Dopaminergic differentiation in defined media was initiated by culturing NSCs for 10 days in neurobasal medium supplemented with NEAA, l-glutamine (2 mM), B27, Shh (200 ng/ml), and FGF8 (100 ng/ml). Shh and FGF8 were then withdrawn and replaced with Brain-derived neurotrophic factor (BDNF) and Glial cell-derived neurotrophic factor (GDNF) (20 ng/ml of each), Transforming growth factor beta-3 (TGFβ3) (1 μM), ascorbic acid (200 μM), and cAMP (1 mM) for 3 weeks (∼30 days after the NSC stage).
