ESCs/iPSCs were treated with CTK dissociation solution for 2 min and feeder cells were removed with PBS. Then ESCs/iPSCs were dissociated to single cells with Accumax, and they were transferred onto Matrigel-coated plates with MN medium containing a 1:1 mixture of Neurobasal medium (Thermo Fisher Scientific) and DMEM/F12 (Thermo Fisher Scientific), supplemented with 0.5% N2 (Thermo Fisher Scientific), 1% B27 (Thermo Fisher Scientific), 1 μM retinoic acid (Sigma-Aldrich), 1 μM smoothened agonist (Enzo Life Sciences), 10 ng/mL brain-derived neurotrophic factor (BDNF; R&D Systems), 10 ng/mL glial cell-derived neurotrophic factor (GDNF; R&D Systems), 10 ng/mL neurotrophin-3 (NT-3; R&D Systems), and 10 μM Y-27632 (Nacalai Tesque). At the same time, the ESCs/iPSCs were infected with SeV-L-N-I; SeV-L-N-I-EGFP; or combinations of SeV-L, SeV-N, and SeV-I (ID Pharma) on day 0. MOIs were 5 or 10. The transduction of SeV vectors to human ESCs/iPSCs was conducted just once. The number of cells per well was 5.0 × 104 in 96-well plates and 1.0 × 106 in 12-well plates. The medium was changed to MN medium without Y-27632 on day 1 and then changed every 3 days.
For phenotype assays, cells were treated with Accumax plus 10 μM Y-27632 and transferred onto poly-L-lysine- and Matrigel-coated glass dishes on day 7. For immunocytochemistry and qPCR analyses, cells were assessed on day 14.
For phenotype assays, cells were treated with Accumax plus 10 μM Y-27632 and transferred onto poly-L-lysine- and Matrigel-coated glass dishes on day 7. For immunocytochemistry and qPCR analyses, cells were assessed on day 14.