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Objective:To establish a method for enriching,culturing and identifying stem-like subpopulation from human hepatic carcinoma cells,and to explore its biological properties.Methods:HepG2cells were cultured in cancer stem cell(CSC)medium to form spheroids.The stem-like HepG2cells obtained from tumor spheroids were then expanded.Flow cytometry was used to detect the expression of CD90 and CD133on the surface of stem-like HepG2 cells.The in vitro colony forming ability and in vivo tumorigenicity were detected by clone formation assay and tumorigenesis assay.Results:HepG2cells could grow in suspension and form spheroids in CSC medium.The stem-like cells had the ability of self-renewal and proliferation.The expression of CD90and CD133on the surface of these stem-like cells were higher than those of parental HepG2cells(P<0.01).The colony formation ability of stem-like cells was higher than that of parental HepG2cells.When injected with 1×106 cells,stemlike cells could form tumors earlier than parental cells in nude mice.The stem-like cells’tumorigenesis rate was higher and the tumor size was larger than those of parental HepG2cells(P<0.01).Conclusion:The suspension sphere culture method could enrich stem-like cells from HepG2cells.The obtained stem-like cells possessed the properties of self-renewal in vitro and tumorigenicity in vivo.