Differentiation of iPSCs into Neural Progenitors

Prior to differentiation, iPSC lines were adapted to feeder-free conditions using Matrigel (BD) and EBs were formed. After 4 days, neural induction was initiated as previously described (32 (link),40 ). Briefly, EBs were plated onto Geltrex-coated plates in Neural Induction medium 1 (DMEM/F12 supplemented with L-Glutamine (2 mM), N2 supplement, bovine serum albumin (BSA) (1 mg/ml), Y27632 (10 μM; Tocris), SB431542 (10 μM, Tocris), Noggin (200 ng/ml) and antibiotic/antimycotic (1% v/v)). After 4 days, medium was changed to Neural Induction medium 2 (as NI1, without SB431542 and Noggin and with addition of SHH C24II (200 ng/ml; SHH C24II; R&D Systems). After 6 days, medium was supplemented with FGF8a (100 ng/ml; R&D Systems), Heparin (5 μg/ml; Sigma), BDNF (20 ng/ml) and Ascorbic Acid (200 μM; Sigma)) and incubated for 7 days, until the appearance of dense neural rosette structures. Neural progenitor cells were manually selected and re-plated onto Poly-D-Lysine/Laminin-coated plates in final differentiation medium (DMEM/F12 supplemented with L-Glutamine (2 mM), N2 supplement, BDNF (20 μg/ml), GDNF (20 μg/ml), N6, 2′ -O-dibutyryladenosine 3′,5′ -cyclic monophosphate sodium salt (dcAMP, 0.5 mM; Sigma), Laminin (1 μg/ml) and antibiotic/antimycotic (1% (v/v)). Neurons were matured for 2 weeks in this medium before experimental procedures were carried out.