The protocol of generating GABAergic human iN cells was described recently 27. Briefly, iPS cells and ES cells were plated as dissociated cells on Matrigel ® Matrix (Corning Life Sciences)-coated dishes in mTeSR (Stem Cell Technologies) medium with 2 ¼M Y-27632 (Stemgent). The following day, the cells were infected with Ascl1, Dlx2 and rtTA lentiviruses for 10-12 hours upon which culture medium was replaced with Neurobasal medium (GIBCO by Life Technologies) with B27 and L-Glutamine supplemented with 2 ¼g/mL of doxycycline (MP Biomedicals) and 2 ¼M Y-compound to induce TetO expression. The protocol for generating lentiviruses expressing different transcription factors was previously described 27. Puromycin and hygromycin selection was conducted for the following 2 days, and on day 5, the iN cells were dissociated with Accutase and plated on glass coverslips with a monolayer of passage three primary astrocytes isolated from p1-3 pups, as described previously 24, 27, 28. Following plating, 50% of the culture medium was replaced every 2-3 days with fresh Neurobasal media containing B27, L-Glutamine, 100 ng/ml of BDNF, NT3 and GDNF.
