Hepatocyte differentiation from mouse liver ductal organoids by transducing 4 liver-specific transcription factors

BACKGROUND: Hepatocyte sources that are expandable in vitro are required for liver regenerative medicine and to elucidate the mechanisms underlying the physiological functions of the liver. Liver ductal organoids (LDOs) comprise liver tissue stem cells with a bipotential capacity to differentiate into hepatocyte and cholangiocyte lineages and can thus serve as a hepatocyte source. However, using current differentiation methods, LDOs differentiate into immature hepatocytes while retaining strong cholangiocyte characteristics. We thus investigated an alternative differentiation method for LDOs to achieve hepatocyte maturation. METHODS: We extracted 12 candidate transcription factors to induce hepatocyte differentiation by comparing their gene expression in LDOs and liver tissues. After evaluating the effects of these transcription factors on LDOs, we analyzed the comprehensive gene expression profile, protein expression, and hepatic function in the transduced organoids. RESULTS: We identified a combination of 4 transcription factors, Hnf4a, Foxa1, Prox1, and Hlf, which upregulated hepatic lineage markers and downregulated cholangiocyte markers. Differentiation-induced LDOs showed more hepatocyte-specific characteristics than those with the conventional method, enhancing the transition from cholangiocyte to hepatocyte lineage and hepatic functions, such as liver-specific protein synthesis, lipid droplet deposition, and ammonia detoxification. CONCLUSIONS: Transduction of the 4 transcription factors (Hnf4a, Foxa1, Prox1, and Hlf) is a promising strategy to promote the differentiation of LDOs to obtain mature hepatocyte-like cells with better functionality.