The adult rat DRG-derived NSCs (DRG-NSCs) previously established in our laboratory (22) were maintained in Dulbecco's Modified Eagle Medium / F-12 Nutrient Mix (D-MEM/F-12) media containing N2 supplement (12μL/mL, Invitrogen, Carlsbad, CA) and penicillin-streptomycin (12μL/mL, Sigma, St. Louis, MO), and grown in a humidified incubator at 37°C and 5% CO2. Media was supplemented with 10ng/mL epidermal growth factor (EGF, Harlan Bioproducts for Science, Indianapolis, IN) and basic fibroblast growth factor (bFGF, PeproTech, Rocky Hill, NJ) twice per week for maintenance of NSCs. The NSCs were maintained in neurosphere form. During the medium changes throughout the years, no passaging (trypsin digestion and cell transferring, except dividing into multiple flasks) was performed. All neurospheres for analyses were screened by size, ≤0.5mm diameter (post-differentiation, round neurosphere), for their robust growth.
