Hepatic stem cells with self-renewal and liver repopulation potential are harbored in CDCP1-positive subpopulations of human fetal liver cells

Ran-Ran Zhang, Yun-Wen Zheng, Bin Li, Yun-Zhong Nie, Yasuharu Ueno, Tomonori Tsuchida, Hideki Taniguchi

Mature human hepatocytes are critical in preclinical research and therapy for liver disease, but are difficult to manipulate and expand in vitro. Hepatic stem cells (HpSCs) may be an alternative source of functional hepatocytes for cell therapy and disease modeling. Since these cells play an import role in regenerative medicine, the precise characterization that determines specific markers used to isolate these cells as well as whether they contribute to liver regeneration still remain to be shown. In this study, human HpSCs were isolated from human primary fetal liver cells (FLCs) by flow cytometry using CDCP1, CD90, and CD66 antibodies. The isolated CDCP1+CD90+CD66- HpSCs were cultured on dishes coated with type IV collagen in DMEM nutrient mixture F-12 Ham supplemented with FBS, human γ-insulin, nicotinamide, dexamethasone, and L-glutamine for at least 2 weeks, and were characterized by transcriptomic profiling, quantitative real-time PCR, immunocytochemistry, and in-vivo transplantation. The purified CDCP1+CD90+CD66- subpopulation exhibited clonal expansion and self-renewal capability, and bipotential capacity was further identified in single cell-derived colonies containing distinct hepatocytes and cholangiocytes. Moreover, in-vivo liver repopulation assays demonstrated that human CDCP1+CD90+CD66- HpSCs repopulated over 90% of the mouse liver and differentiated into functional hepatocytes with drug metabolism activity. We identified a human hepatic stem/progenitor population in the CDCP1+CD90+CD66- subpopulation in human FLCs, indicating CDCP1 marker could potentially be utilized to identify and isolate HpSCs for further cytotherapy of liver disease.