Rapid changes in chromatin structure during dedifferentiation of primary hepatocytes in vitro

Primary hepatocytes are widely used in the pharmaceutical industry to screen drug candidates for hepatotoxicity, but hepatocytes quickly dedifferentiate and lose their mature metabolic function in culture. Attempts have been made to better recapitulate the in vivo liver environment in culture, but the full spectrum of signals required to maintain hepatocyte function ex vivo remains elusive. To elucidate molecular changes that accompany, and may contribute to dedifferentiation of hepatocytes ex vivo, we performed lineage tracing and comprehensive profiling of alterations in their gene expression profiles and chromatin landscape during culture. First, using genetically tagged hepatocytes we demonstrate that expression of the fetal gene alpha-fetoprotein in cultured hepatocytes comes from cells that previously expressed the mature gene albumin, and not from a population of albumin-negative precursor cells, proving mature hepatocytes undergo true dedifferentiation in culture. Next we studied the dedifferentiation process in detail through bulk RNA-sequencing of hepatocytes cultured over an extended period. We identified three distinct phases of dedifferentiation: an early phase, where mature hepatocyte genes are rapidly downregulated in a matter of hours; a middle phase, where fetal genes are activated; and a late phase, where initially rare contaminating non-parenchymal cells proliferate, taking over the culture. Lastly, to better understand the signaling events that result in the rapid downregulation of mature genes in hepatocytes, we examined changes in chromatin accessibility in these cells during the first 24 h of culture using Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). We find that drastic and rapid changes in chromatin accessibility occur immediately upon the start of culture. Using binding motif analysis of the areas of open chromatin sharing similar temporal profiles, we identify several candidate transcription factors potentially involved in the dedifferentiation of primary hepatocytes in culture.