Generation and characterization of mature hepatocyte organoids for liver metabolic studies.

Hepatocyte organoids (HOs) generated in vitro recently are powerful tools for liver regeneration. However, the reported HOs were mostly fetal in nature with low expression levels of metabolic genes characteristic of adult liver functions, hampering their application in studying metabolic regulations and therapeutic testing for liver disorders. We report development of novel culture conditions that contains optimized levels of Triiodothyronine (T3) with the removal of growth factors, enabled successful generation of mature hepatocyte organoids (MHOs) with metabolic functions characteristic of adult livers of both mouse and human origins. We showed that the MHOs can be used to study various metabolic functions including bile and urea production, zonal metabolic gene expression, and metabolic alterations in both alcoholic and non-alcoholic fatty liver diseases as well hepatocyte proliferation, injury, and cell fate changes. Notably, the MHOs derived from human fetal hepatoblasts also showed improved hepatitis B virus (HBV) infection. Therefore, these MHOs provide a powerful in vitro model for studies of human liver physiology and diseases. The human MHOs are potentially a robust research tools for therapeutic development as well.