Fig. 4. Isolated cells mimic neurogenesis observed in whole EBs (d7+7). (A,B) Differentiating neurons with bipolar-(arrow) and multipolar (arrowhead) morphology were seen after plating of isolated cells A: combined transmission and fluorescence; B: fluorescence. (C,D) A high overlap (D, yellow) between EGFP expression (C) and nestin labelling was observed. Besides the generation of neurons, EGFP-positive and -negative glial cells (E) were identified based on GFAP staining (F, superimposed). Flat glial cells were weak/negative for EGFP (arrowhead), while elongated fibre-like glial cells were EGFP positive (arrow). (G,H) The formation of synaptic connections between isolated neurons was confirmed by synaptophysin staining (H). As expected for differentiated neurons, low or no EGFP signal (G) was detected in synaptophysin-positive cells. Little overlap (K, superimposed) between MAP-2 staining (K) and EGFP expression (J) was detected. The flat glial cells (green) and differentiating neurons (red) were found to closely associate. Bars, 25 µM.