Efficient Directed Differentiation of PSCs into NPCs

Primed PSCs were differentiated into expandable NPCs by using the STEMdiff SMADi Neural Induction Kit (Stem Cell Technologies) as previously described [34 (link)–36 (link)]. In brief, primed PSCs were maintained on a Matrigel (Corning)-coated plate in mTeSR1 media (Stem Cell Technologies) prior to the NPC induction. The cells were harvested using Accutase (EMD Millipore) and transferred at 3 x 10⁶ cells to a well of an AgrreWell800 plate (Stem Cell Technologies) in STEMdiff Neural Induction Medium + SMADi (Stem Cell Technologies) supplemented with 10 μM Y-27632. Five days later, uniformly sized aggregates were collected using a 37 μm Reversible Strainer (Stem Cell Technologies) and plated onto a Matrigel-coated 6-well plate in STEMdiff Neural Induction Medium + SMADi. Seven days later, neural rosette structures were selectively removed by using STEMdiff Neural Rosette Selection Reagent (Stem Cell Technologies) and plated onto a new Matrigel-coated 6-well plate in STEMdiff Neural Induction Medium + SMADi. After that, the cells were passaged every 2–3 days until day 30 post-differentiation. The established NPCs were maintained on a Matrigel-coated plate in STEMdiff Neural Progenitor Medium (Stem Cell Technologies) and passaged every 3–4 days.