Fig. 3. Regulation of nestin expression by rSCs, by the number of passages. rSCs express nestin weakly between passage 0 and passage 6 (A, rSCs passage 4). The expression increases between passage 6 and passage 10 (B, rSCs passage 10). Beyond passage 11, 75% of rSCs express nestin in serum-free medium (C, rSCs passage 15). Cultures were labeled with nestin antibody (green labelling) and cell nuclei were counterstained with ethidium homodimer (red labelling). (D) The evolution of nestin expression as function of the number of passages of rSCs. This graphic was established from results of immunocytochemical analysis on passage 4, 6, 7, 8, 9, 11, 14 and 15. In this experiment, results are expressed as percentages of nestin-positive cells in each passage in serum-free condition (n=4, representing a minimum of 1500 counted cells in each passage). These results were confirmed by western blotting on passage 4 (P4), 10 (P10) and 15 (P15) (E). Radial glial cells (RGCs) were used as nestin-positive control and actin was used as protein-loading control. No alteration of the capacity of differentiation: (F) P10 adipocyte, (G) P18 adipocyte, (H) osteocytes can be observed even in the long-term cultures. Scale bar: (A-C,F,G) 40 µm.

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