The undifferentiated and differentiated neurospheres were defined previously (Singh et al., 2009). In brief, the undifferentiated neurospheres were obtained from the above culture medium minus EGF+bFGF for two days. The neurospheres allowed to differentiate were withdrawn from the EGF+bFGF supplementation and equilibrated for 5-10 minutes in Neurobasal media (no supplements) before being plated into a 10 mm plastic Petri dish (for MeDIP analysis) or in 16-well chamber slides (Nunc, Rochester, NY; for immunocytochemistry), both coated with poly-D-lysine (50μg/mL, Sigma, St. Louis, MO) and laminin (50μg/mL, Sigma). Neurospheres were allowed to differentiate for 2 (for MeDIP or morphological analysis) or 4 days (for morphological analysis only) in culture (2 or 4 DIC) with differentiation media consisting of Neurobasal media supplemented with 10% fetal bovine serum, 1.2% B27, and 1.2% penicillin-streptomycin.
