Figure 4From: Regulation of nestin expression by thrombin and cell density in cultures of bone mesenchymal stem cells and radial glial cellsCell density regulation of nestin expression by rat MSCs in culture. Rat MSCs were seeded at different cell densities (2.5 to 100·103 rMSCs/cm2) and maintained during 3 days in serum-free medium. These cells were then fixed, labelled with anti-nestin antibody (green), counterstained with TOPRO-3 (red) and positive cells were counted in triplicate cultures. A and B show typical results of anti-nestin immunostaining in culture seeded at initial densities of 20 and 50·103 cells/cm2. From 50000 rMSC/cm2, nestin expression decreases rapidly (C). In this experiment, results are expressed as percentages of nestin-positive cells in each culture conditions (n = 3, ANOVA1). These results were confirmed by Western blotting analysis. Rat MSCs were plated during 3 days in serum-free medium at different densities (20 (D), 50 (E) or 100 (F)·103 cells/cm2) and representative images were taken with a phase contrast microscope. Protein extracts were separated and revealed with anti-nestin and anti-actin antibodies. A decrease of nestin expression is apparent from an initial density of 50·103 cells/cm2 (G). Actin immunostaining controls the protein amounts loaded on the gel in each condition (H). Rat MSCs were cultivated at a density of 10,000 rat MSCs/cm2 together with different densities of rat MSCs in a physically separated millicell co-culture device. No significant difference was observed when the proportion of rat MSCs immunolabelled for nestin was compared in each condition (I) (n = 3, ANOVA1). Serum added in a millicell device as a positive control strongly inhibits nestin expression by rMSCs in the same setup. Scale bar A, B, D, E, F = 40 μm.Back to article page