Directed Differentiation of mESCs to NPCs

ES to NPC differentiation was performed according to standard protocols with small modifications as previously described (Conti et al, 2005 (link); Splinter et al, 2011 (link); Bowness et al, 2022 (link)). First, mESCs were extensively isolated from feeder cells by four rounds of pre-plating, each for 35–40 min. 0.5 × 10⁶ mESCs were plated to gelatin-coated T25 flasks in N2B27 media (50:50 DMEM/F-12:Neurobasal (Gibco) supplemented with 1× N2 and 1× B27 (ThermoFisher), 1 mM l-glutamine, 100 μM β-mercaptoethanol, 50 U/mL penicillin/50 μg/mL streptomycin (all from Life Technologies) with 1 μg/ml doxycycline for continuous Xist induction. Media changes were performed every day except day 1. After 7 days, cells were detached with Accutase (Merck Life Sciences) and 3 × 10⁶ cells were plated to grow in suspension in 90 mm bacterial petri dishes containing N2B27 media supplemented with 1 μg/ml doxycycline and 10 ng/ml EGF and FGF (Peprotech). Embryoid-body-like aggregates were collected by mild centrifugation (100× g for 2 min) on day 10 and re-plated to 90 mm gelatinised cell culture dishes in N2B27 + dox + FGF/EGF media, with media changes performed every other day. Upon reaching ~80% confluency, NPC outgrowths were passaged using Accutase at a 1:3–1:4 ratio to new cell culture dishes until the establishment of a homogenous cellular population of NPCs from approximately day 15–17.