Expression and Role of Oct3/4 in Injury-Repair Process of Rat Alveolar Epithelium after 5-Fu Treatment.

OBJECTIVE: We aimed to investigate how the embryonic stem cell-related gene Oct3/4 changes during the injury-repair process of distal pulmonary epithelium induced by 5-fluorouracil (5-Fu). METHODS: We have developed the lung injury model induced by 5-Fu and observed the dynamic changes of Oct3/4 by indirect immunofluorescence, Western blot, and quantitative real-time PCR. Immunofluorescence double staining was used to compare the positions of Oct3/4(+) cells and other reported alveolar epithelial stem cells. RESULTS: Oct3/4(+) cells were not found in normal rat lung epithelial cells. However, after treatment with 5-Fu, Oct3/4(+) cells appeared at 12 h, reached the peak at 24 h, then decreased at 48 h, and eventually disappeared at 72 h. Oct3/4 was localized in the nucleus. We found that the sites of Clara cell secretory protein and surfactant protein-C dual positive cells were apparently different from Oct3/4(+) cells. CONCLUSIONS: Our results revealed that, in rat alveolar epithelium, expression of Oct3/4 could be induced after treatment with 5-Fu, then decreased gradually, and was silenced following the alveolar epithelial differentiation. We hold that Oct3/4(+) cells are lung stem cells, which can provide new evidence for identification and isolation of lung epithelial stem cells.

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 ß-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 ß-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.