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

The ever-increasing therapeutic and pharmaceutical demand for liver cells calls for systems that enable mass production of hepatic cells. Here we describe a large-scale suspension system that uses human endoderm stem cells (hEnSCs) as precursors to generate functional and transplantable hepatocytes (E-heps) or cholangiocytes (E-chos). hEnSC-derived hepatic populations are characterized by single-cell transcriptomic analyses and compared with hESC-derived counterparts, in-vitro-maintained or -expanded primary hepatocytes and adult cells, which reveals that hepatic differentiation of hEnSCs recapitulates in vivo development and that the heterogeneities of the resultant populations can be manipulated by regulating the EGF and MAPK signaling pathways. Functional assessments demonstrate that E-heps and E-chos possess properties comparable with adult counterparts and that, when transplanted intraperitoneally, encapsulated E-heps were able to rescue rats with acute liver failure. Our study lays the foundation for cell-based therapeutic agents and in vitro applications for liver diseases. Human endoderm stem cells (EnSCs) were expanded and used as precursors for stepwise directed differentiation into hepatoblasts, as well as functionally mature and transplantable hepatocytes (E-heps) or cholangiocytes (E-chos) in a spinner-flask-based large-scale serum-free 3D culture system. Thorough functional assessment of E-heps and E-chos was performed to compare them with the adult cells. Single cell transcriptomic analysis was applied to characterize the EnSC-derived hepatic populations and to compare them with the pluripotent stem cell-derived counterparts, the in vitro maintained or expanded primary hepatocytes and the adult hepatic cells.