Morphology and immunofluorescence analysis of NTCSC-derived neurons. (a) Representative image of the cells at 80% confluence. (b) Representative image of cells cultured in serum-free medium. (c) The cells aggregated into groups following RA/SHH induction. (d) H&E staining revealed that the cells possessed neuron-like morphology following induction. After 48 hours of induction, the expression of Nestin ((e), (f)) and β-III tubulin ((g), (h)) was observed. Synapsin I was also expressed in aggregated cells (i), and NeuN was present in the nuclei (j). The uninduced cells were negative for these markers (Nestin, (k); β-III tubulin, (l); synapsin I, (m)). Nuclei ((f), (h), (k), (l), (m)) was staining with DAPI. Scale bar: ((c), (j), (k), (l), (m)): 100 μm; ((a), (b), (d), (e), (f), (g), (h), (i)): 200 μm.