Differentiating human ESCs into neurons

Human embryonic stem cell line H8 was obtained from Prof. Wei Jiang (Wuhan University). Cells were maintained by mTeSR1 medium with feeder cell free. Neural induction was conducted according to the protocol as reported with minor modification (Chambers et al., 2009 (link)). Briefly, human ESCs were digested using accutase and cultured in suspension using neural induction medium (NIM) for 7 days. After embryonic body (EB) formation, EBs were transferred to 6-well plates pre-coated with matrigel. Cells were cultured with NIM until rosette formation. Rosettes were digested by neural rosette selection reagent (Cat#05832, STEMCELL Technologies, Canada) and seeded onto coverslips pre-coated with matrigel and cultured with 50% neuron-differentiation medium (NDM), and 50% neurobasal containing 2% B27, 100 nmol/L brain-derived neurotrophic factor (BDNF, Cat. 45002, Peprotech) and 100 nmol/L glia-derived neurotrophic factor (GDNF, Cat. 45010, Peprotech) for 3 days. Then, cells were treated with VPA (1 mM, P4543, Sigma) or vehicle solution for 3 days as described (Meng et al., 2022 (link)).
For evaluating neuronal differentiation, cells were collected at 8 d after VPA treatment. For assessing metabolic changes, cells were analyzed at 3 d following VPA treatment. For interfering glycolysis, 2-DG was added at 3-5 d post VPA treatment.