Neural stem cell (NSC) culture

Neural stem cells NCRM1 (derived from cord blood, NIH) were thawed and cultured as previously described, NCRM‐1 (RRID:CVCL_1E71) (Wang et al, ^2013b). Briefly, we coated six‐well tissue culture plates using hESC‐qualified Matrigel (Corning Life Sciences, Tewksbury, MA, USA) for 30 min at room temperature. Cells were thawed from liquid nitrogen in a 37°C bath, spun at 1,000 rpm for 5 min, and plated at approximate concentration of 1 × 10⁵ cells/ml with StemPro™ NSC SFM medium (Gibco, ThermoFisher Scientific, USA). Cells were incubated for 2–3 days and passaged near confluence (80%). Passaging of the cells consisted of washing cells with D‐PBS, adding appropriate volume of accutase (ThermoFisher Scientific, USA) depending on plate surface area and incubating at 37°C. Dissociated cells were collected and spun down at 1,000 rpm for 5 min. Supernatant was removed carefully, and 10 ml of NSC medium was added. Cells were counted using Cellometer Auto 2000 cell counter (Nexcelom Biosciences, Lawrence, MA, USA) and plated onto a Matrigel‐coated 6‐well plate at 20,000–30,000 cells per cm². For luciferase reporter assay, NCRM‐1 cells were seeded at ~1.0 × 10⁵ cells/well on Matrigel‐coated 24‐well plates. The cells reached 90% confluence after 24 h, ready for the transfection experiments. NCRM1 cells were tested for contamination with mycoplasma. Cells were not authenticated via STR or other genetic methods because they were derived from cord blood and no previous genetic analyses were conducted that could be used for a comparison.