Characteristics of SP cells on ex vivo expansion. (A-a) Phase-contrast images of cultured SP cells. Scale bar = 100 μm. (A-b) Colony-forming assay with the SP cells. SP cells were seeded at a density of 500 cells per a fibronectin coated 35-mm dish. (A-c) Cell proliferation speed was markedly promoted by addition of growth factors and fibronectin addition. Values are the mean and SEM of 3 independent cell lines. (B) Ho-staining and FACS analysis of ex vivo expanded SP and NSP cells. Open triangles indicate the populations possessing Ho-efflux properties. The NSP cells did not represent the Ho-efflux property. All experiments were performed using 6 times passaged cells, and replicated 3 times using 3 independent cell lines. (C) Analysis of the stem cell marker gene expressions based on quantitative RT-PCR in ex vivo expanded SP cells. Y-axis represents the relative values for SP cells of primary culture (correspond to the mean values of cS in figure 3A). Significant differences between primary SP cells and 7 times passaged SP cells were observed by Dunnet's test. Significant differences between passaged SP cells and passaged NSP cells were observed by student t-test.