DNA methylation during HepaRG hepatocyte differentiation

During embryonic development DNA methylation is highly dynamic, although less is known about the stability and fine-tuning of DNA methylation at later stages of differentiation. To understand the role of DNA methylation during hepatocyte differentiation, we profiled approximately 450k methylation sites at different time points in the progression from hepatoblast to hepatocyte stages using the bipotent liver progenitor HepaRG cell line. Progressive demethylation of HNF4A P1 was highly correlated with increased expression of the shorter isoforms of HNF4A. In addition, the absence of cell division at later stages of differentiation and the increased expression of TET1 and TET2 transcripts indicates that a process of active demethylation is taking place at this specific locus. These data suggest that liver progenitors are poised for targeted demethylation at specific genomic locations involved in terminal stages of hepatocyte differentiation. HepaRG cells were seeded in culture six-well plates using William’s Medium E and incubated at 37°C, 5% CO2. The medium was renewed every 3 days. Once a maximal confluence was reached (85 to 100%), culture medium was supplemented with EGF (90 ng/mL) during 1 week then with EGF and 2% dimethyl sulfoxide (DMSO) for another week to stimulate HepaRG cells differentiation. Both EGF and DMSO were kept thereafter until final differentiation (week 4).