Differentiation of Neural Progenitor Cells

The neurons were differentiated according to Zhang et al. (2017 (link)). When the NPCs reached 70% confluency, they were plated on Matrigel in a neural expansion media (NEM). The NEM was supplemented with 20 ng/ml of bFGF, and EGF. The NPCs were re-plated several times until passage three. Then, they (for last passage) were plated on 0.001% Poly-L-Ornithine (P4957; Sigma-Aldrich, St. Louis, MO, United States) and a 5 μg/ml laminin (L2020; Sigma-Aldrich, St. Louis, MO, United States) coating in a neural maturation media (NMM) for 10 days. The NMM was supplemented with 20 ng/μl brain-derived neurotrophic factor (BDNF) (CYT-207; Prospec, Rehovot, Israel), 10 ng/μl glial cell line-derived neurotrophic factor (GDNF) (CYT-305; Prospec, Rehovot, Israel), 200 μM L-ascorbic acid 2-phosphate (A8960, Sigma-Aldrich, St. Louis, MO, United States), and 50 μM dibutyryl-cyclic adenosine monophosphate (db-cAMP) (A6885; Sigma-Aldrich, St. Louis, MO, United States). Half media change was performed every 3 days. Ten-day immature neurons were passaged a last time on PLO-Laminin coated plates with the NMM at a seeding density of 50,000 cells/cm² until they reached maturation for the metabolic assays.