To date, most of the pluripotent stem cell (PSC)-based liver modeling systems have initiated differentiation from highly purified definitive endoderm (DE) and have generated monolayer hepatocyte-like cells. However, preclinical predictions from such models are usually difficult to interpret, and may be misleading, because homogeneous DE populations are insufficient to accurately replicate the complex regulation of signals among cells and tissues during liver organogenesis.

A recent study, which mimicked liver development by combining hepatic endoderm cells with endothelial cells and mesenchymal progenitors, resulted in the generation of a liver bud-like structure with improved function. Similarly, co-culture of fetal liver progenitor cells and liver extracellular matrix triggered the formation of hepatocytes with bile duct-like structures. These findings highlighted the importance of multicellular interactions during early liver development. However, these studies combined cells that were isolated from multiple and postnatal individuals, suggesting a severe limitation of the transplanting applications based on these strategies.

Here, we reported a method to generate functional hepatobiliary organoids (HBOs) from hiPSCs. To achieve this goal, we learned from early hepatogenesis and simultaneously induced endoderm and a small part of mesoderm by the inclusion of 25% mTeSR into hepatic differentiation medium. This treatment mildly suppressed/delayed early hepatic differentiation, but turned on biliary specification by activating the NOTCH2 and TGF-ß signaling pathway. Then we achieved co-differentiation of hepatocyte-like cells and cholangiocyte-like cells. Finally, our uniquely prepared cholesterol+ MIX (mainly comprised of cholesterol and other small molecules) promoted the HBO formation and functional maturation.