Liver exhibits robust regeneration following mechanical damage due to immediate proliferation of hepatocytes. It has been recently reported that hepatocyte replenishment following liver damage could also occur via a reversible transition between hepatocytes and duct-like progenitor cells1. Hepatocyte-derived duct-like progenitor cells were observed to expand extensively in the presence of toxic insults and re-differentiate into functional hepatocytes upon cessation of injury1. This process was identified as ductal metaplasia of adult hepatocytes in vivo and described as an alternative mechanism for hepatocytes to escape from various chronic injuries1,2. Given the highly proliferative potential of the in vivo hepatocyte-derived progenitor cells, we sought to determine whether an optimal culture condition mimicking the in vivo milieu could be developed to extensively expand functional hepatocytes through reversible transition in vitro.

With this established reporter system for screening, we optimized the culture condition for maintenance and expansion of the hepatocyte-derived ductal cells over a longer timeframe. A pool of candidate growth factors and bioactive small molecules were selected for screening based on previous findings regarding the survival and growth requirements for hepatic epithelial cells in vivo (Supplementary information, Table S1). Candidates were selected based on their ability to (1) activate particular cytokine- and growth factor-mediated pathways involved in regulating liver regeneration; (2) mimic Wnt signals for liver repair and activation of progenitor-like cells after liver damage; (3) regulate Hippo/Yap or Notch signaling pathways for controlling liver cell fate and hyperplasia; and (4) inhibit TGF-β for further promotion of liver regeneration and cell expansion. After systematic testing, a chemically defined culture medium optimal for hepatocyte-to-ductal cell transition and expansion of duct-like cells was eventually established (Supplementary information, Data S1). The transition and expansion medium (TEM) contained growth factors EGF and HGF, agonist of Wnt signaling (CHIR99021), Yap signaling activators lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P)6, inhibitor of TGF-β signaling (A83-01), and inhibitor of ROCK kinase (Y27632) (Supplementary information, Table S1). Removal of any of these individual components negatively influenced the proliferation of duct-like cells, suggesting a synergistic relationship among these factors (Supplementary information, Figure S1H).

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