To confirm that NSCs can differentiate into astrocytes, NSCs were cultured in DEMEM/F12 medium supplemented with 1× NEAA, l-glutamine (2 mM), 1× N2, and 1× B27 for 2 weeks and processed for immunostaining. To initiate oligodendrocyte differentiation, NSCs were cultured in medium containing DMEM/F12, 1× NEAA, l-glutamine (2 mM), 1× N2, 1× B27, Shh (200 ng/ml, R&D Systems), and retinoic acid (RA; 2 μM) for 10 days, and then with NT3 (30 ng/ml, R&D Systems) and platelet-derived growth factor alpha (PDGFα) (10 ng/ml, R&D Systems) but without Shh and RA for an additional 2 weeks.
