Human iPSC forebrain neuron differentiation

To generate telencephalic neurons from iPSCs, stem cells were cultured on a feeder layer of irradiated MEFs in 6-well tissue culture-treated plates for around 6 days, with the human ESC media (+10 ng/ml fibroblast growth factor [FGF]-2) changed daily. When nearly confluent, cells were detached from the feeder layer to initiate neural differentiation, as described previously (36–38). Briefly, iPSC aggregates were cultured in suspension for 4 days in human ESC media and then transferred to neural induction media (NIM). After an additional 3 days in suspension, iPSC aggregates were plated onto 6-well tissue culture plates in NIM with 10% fetal bovine serum. After 12 h, the media was replaced with fresh NIM. Media was then changed every other day until day 17, when the generated neuroepithelial (NE) cells were isolated. Mechanically isolated NE cells were cultured in suspension with NIM (+B27, +cAMP, +insulin-like growth factor 1 [IGF-1]) to generate neurospheres for at least 10 additional days. On about day 28, neurospheres were dissociated and plated onto polyornithine- and laminin-coated coverslips in neural differentiation media (NDM) containing N2, B27, ascorbic acid, cAMP, laminin, IGF-1, brain-derived neurotrophic factor and glial-derived neurotrophic factor. Half of the media was changed every other day for 6–12 weeks, depending on the analysis to be performed. For treatment of cells with mdivi-1, the media was replaced with standard neural differentiation media supplemented with 10 µM mdivi-1 (Sigma-Aldrich) dissolved in DMSO.