24-well plate). On day 7, iN cells were dissociated using Accutase and plated on coverslips (~3 × 105 cells/well of 24-well plate) in Neurobasal medium supplemented with B27/Glutamax (Invitrogen) containing BDNF (20ng/ml, Peprotech). Around day 10, Ara-C (2 g/l, Sigma) was added to the medium to inhibit astrocyte proliferation. After day 7, 50% of the medium in each well was exchanged every 2–3 d. FBS (1%) was added to the culture medium on day 10 to support astrocyte viability, and iN cells were assayed on day 35 (5 weeks) in most experiments. The efficiency of conversion of ES and iPS cells into iN cells was calculated from counts of cell densities in ten random fields on each coverslip (Fig. 1b) as the percentage of HuNu-positive cells that expressed MAP2 as revealed by immunofluorescence assay.
