Fig. 5. Effect of co-culture on neural differentiation. Heterogenous spheres of nestin-positive rSCs stained with DiD Vybrant^TM cell-labelling solution (red) (Molecular Probes) and GFP-positive NSCs (M.1,M.2) are placed on polyornithine-coated dishes for 5 days. Some rSCs have differentiated into GFAP-positive cells (indicated by arrowheads). GFAP (red) is expressed by a large fraction of cells (A), of which some also contained green fluorescent protein (B), and thus originate from mNSC. Triple labelling (including nuclei stained by EtD1 in blue) allow the identification of non-green rSCs that express GFAP (C). Co-culture of passage 4 nestin-negative rSCs with GFP-positive mNSC demonstrated that only a small percentage of GFAP-positive cells were derived from MSCs (D-F). Astroglial differentiation of passage 15 rSCs were confirmed with Glast marker (red) (G-I). Pure GFP-positive mNSC were used as a control to demonstrate that all GFAP (red)-positive cells from mNSC remain GFP-positive (green) (J-L). A double-labelling with M2 (blue) and GFAP (red) antibodies allows the confirmation of the mesenchymal origin of some GFAP-positive cells (O). rSC-derived GFAP-positive cells are not recognized by the M2 antibody. The GFP-positive astrocytes (green astrocytes) that differentiate from mNSC are recognized by the M2 antibody (N). rSCs, mNSC and co-cultured rSCs and mNSC were stained with propidium iodide and subjected to FACS analysis (P). Arrowheads in C,E,H,O indicate the mesenchymal-derived cells that express neural markers. Scale bars: (A,B,D,F,G,I,M.1) 150 µm; (C,E,H,J,K-M.2) 40 µm; (N,O) 60 µm.