Serum-free Differentiation of Cells

Throughout long-term culture, a population of cells at passages 2, 4, and 6 were cultured to confluency in serum-containing or serum-free conditions, and media was changed to a previously described serum-free differentiation media consisting of Neurobasal (Invitrogen #21103049, Carlsbad, CA, USA) and L15 (Invitrogen #11415064) basal media (1:1) supplemented with 1% antibiotic/antimycotic, 10 ng/mL insulin-like growth factor 1 (IGF-1; Shenandoah Biotechnology #100-34AF-100UG, Warminster, PA, USA) and 100 ng/mL epidermal growth factor (EGF; Shenandoah Biotechnology #100-26-500UG)^{10 (link)}. Cells were differentiated for 4-6 days (changing media every two days).