Large-scale Generation of Functional and Transplantable Hepatocytes and Cholangiocytes from Human Endoderm Stem Cells

The ever-increasing therapeutic and pharmaceutical demands for liver cells and tissues call for mass production systems that enable efficient in vitro generation of hepatic cell lineages. Here, we describe the use of human endoderm stem cells (EnSCs) as precursors for generating hepatoblasts, as well as functionally mature and transplantable hepatocytes or cholangiocytes in a large-scale serum-free 3D differentiation system. Single-cell transcriptomic analysis is used as a tool to characterize the hepatic populations and guide improvements to the protocol. We demonstrate that the hepatic differentiation of EnSCs recapitulates the in vivo developmental trajectory and find that the regulation of the EGF and MAPK signaling pathways is important for proper lineage progression for the generation of functionally mature hepatocytes or cholangiocytes. When transplanted into liver disease animal models, the resultant hepatocytes are able to rescue the animals and repopulate the host livers. The establishment of this large-scale system for hepatic differentiation of EnSCs lays the foundation for cell-based therapeutic applications for liver diseases and for in vitro liver reconstruction.