 3 media containing various combinations of cytokines and growth factors and evaluated the proliferation of HepaRG™ cells. These cells were first cultured at day 1 in medium without DMSO (MIL700 + ADD710) in order to trigger the cell cycle priming [ 41 ] ( Figure 3 B). Then, the “primed” cells were cultured at day 2 in the mitogenic media, switched back to the medium without mitogenic cocktail at day 3 and DMSO-containing medium at day 4 ( Figure 3 B) to maintain the differentiation status.…”

“…As previously observed ( Figure 3 A), in HepaRG™ cells maintained in DMSO-containing medium, transfection efficiency remained low with less than 10% of GFP+ cells regardless of the charge ratio. In cells cultured for 3 days in absence of DMSO, the transfection efficiency was significantly higher than that of cells maintained in DMSO-containing medium, possibly because DMSO-removal induced a moderate cell proliferation [ 41 ]. In this culture condition, the number of GFP+ cells increased with higher charge ratio with up to 25% of transfected cells at R = 2.…”

Physiomimetic In Vitro Human Models for Viral Infection in the Liver

“…DMSO (184,185) may improve both the infectability and support maintenance of differentiation of more sophisticated in vitro liver platforms.…”