Generating Neurons from iPSCs

An existing protocol, based on the synergistic action of two inhibitors of SMAD signaling, Noggin and SB431542, was adapted to generate neural progenitor cells (NPCs), which were then further induced to differentiate into neurons (not enriched in DAn) (Chambers et al, 2009 (link)). Briefly, the iPSCs were maintained in mTeSR™ medium until confluence. EBs were generated and maintained in suspension for 48 h. Subsequently, the medium was changed to a proneural medium (PN) and maintained for 5 days. PN medium consists of DMEM/F12 medium (GIBCO), neurobasal medium (GIBCO), 0.5× B27 minus vitamin A supplement (GIBCO), 0.5× N2 supplement (GIBCO), 2 mM Ultraglutamine (Lonza), β‐mercaptoethanol (Life Technologies), and penicillin–streptomycin (Lonza). To obtain neural rosettes, EBs were seeded on poly‐l‐ornithine/laminin (POLAM)‐coated plates with PN medium supplemented with Noggin (200 ng/ml; Peprotech) and SB431542 (10 μM; Tocris). After 8–12 days, the neural rosettes were picked and expanded in new POLAM‐coated dishes. Neural rosettes were enzymatically dissociated and seeded in new POLAM‐coated plates with PN medium supplemented with FGF2 (10 ng/ml; Peprotech) and EGF (10 ng/ml; Peprotech) to promote the generation and proliferation of neural progenitor cells (NPCs). After forming a homogenous cell population, the NPCs were further differentiated into GABA and glutamatergic neurons on POLAM‐coated plates with PN medium during 3–5 weeks.