Figure 1

Repression of nestin expression in rat MSCs cultures by serum, thrombin and TRAP6. Rat MSCs and mouse MSCs of at least 25 population doublings were cultured for 3 days in serum-free medium to induce nestin expression. Then, the cells were renewed in DEM supplemented with different concentrations of thrombin (0 (A, rat MSCs), 90, 180, 450 (B, rat MSCs) and 900 pM), in DEM + 10% FBS or MEM + 10% horse serum (L) or in DEM + 900 pM of thrombin with 10^-5M of FUdR (K) for a further 3 days period and then fixed and labelled with anti-nestin antibody (green). Nuclei were counterstained with TOPRO-3 (red) and then counted. In these conditions, there is a significant decrease of nestin expression both in rat MSCs (C) with 180 pM (*, p < 0,05), 450 and 900 pM of thrombin (**, p < 0,01) and in mouse MSCs (F) from 180 pM of thrombin (**). (L) There is however no difference when HS in MEM (18% of cell expressing nestin) is used instead of FBS in DEM (10% of cell expressing nestin). These results were confirmed by Western blotting (D), where nestin expression in rat MSCs cultivated in DEM, DEM + 1 nM of thrombin and DEM + 10% FBS was compared after 3 days of induction of nestin expression. The actin immunostaining controls the amount of protein loaded on the gel in each condition and allows to calculate the ratio between the intensity of the nestin and actin signals and to normalize data to the level of nestin expression in rat MSCs treated with DEM only (E). Rat MSCs were cultured for 3 days in serum-free medium to induce nestin expression. Then, those cells were renewed in DEM supplemented with different concentration of TRAP6, for a further 3 days period and then fixed and labelled with nestin antibody. Nuclei counterstained with TOPRO-3 and nestin-positive MSCs were then counted. (G) In these conditions, there is a significant decrease of nestin expression from 1 μM TRAP6 (*, p < 0,05; **, p < 0,01 from 30 μM, n = 3, ANOVA1). (H) To measure the effect of thrombin on the apoptosis of rat MSCs, cells were cultivated for 3 days in serum-free medium to induce nestin expression. Then, those cells were renewed in DEM with various concentrations of thrombin. After a new 3 days period, cells were fixed and submitted to a TUNEL labelling (green). Cell nuclei were counterstained with DAPI. For the positive control of the TUNEL assay, rat MSCs previously treated with DNAse were used (I). (J) To measure the effect of thrombin on cell density, cells were treated with the indicated concentrations of thrombin, then fixed, labelled with DAPI and counted in 10 microscopic fields at 10× magnification. Scale bar in A, B, H, I = 40 μm.