title: Enrichment of Oct3/4-positive cells from a human bronchial epithelial cell line.
abstract: 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.
authors.affiliation: Department of Pathology, the First Affiliated Hospital and College of Basic Medical Sciences of China Medical University, Shenyang, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.forename: Lanling
authors.initials: L
authors.lastname: Jia
authors.forename: Xinshan
authors.initials: X
authors.lastname: Jia
authors.forename: Mumu
authors.initials: M
authors.lastname: Shi
authors.forename: Xiaolei
authors.initials: X
authors.lastname: Li
authors.forename: Xulv
authors.initials: X
authors.lastname: Ye
authors.forename: Ruiyue
authors.initials: R
authors.lastname: Wang
authors.forename: Yanlei
authors.initials: Y
authors.lastname: Xiong
authors.forename: Enhua
authors.initials: E
authors.lastname: Wang
authors.forename: Fang
authors.initials: F
authors.lastname: Li
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000139:chemically induced; D042002:Chromatin Assembly and Disassembly; D053595:Embryonic Stem Cells / Q000378:metabolism; D044127:Epigenesis, Genetic; D005472:Fluorouracil / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism*; D020545:Respiratory Mucosa / Q000187:drug effects* / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D014410:Tumor Stem Cell Assay
chemical_list: D000970:Antineoplastic Agents; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; D005472:Fluorouracil