previously described (Yang et al., 2017). Puromycin (1 μg/mL; Sigma Aldrich, catalog# P8833) and hygromycin (50 ng/μL; Sigma Aldrich, catalog# H9773) selection was conducted for the following 2 days, and on day 5, the iN cells (~200,000 iNs/coverslip) were dissociated with Accutase™ and plated on glass coverslips with a monolayer of passage three primary mouse astrocytes (~50,000 cells/coverslip) isolated from postnatal day 1–3 pups, as described (Maximov, Pang, Tervo, & Südhof, 2007; Oni et al., 2016; Yang et al., 2017). On day 6, fresh Neurobasal™ media containing B27, l-glutamine, Ara-C (4 mM; Sigma Aldrich, catalog# C6645) 10 ng/mL of BDNF, NT3, and GDNF (Peprotech™). Following day 6, 50% of the culture medium was replaced every 5 days with fresh Neurobasal™ medium containing B27, l-glutamine, BDNF, NT3, and GDNF. Molecular, morphological, and functional analyses were conducted ~5 weeks (days in vitro [DIV] 40) following initial induction with doxycycline.
