Conversion of terminally committed hepatocytes to culturable bipotent progenitor cells with regenerative capacity

A challenge for advancing approaches to liver regeneration is loss of functional differentiation capacity when hepatocyte progenitors are maintained in culture. Recent lineage-tracing studies have shown that mature hepatocytes (MHs) convert to an immature state during chronic liver injury, and we investigated whether this conversion could be recapitulated in vitro and if such converted cells could represent a source of expandable hepatocytes. We report that a cocktail of small molecules, Y-27632, A-83-01 and CHIR99021, can convert rat and mouse MHs in vitro into proliferative bipotent cells, which we term chemically induced liver progenitors (CLiPs). CLiPs can differentiate into both MHs and biliary epithelial cells that can form functional ductal structures. CLiPs in long-term culture did not lose their proliferative capacity or their hepatic differentiation ability, and rat CLiPs were shown to extensively repopulate chronically injured liver tissue. Thus our study advances the goals of liver regenerative medicine. Transcriptomic analyses for freshly isolated MHs and cells cultured for the designated periods with or without YAC stimulation (Matrix 1). Transcriptomic analysis for CLiPs which underwent hepatic induction (Matrix 2). Transcriptomic comparison of hepatic inducibility between CLiPs at early passage and those at late passages (Matrix 3). Transcriptomic comparison between chimera-derived rat cells (designated as “2nd”) and primary rat MH-derived cells (designated as “1st”) (Matrix 4). Freshly isolated hepatocytes were cultured in the presence or absence of YAC. Hepatic induction was performed for P1 CLiPs or clonal CLiPs.