Abstract
Background and Aims: Recent evidence suggests that some solid cancers may originate from cancer stem cells. We have identified subset of candidate stem cells termed side population (SP) cells in Huh7, a hepatocellular carcinoma cell line (Stem Cells 2006). On the other hand, stem cells are considered to exist at G0 phase of cell cycle. In this study, we investigated the relationship between SP cells and G0 cells, and clarified the biological characteristics of G0 phase cells. Study Design: Huh7 cells were sorted by flow cytometry using Hoechst 33342 and pyronin Y. Then, the cells were divided into G0, G1, and G2/M phase, respectively, and also into SP and non-SP fractions. The cells were cultured under a low attached condition to obtain spheroid cell clusters, and SP and G0 cells were counted. The cells in each cell-cycle phase were extracted respectively, and analyzed with respect to DNA synthesis condition (Ki67), hepatocytic (albumin expression) or cholangiocytic (keratin19 expression) differentiations. Results: The proportion of each cell-cycle phase of the Huh7 cells was 0.7%, 63.8%, and 34.5%, in G0, G1, and G2/M, respectively. On the other hand, SP and non-SP fraction were recognized in 0.9% and 98.8%, respectively. Interestingly, the G0 phase cells were located at a neck of SP fraction. Under a condition of spheroid cell culture, the ratio of SP fraction and that of G0 fraction were significantly increased. The G0 cells showed negative Ki67 expression, weakly positive albumin expression, and positive keratin19 expression, whereas, the G1 and G2/M cells did positive Ki67 expression, albumin expression, but negative keratin19 expression. Conclusion: It is demonstrated that Huh7 cells in G0 phase were included in the SP fraction and that they showed less proliferation activity. These cells, however, showed bidirectional differentiation to hepatocyte and cholangiocyte lineages. The findings suggest the G0 cells of Huh7 are promising candidate of cancer stem cells in hepatocellular carcinoma.
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