Lentiviruses were harvested with the medium 46 hr after transfection, pelleted by centrifugation (49,000 × g for 90 min), resuspended in MEM, aliquoted, and snap-frozen in liquid N2. Only virus preparations with > 90% infection efficiency as assessed by EGFP

expression or puromycin resistance were used for experiments. For IDO inhibitor details of lentiviral constructs, see Supplemental Experimental Procedures. ESCs and iPSCs were treated with Accutase (Innovative Cell Technologies) and plated as dissociated cells in 24-well plates (H1: 1 × 104 cells/well; iPSCs: 1.5 × 104 cells/well) on day −2 (Figure 1B). Cells were plated on matrigel (BD Biosciences)-coated coverslips in mTeSR1 containing 2 μM thiazovivin (Bio Vision). On day −1, lentivirus prepared as described above (0.3 μl/well of 24-well plate) was added in fresh mTeSR1 medium containing polybrene (8 μg/μl, Sigma). On day 0, the culture medium was replaced with N2/DMEM/F12/NEAA (Invitrogen) containing

human BDNF (10 μg/l, PeproTech), human NT-3 (10 μg/l, PeproTech), and mouse laminin (0.2 mg/l, Invitrogen). Doxycycline (2 Selleckchem BLZ945 g/l, Clontech) was added on day 0 to induce TetO gene expression and retained in the medium until the end of the experiment. On day 1, a 24 hr puromycin selection (1 mg/l) period was started. On day 2, mouse glia cells were added in Neurobasal medium supplemented with B27/Glutamax (Invitrogen) containing BDNF and NT3; Ara-C (2 g/l, Sigma) was added to the

about medium to inhibit astrocyte proliferation. After day 2, 50% of the medium in each well was exchanged every 2 days. FBS (2.5%) was added to the culture medium on day 10 to support astrocyte viability, and iN cells were assayed on day 14 or 21 in most experiments. The efficiency of conversion of ESCs and iPSCs into iN cells was calculated by two approaches from counts of cell densities in four random fields on each coverslip (Figure 2D): (1) as the percentage of EGFP-positive lentivirally transduced cells that also express MAP2 or NeuN; (2) as the percentage of starting cells that become NeuN positive. Immunofluorescence experiments were performed essentially as described (Pang et al., 2011). Briefly, cultured iN cells were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature, washed three times with PBS, and incubated in 0.2% Triton X-100 in PBS for 10 min at room temperature. Cells were blocked in PBS containing 10% goat serum for 1 hr at room temperature. Primary antibodies were applied overnight, cells were washed in PBS for three times and blocked with 10% goat serum for 15 min, secondary antibodies were applied for 1 hr. Transplanted iN cells in mouse striatum were analyzed by immunofluorescence staining after mice were transcardially perfused with saline followed by 4% paraformaldehyde.