Enrichment of Oct3/4‐positive cells from a human bronchial epithelial cell line
Most adult stem cells are in the G0 phase of the cell cycle, accounting for only a small percentage of the cells in the tissue. Thus, isolation of stem cells from tissues for further study represents a major challenge. The anti‐tumor drug 5‐fluorouracil (5‐FU) selectively kills proliferating cells, sparing cells in the G0 phase. Thus, the objective of this study was to determine whether 5‐FU can be used to enrich stem cells in a human bronchial epithelial (HBE) cell population in vitro. Side population (SP) cells were isolated from untreated HBE cells or HBE cells treated with 5‐FU, and the resulting cells were subjected to colony formation assays, culturing of cell spheres, and tumorigenicity assays. Expression of Oct3/4, Sox2, PCK, and β‐catenin were examined by Western blot analysis and immunofluorescence. Treatment with 5‐FU increased the percentage of SP cells from 0.3% to 1.5%, and the clonogenic ability of 5‐FU‐treated cells was more than twofold higher than that of HBE cells. Cells that survived after 5‐FU treatment exhibited a higher capacity for sphere formation. Furthermore, spheres formed from 5‐FU‐treated cells possessed the capacity to generate differentiated progenies. Cells treated with 5‐FU also exhibited tumorigenic potential, based on tumor formation assays in nude mice, and Oct3/4‐positive cell aggregates were identified in the resulting tumors. In this study, we have shown that 5‐FU treatment enriched the population of cells expressing the putative embryonic markers Oct3/4 and Sox2 and exhibiting nuclear accumulation of β‐catenin. Furthermore, 5‐FU‐treated cells expressed low levels of the epithelial differentiation marker PCK. Analysis of epigenetic modifications suggested that Oct3/4‐positive cells possessed characteristics of stem cells. These results demonstrate that treatment with 5‐FU can enrich the stem cell population present in a human bronchial epithelial cell line, and implicate combined treatment with 5‐FU and serum‐free medium as a new method for isolation of stem‐like cells from the HBE cell line.
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Document Type: Research Article
Publication date: July 1, 2013