Humoral effect of nestin-positive MSCs. Neurosphere-derived GFP-expressing cells were cultivated with npMSC or nnMSC conditioned media, on polyornithine-coated dishes. After 5 days, the cells were fixed and immunostained with antibodies raised against GFAP (A, D, green), Tuj1 (B, E, green) and O4 (C, F, green). Nuclei were counterstained with EtD1 (red). Cultures with npMSC conditioned medium are illustrated in A, B and C. As observed in co-culture conditions, there is a significant increase of the number of GFAP-positive cells and significant decreases of the number of Tuj1-positive cells and O4-positive cells (n = 8, ***Student T test, P < 0.0001). Cultures with nnMSC conditioned medium are illustrated in D, E and F (n = 7, ***Student T test, P < 0.0001). Stained cells counting results are shown in G and include the control condition of neurosphere-derived cells without conditioned medium. No significant difference is observed between neurosphere differentiated in co-culture and in conditioned medium. (Student T test, P > 0.05). Scale bars = 20 μm in A-F.