Stemdiff forebrain neural differentiation medium

iPSCs were differentiated into neural progenitor cells (NPCs) using STEMDiff Neural Induction Medium (NIM) from StemCell Technologies. iPSCs were placed into a single cell suspension in NIM with SMADi/ROCKi (SMAD inhibitor and ROCK Inhibitor) in an AggreWell 800 plate. Embryoid bodies were cultured in the AggreWell plate for 5 days with NIM/SMADi partial medium changes daily. Embryoid bodies were plated onto Matrigel (Corning) coated plates and fed daily with NIM/SMADi medium until day 12 to allow neural rosette formation. Neural rosettes were selected using Neural Rosette Selection Reagent (StemCell Technologies) and plated onto Matrigel coated dishes with NIM/SMADi. The medium was changed daily for 7 days, after which neural progenitor cells were cryopreserved and split into defined Neural Progenitor Medium (StemCell Technologies). NPCs were plated on PLO/Laminin (Sigma) coated dishes in Neural Progenitor Medium. The following day the medium was changed to StemDiff Forebrain Neural Differentiation Medium (StemCell Technologies), which was changed daily for 7 days [25 (link), 26 (link)]. Cells were then plated onto PLO/Laminin coated dishes in defined Brain Phys Medium (with N2A, SM1, BDNF, GDNF, cAMP, and ascorbic acid) for neuronal maturation. Neurons were matured for 7 days for downstream experiments.