Cell 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.