Project description:Inorganic arsenic, a ubiquitous environmental contaminant of food and drinking water, is a human carcinogen associated with lung, liver, prostate, renal, and bladder cancers. It has been postulated that inorganic arsenic targets stem cells or partially differentiated progenitor cells, causing their oncogenic transformation. This is proposed to be one of the key mechanisms in arsenic-associated carcinogenesis; however, the underlying mechanisms for this process remain largely unknown. To address this question, human liver HepaRG cells, at progenitor and differentiated states, were continuously treated with a non-cytotoxic concentration of 1 μM sodium arsenite (NaAsO2). Briefly, in Experiment 1, three days after the initial seeding, 1 μM NaAsO2 was added to the media and the cells were maintained in the NaAsO2-containing media for an additional 25 days. In Experiment 2, fourteen days after the initial seeding, 1 μM NaAsO2 was added to the media and the cells were maintained in the NaAsO2-containing media for an additional 14 days. In Experiment 1 and Experiment 2, control and NaAsO2-treated cells were harvested on the 28th day after the initial seeding. In Experiment 3, twenty-eight days after the initial seeding, the terminally-differentiated cells were treated continuously with 1 μM NaAsO2 for an additional 14 days and then harvested. Transcriptomic analysis of NaAsO2-treated progenitor-like HepaRG cells (Experiment 1 and Experiment 2) identified 743 and 639 differentially expressed genes, respectively, among which 343 genes were expressed in common. Pathway analysis of common differentially expressed genes demonstrated a substantial inhibition of cellular metabolic pathways, mainly lipid and xenobiotic metabolism, and cell death pathways. In contrast, cell proliferation, cell survival, and inflammation, were substantially activated. Treatment of differentiated HepaRG cells with NaAsO2 (Experiment 3) resulted in prominent gene expression changes, with a total of 1058 transcripts being significantly different from the control HepaRG cells. Pathway analysis of differentially expressed genes in NaAsO2-treated cells differentiated HepaRG cells showed activation of cellular death-associated pathways and inhibition of cell survival and cell proliferation.

Project description:Rifampicin and efavirenz are commonly used medicines to treat tuberculosis (rifampicin) and HIV/AIDS (efavirenz). Rifampicin and efavirenz are metabolized in the liver and exposure to these medicines can alter microRNA expression in hepatocytes. Changes in microRNA expression may affect metabolism of these medicines and potentially influence how patient's respond to therapy. It is important to understand changes in microRNA expression in a hepatic cell-based model. Differentiated HepaRG cells were used because mRNA expression and induction of drug metabolizing enzymes are comparable to that of primary human hepatocytes yet differentiated HepaRG cells have an extended lifespan. Differentiated HepaRG cells were obtained from Merck Millipore and cultured as an adherent cell line at a density of 315000 cells/well using collagen I coated 24-well plates. Clinically relevant concentrations of rifampicin and efavirenz were selected as 6.4uM efavirenz (dissolved in 100% dimethyl sulfoxide) and 24.4uM rifampicin (dissolved in 100% dimethyl sulfoxide), while 0.02% dimethyl sulfoxide was used as control to minimize toxicity. HepaRG serum-free induction medium was used to dilute efavirenz and rifampicin. On day 7 since thawing differentiated HepaRG cells, these cells were treated with 6.4uM efavirenz or 24.4uM rifampicin or 0.02% dimethyl sulfoxide for a period of 24 hours. Three biological replicates were available for each treatment condition. Total RNA was isolated from the cells, after treatment, using the Quick-RNATM MiniPrep Kit from Zymo Research Corporation. MicroRNA expression profiling was performed using the TaqMan® OpenArray® Human MicroRNA Panel and QuantStudioTM 12K Flex system. The R/Bioconductor package “Automated Analysis of High-Throughput qPCR Data” were used for data analyses. CT values were used for differential expression analysis and microRNAs with a CT value > 35 was considered undetected. MicroRNAs undetected in any of the replicate samples or microRNAs with AmpScore < 1.24 or CqConf < 0.8 were excluded. Quantile normalization was followed by limma analyses to identify differentially expressed microRNAs for efavirenz versus dimethyl sulfoxide and rifampicin versus dimethyl sulfoxide.

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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Single-cell characterization of differentiated HepaRG cells

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