Figure 2

Morphological and immunocytochemical characteristics of NE-4C cells at defined stages of RA induced neural differentiation. A-E: Phase contrast views of differentiating NE-4C cultures from stage 1 to stage 5. Non-induced (stage 1) NE-4C cells show epithel-like morphology (A) and immune-reactivity for nestin and SSEA-1 (F1–F3). RC2 immune-positive cells appear at stage 2 (B), and their frequency increases with formation and compaction of the aggregates (C, G) at stage 3. The first βIII-tubulin-positive neuronal precursors (H) appear inside the compact aggregates (C). At stage 4, cells migrate out from the aggregates (D), and form a two-layer arrangement with a basal substrate-attached monolayer and loose neuronal aggregates on the top of it (J). Morphologically "mature" neurons appear by the end of the first week of induction (stage 5) and form dense networks on the surface of a substrate-attached monolayer (E, K). By the time neurogenesis reaches its highest level, the number of RC2 immune-positive radial-glia like cells decreases (L). Averages of fluorescence area-values for RC2 and βIII-tubulin (see M&M) staining were related to the DAPI-stained area-value representing all cell nuclei, on the same microscopic field. Data are presented as percentages of the related area-values obtained from stage 4 cultures (100%; n = 4). Bars: 10 μm