Neural markers and glial markers were expressed in early spheres. The Sox1-positive floating spheres (A, Sox1-GFP) were positive for βIII-tubulin (B, red) and neurofilament M (C, blue) indicating early upregulation of neural genes in the neural progenitor cells. The tubulin and neurofilament labeling overlap (D, merged). GFAP (G, in red) is expressed at the same time as β-III tubulin (F, in blue) in these Sox1-GFP containing spheres (E, in green), indicating that differentiation of both neural and glial lineages has been initiated in the neural progenitors. Most of these cells were non-overlapping (H, merged images). Scale bar is 50 microns.