Differentiation of Human Neural Progenitor Cells

The generation and culturing of human NPCs has been previously described (37 (link)). NPCs were grown in N2B27 medium (DMEM-F12/Neurobasal at 50:50 supplemented with 1% penicillin/streptomycin/glutamine, 1% B27 supplement without vitamin A, and 0.5% N2 supplement) containing 3 μM CHIR99021 (Cayman), 150 μM L-ascorbic acid (Sigma-Aldrich), and 0.5 μM Smoothened Agonist (SAG) (Cayman). For differentiation, the medium was replaced with N2B27 containing 1 ng/ml BDNF (Miltenyi Biotec), 0.2 mM L-ascorbic acid, 1 μM retinoic acid (Sigma-Aldrich), 1 ng/ml glial cell line-derived neurotrophic factor (GDNF) (Miltenyi Biotec), and 0.5 μM SAG. On day 8, the medium was changed for inducing neural maturation to N2B27 containing 5 ng/ml activin A (Miltenyi Biotec), 0.1 mM dbcAMP (Sigma-Aldrich), 2 ng/ml BDNF, 0.2 mM L-ascorbic acid, 1 ng/ml TGFβ-3 (Peprotech), and 2 ng/ml GDNF. On day 10, the cells were seeded on a four-well plate for immunofluorescence and 2.5 μM N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT) (Sigma-Aldrich) and activin were added to the maturation medium. After 2 d, DAPT and acitivin were removed and the cells were further grown in the maturation medium for neural maturation until day 30.