Neuronal differentiation

Single cell-dissociated hESCs (1 × 10⁵) were plated onto Matrigel-coated 24-well plates in mTeSR™1 supplemented with 5 µM of Y27632 (day − 4). On day − 3, the medium was replaced with mTeSR™1 without Y27632, and the medium was changed daily. On day 0, the mTeSR™1 medium was replaced with neuronal cell induction medium (1% MEM-NEAA, 1% GlutaMX™, 2% B27 supplement, 1% N2 supplement, and 20 µg/mL of human insulin in a 1:1 mixture of neurobasal medium and DMEM/F-12), and the medium was replaced every day until day 10. On day 11, the dissociated cells were resuspended in a neuronal cell induction medium supplemented with 5 µM of Y27632 and replated onto a Matrigel-coated 24-well plate (3 × 10⁵ cells/well). On day 13, the medium was changed to neuronal cell induction medium supplemented with 100 nM of LDN-193,189, 10 µM of SB431542, and 2 µM of XAV 939. The medium was refreshed every other day until day 17. On day 19, the medium was changed to a neuronal cell maturation medium (1% N2 supplement, 2% B-27 supplement, and 25 ng/mL of BDNF in neurobasal medium). The medium was then changed every other day. On day 25, the cells were dissociated and replated onto a Matrigel-coated 24-well plate (1 × 10⁵ cells/well), and the medium was replaced every other day.