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Stable lentiviral-mediated expression of Cytochrome P450 2D6 in HepaRG cells: New means for in vitro assessment of xenobiotic biotransformation and cytotoxicity.

ABSTRACT: The human hepatic HepaRG cell line is often considered as the closest surrogate to primary culture of normal human hepatocytes (PHH) for toxicity studies. However, differentiated HepaRGcells express very low levels of the cytochrome P450 2D6 (CYP2D6) protein, which is essential for the biotransformation of nearly 25% of drugs on the market. To overcome this limitation, infection of progenitor HepaRG cells were performed using lentiviral particles containing a transgene encoding a single mRNA translated into a polypeptide undergoing proteolytic cleavage via the T2A peptide to produce both human CYP2D6 and GFP proteins. Differentiated HepaRG cells transduced with this lentivirus stably expressed GFP and catalytically active CYP2D6 enzyme at levels close to those found in PHH with high CYP2D6 activities. Using the CYP2D6 transgenic HepaRG cells, we showed that tramadol was metabolized into both, N- and O-desmethyl tramadol as observed in human serum in contrast with the production of N-desmethyl tramadol only in parental HepaRG cells via the CYP3A4 activity. Similarly, after perhexiline (PHX) treatments, higher IC50 were found in CYP2D6 expressing HepaRG cells associated to lower mitochondrial damages compared to those found in parental cells for the same PHX concentrations. Gene profiling between parental and transgenic cells demonstrated that the CYP2D6 expressing HepaRG cells had kept their ability to proliferate and differentiate with low impact on the expression of the hepatocyte specific functions, however, we identified a limited set of genes including NXF3 and TRIM63 that were up-regulated in CYP2D6 expressing HepaRG cells. By knocking-down GFP and CYP2D6 expression using CRISP/Cas9 technology in the transgenic cell line, we demonstrated that NXF3 and TRIM63 induction was triggered by the mRNA encoding GFP and CYP2D6 but not by the lentiviral transgene inserted into the HepaRG cell genome. Together, these data confirmed that the CYP2D6 transgenic HepaRG cells represent a suitable optimized transgenic model of HepaRG cells to evaluate biotransformation and toxicity of specific compounds metabolized by CYP2D6.

ORGANISM(S): Homo sapiens

PROVIDER: GSE297830 | GEO | 2025/09/30

REPOSITORIES: GEO

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Project description:Primary hepatocytes are widely utilized for investigating drug efficacy and toxicity, yet variations between batches and limited proliferation capacity present significant challenges. HepaRG cells are versatile cells, capable of maintaining an undifferentiated state and differentiating through dimethyl sulfoxide treatment, allowing for molecular analysis of hepatocyte plasticity. To elucidate the underlying molecular mechanisms of HepaRG cell plasticity, we used CYP3A4G/7R HepaRG cells engineered to express DsRed under the control of the fetus-specific CYP3A7 gene and EGFP under the adult-specific CYP3A4 gene promoter. In time-lapse imaging of CYP3A4G/7R HepaRG cells, we observed CYP3A7-DsRed expression transitioning from negative to positive during proliferation period and CYP3A4-GFP expression activating during differentiation. The de-differentiation potency of differentiated CYP3A4G/7R HepaRG cells was assessed using inhibitors and cytokines. It was found that Y-27632 (Y), A-83-01 (A), and CHIR99021 (C) (collectively referred to as YAC), which are known to promote liver regeneration in mice, did not induce CYP3A7-DsRed expression. Instead, these inhibitors increased CYP3A4-GFP expressing population. Furthermore, CHIR99021 alone increased CYP3A4-GFP-positive cells, while Wnt3a treatment increased CYP3A7-DsRed-positive cells, suggesting that Wnt signaling plays distinct roles in HepaRG cells. It was apparent that de-differentiated cells had increased CYP3A4 activity after a second round of differentiation, compared to differentiated cells after the first round. Transcriptomic analysis of HepaRG cells revealed distinct profiles between proliferative, differentiated, and de-differentiated states, highlighting their robust plasticity. Notably, hepatoblastic cells de-differentiated by YAC or C displayed transcriptome patterns similar to undifferentiated cells, whereas CYP3A7-DsRed and CYP3A4-GFP exhibited expression patterns different from those of undifferentiated cells. These findings underscore the dynamic nature of HepaRG cells while cautioning against solely relying on CYP3 family gene expression as a marker of differentiation.

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Stable lentiviral-mediated expression of Cytochrome P450 2D6 in HepaRG cells: New means for in vitro assessment of xenobiotic biotransformation and cytotoxicity.

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