Background: Recent evidence suggests that some solid cancers originate from cancer stem cells. We have identified a subset of candidate stem cells, which are termed side population (SP) cells, in the hepatocellular carcinoma cell line Huh7. Because most stem cells reside in the G0 phase of the cell cycle, G0 cells were isolated, and the relationship between SP cells and G0 cells was investigated to clarify the biological characteristics of G0 cells. Methods: Huh7 cells were sorted using Hoechst 33342 and Pyronin Y. The cells were then divided into G0, G1, and G2/M fractions and cultured under low-attachment conditions to obtain cellular spheres. Tumorigenetic ability was investigated using subcutaneous transplantation to NOD/SCID mice. G0 and G1 cells were analyzed for markers indicative of hepatocytic (albumin expression) and cholangiocytic (keratin19 expression) differentiation and DNA synthesis (Ki67). Results: The cell-cycle distribution of cultured Huh7 cells was 0.7% (G0), 63.8% (G1), and 34.5% (G2/M, S). The G0 cells were located within the neck of the SP fraction. The G0 cells showed spheroid formation and 3-dimensional growth. Those cells showed marked tumorigenesis in NOD/SCID transplantation. G0 cells, which did not express Ki67, were weakly positive for expression of albumin and were clearly positive for the expression of keratin19. In contrast, G1 cells were positive for Ki67 and albumin expression but negative for keratin19. Conclusion: G0 cells are present in the SP fraction of Huh7. They show self-renewal, tumorigenesis, and bidirectional lineage. These findings suggest that the G0 cells within the Huh7 cell line are promising candidates as cancer stem cells for future studies of hepatocellular carcinoma.
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