NPCs were differentiated into neurons and glia by removing FGF and culturing in NGD base levels of insulin (5ug/mL) without retinoic acid addition (NGD contains no additional retinoid). To initiate differentiation, NPCs were plated in a 35mm dish on 1% matrigel at 5*105/cm2 and fed 5mL of NGD every 2–3 days. At 4 weeks, neurons appeared in the culture and a final dissociation was performed. The culture was dissociated in the presence of 0.05% DNase I by incubation with Accutase (Stem Cell Technologies) for 30′ at 37degC with gentle agitation (bacterial rotator), re-suspended in chilled HBSS/0.1% BSA and filtered through a 40μM mesh before being centrifuged through a cushion of 4% BSA to remove cellular debris. For 2D cultures and for medium conditioning, cells were re-plated at 5*105/cm2 on 0.1% PEI-coated plastic or glass. For re-aggregation with pMGLs, 3D cultures were initiated by plating 1.5×106cells in a 0.3cm2 PET transwell with 0.4mm pores coated with 0.1% PEI. Cells were mixed with transduced pMGLs in a 1:10 ratio. Spheroid formation was initiated by re-aggregating 3*104 NPCs per well in 96-well ultra-low attachment plate (corning), mixed with pMGLs at 1:10 ratio.
