The lentivirus generation protocol was previously outlined by Wang et al. (88). Selection with puromycin (1 mg/ml; Sigma-Aldrich, catalog #P8833) was carried out for the next 2 days. On day 5, the Ngn2-iNs (approximately 200,000 iNs per coverslip) were dissociated using Accutase and plated on glass coverslips coated with primary mouse glia (approximately 50,000 cells per coverslip). On day 6, fresh Neurobasal medium containing B-27 supplement, l-glutamine, Ara-C (4 mM; Sigma-Aldrich, catalog #C6645), brain-derived neurotrophic factor (BDNF) (10 ng/ml; PeproTech, catalog #450-02), NT3 (10 ng/ml; PeproTech, catalog #450-03), and glial cell line–derived neurotrophic factor (GDNF) (10 ng/ml; PeproTech, catalog #450-10) were added. Subsequently, every 5 days, starting from day 6, 50% of the culture medium was replaced with fresh Neurobasal medium supplemented with B27, l-glutamine, BDNF, NT3, and GDNF.
