Primary neural stem cells were isolated from the embryonic forebrain at E14.5 and were cultured in the presence of growth factors to generate primary neurospheres. After 7 days in culture, primary neurospheres were dissociated and cultured under differentiation conditions for 8 days, reported to be sufficient for differentiation of both neuronal and glial cells [35,37] (Figure 2A). In agreement with our previous report [37] the proliferating primary neurospheres expressed NESTIN and KI67 (Figure 2B). Similar to our previous reports [35,37] differentiated NSC at D8 consisted of a mixed population of neurons, astrocytes, and oligodendrocytes (Figure 2C). The composition of the D8 population was determined by detection of cell type-specific markers (TUB III (4.7% ± 0.8 mean ± standard error of the mean (SEM)), Glial fibrillary acidic protein (GFAP) (54.4% ± 1.1), S100B (15.9% ± 2.7), 2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNPase) (1.6% ± 0.2), Myelin basic protein (MBP) (2.6% ± 0.42), Oligodendrocyte lineage transcription factor 2 (OLIG2) (36.2% ± 1.8) and KI67 (92.4% ± 1.3)) by immunofluorescence (Figure 2C). KI67 is not a cell type-specific marker, but rather reflects the fraction of cycling
