Generation of liver bipotential organoids with a small-molecule cocktail

Understanding the mechanismof how cholangiocytes (liver ductal cells) are activated upon liver injury and specified to hepatocytes would permit liver regenerative medicine. Here we achieved long-term in vitro expansion of mouse liver organoids by modulating signaling pathways with a combination of three small-molecule compounds. CHIR-99021, blebbistatin, and forskolin together maintained the liver organoids in bipotential stage with both cholangiocyte- and hepatocyte-specifigene expression profis and enhanced capacity for further hepatocyte differentiation. By employing a chemical approach, we demonstrated that Wnt/beta-catenin, NMII-Rac, and PKA-ERK are core signaling pathways essential and suffint for mouse liver progenitor expansion. Moreover, the advanced small-molecule culture of bipotential organoids facilitates the ex vivo investigation of liver cell fate determination and the application of organoids in liver regenerative medicine.