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 twice per week (bFGF, PeproTech, Rocky Hill, NJ) for maintenance of NSCs in neurosphere form. During the medium changes throughout the years, no passaging (trypsin digestion and cell transferring, except dividing into multiple flask) was performed. All neurospheres for analyses were screened by size <1mm shape (round neurosphere) for their robust growth.
