The iPS cell differentiation and iN seeding and analysis procedures are summarized in Fig. 2. iPS cells were passaged into a 6 well plate at a density of 500 000–750 000 cells per well on day 1 using mTeSR™1 medium (STEMCELL Technologies) with 5 μM Y-compound (Y-27632, a ROCK1 and ROCK2 inhibitor which promotes the survival of stem cells).28 On day 0, iPS cell medium was changed to mTESR containing 5 μM Y-compound and lentivirus specific to the desired neuronal phenotype. Excitatory neurons were generated by the addition of Ngn2 lentivirus and inhibitory neurons were generated by the addition of Dlx2 and Ascl1 lentiviruses.26 Since Ngn2, Dlx2, and Ascl1 virus use the Tet-on operator, all cells were also infected with rtTa lentivirus at a ratio of 1.5× the volume of the other viruses.
