Figure 3

Nes-GFP⁺ cells showed self-renewal property. (A) Cultured Nes-GFP⁺ spheres expressed PDGFR-α, LIFR, PH3, SSEA-1, SSEA-4, and GATA-4, but did not express 3β-HSD or LHR. Nuclei were counterstained with DAPI (blue). Scale bar, 25 μm. (B) Schematic of the experimental procedure used to generate the Nes-GFP⁺ clonal sphere cultures. (C) Representative images showed clonal sphere growth from single cells observed using a bright field (top panel) or fluorescence (bottom panel) microscope. Scale bar, 10 μm (days 1 and 2) and 25 m (days 4 and 10). (D) A comparison of the sphere formation in wells containing a single cell showed that the frequency of sphere formation was equivalent in the primary and secondary spheres (n = 4). Data are expressed as the mean ± SEM. (E) The proliferation rates of the Nes-GFP⁺ cells at different passages (P₇, P₁₅ and P₃₀) were assessed using the CCK8 assay and were shown to be similar (n = 6). Data are expressed as the mean ± SEM.