Fig 5. Sphere forming ability and properties of human cells.

The majority of ATSC, BMSC and SKSC were able to grow as sphere, in specific culture medium. Using light and transmission microscope, we were not able to distinguish adherent cells from re-adherent sphere-derived cells (a and b). Spheres presented a diameter between 80 and 350 microns (c). We quantified the ability to each cell type to grow as spheres, after 12 days of culture compare to the initial number of plated cells (75 000 cells in 25 cm² T-flask). SKSC presented a significantly higher ability to grow as spheres compared to BMSC and ATSC (respectively p<0.0001 and p<0.05 one way ANOVA followed by HSD post hoc test) (d). Similar immunostaining characterizations as performed in Fig 4 were performed on sphere-derived cells. In this case, human sphere-derived ATSC, BMSC and SKSC expressed NESTIN (e-h) and did not express SOX10 (m-p). However, unlike adherent cells, sphere-derived ATSC, BMSC and SKSC expressed P75^NTR (i-l). Quantitative RT-PCR were performed on adherent and sphere-derived cells and confirmed immunological observation made at mRNA level (h, l, p). Data were normalized to SKSC adherent cells expression level which was set as 1. Statistical analysis: one way ANOVA followed by HSD post hoc test. **** means p<0.0001. (Scale bars = 50μm).