In Vitro Expansion of Primary Human Hepatocytes with Efficient Liver Repopulation Capacity

Transplantation of human hepatocytes (HHs) holds significant potential for treating liver diseases. However, the supply of transplantable HHs is severely constrained by limited donor availability and compromised capacity for in vitro expansion. In response to chronic injury, some HHs are reprogrammed into proliferative cells that express both hepatocyte and progenitor markers, suggesting exploitable strategies for expanding HHs in vitro. Here, we report defined medium conditions that allow 10,000-fold expansion of HHs. These proliferating HHs are bi-phenotypic, partially retaining hepatic features while gaining expression of progenitor-associated genes. Importantly, these cells engraft into injured mouse liver at a level comparable to primary HHs, and they undergo maturation following transplantation in vivo or differentiation in vitro. Thus, this study provides a protocol that enables large-scale expansion of transplantable HHs, which could be further developed for modeling and treating human liver disease. [Display omitted] •Human hepatocytes can be serially passaged and expanded 10,000-fold•Proliferating HHs (ProliHHs) display hepatic features and express progenitor markers•ProliHHs can mature following in vitro differentiation or transplantation•ProliHHs repopulate injured livers with comparable efficiency to primary hepatocytes Hui et al. developed a defined medium to expand primary human hepatocytes to a large quantity. Proliferating human hepatocytes show both hepatocyte and progenitor features and could be reverted to mature phenotypes in an organoid culture and notably can repopulate mouse livers with high efficiency.