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Pancreatic cancer is among the most aggressive solid malignancies. It is possible that pancreatic cancer contains cancer stem cells responsible for its malignancy. The purposes of this study were (1) to establish an assay in which a subset of pancreatic cancer cell line (PANC-1) cells with stem cell properties can propagate, and (2) to identify the cells obtained from this assay.The PANC-1 cells were cultured in Dulbecco modified eagle medium F12 supplemented with epidermal growth factor, basic fibroblast growth factor, insulin, transferrin, selenium, and bovine serum albumin at a density of 1000 cells/mL for 10 to 14 days to form spheres. Cells of spheres were cultured in different conditions to evaluate their ability of self-renewal and differentiation. Clone formation assay and tumor formation assay were used to identify the ability of propagation in vitro and in vivo. The cells and spheres were also stained by using Hoechst 33342 dye to evaluate their capacity of excluding Hoechst dye. Real-time polymerase chain reaction was used to detect expressions of LY6E, c-Met, TACSTD1, CD34, and CD44 mRNA.A subpopulation of PANC-1 cells could propagate to form spheres in this assay. Cells of obtained spheres had the hallmark of excluding Hoechst 33342 dye. Cultured in serum-free medium, the dissociated single cells of primary spheres could form filial spheres again; cultured in serum-containing medium, these cells generated both the cells with the ability of excluding Hoechst 33342 dye and the cells without that ability. The propagation capacity of PANC-1 spheres was higher than that of cells cultured in serum-containing medium both in vitro and in vivo. In addition, LY6E, TACSTD1, and CD44 mRNA were overexpressed in PANC-1 spheres.A subpopulation of PANC-1 cells can propagate to form spheres with properties of stem cells in this assay; enough of these cells can be obtained for further study. Considering the overexpression of mRNA, it was tentatively suggested that LY6E, TACSTD1, and CD44 proteins may act as surface markers for sorting pancreatic cancer stem cells with fluorescence-activated cell sorter/magnetic-activated cell sorter.