Figure 3.

Coculture of cortical neurons with mature IDS-ko NSC-derived astrocytes. (a) Wt and IDS-ko NSC-derived mature astrocytes (21 div) were infected with lentiviral vector carrying the rfp reporter gene and cocultured for further 40 div with rat primary neurons. Confocal microscopy images showing the immunostaining of the neuronal marker MAP2 (green) and of RFP (red). Scale bars: 75 μm. (b and c) Densitometric analysis of MAP2 and RFP intensity at 11, 20 and 40 div of coculture shows that a remarkable reduction of MAP2 neuronal expression (b) is accompanied by a massive increase of RFP intensity (c) where primary neurons are cocultured with mutant astrocytes. (d) Quantitative analysis of NeuN+ cells in cocultures of primary neurons with NSC-derived astrocytes previously differentiated for 7, 14 and 21 div. The reduction of NeuN+ cells over total DAPI+ nuclei is evident only with mature astrocytes (21 div). (e) Quantitative analysis of NeuN+ neurons cocultured for 40 div with NSC-derived astrocytes, differentiated for 21 div in areas with 25–30% GFAP+ cells over total DAPI+ nuclei. The reduction of neuronal cells is enhanced by high density of GFAP+ astroglial cells suggesting that neuronal demise is driven by cocultured astrocytes in a dose-dependent manner. (b and e) t-Test for all experiments was applied. Data are reported as mean±S.E.M.