Figure 2.

EGFP-ScNs formed connections with wild type mouse CN neurons. (a) Primary culture of postnatal day 3 EGFP-mouse SGN-derived cells formed spherical structures in suspension culture during culture day 0–5 (D0-D5). (b) Passage 3 EGFP-mouse SGN-derived neural spheres expressed NSC proteins Sox2 and nestin in suspension culture. (c) EGFP-mouse SGN-derived NSCs were induced into neurons (ScNs) expressing neuronal protein TUJ1, otocyst-derived cell protein Gata-3, and glutamatergic protein VGlut1 after seeding to the culture wells for 8–12 days. (d) EGFP-ScNs were co-cultured with dissociated wild type postnatal mouse CN cells for 5 days. EGFP indicated the cells originated from EGFP-mouse SGN-derived cells, whereas CN cells were observed by DIC microscopy without EGFP expression. (e) After co-culturing for 4–6 days, TUJ1 positive connections were observed between EGFP-ScNs (asterisks) and wild type CN neurons (arrowheads). In the control group (co-cultures without ACM supplement), a few SV2 positive puncta (arrows) were observed along connections between the ScN and CN neuron. In contrast, a number of SV2 puncta were found along connections in the ACM group (co-cultures supplied with ACM). (f) The quantitative study showed percentage of NSC proteins Sox2, nestin, and double-labeled cells in passage 3 SGN-derived neural spheres. The quantitative study showed that relative number and area of SV puncta per connection were significantly larger in the ACM group (mean ± standard error shown in the figure; **indicates P < 0.01, Mann-Whitney U test). Scale bar: 50 µm in (a and d), 25 µm in (b and e), and 10 µm in (c).