Glutamatergic Neuron Differentiation from ESCs

Neuronal differentiation of ESCs into cortical glutamatergic neurons was carried out as previously described^{92 (link)}. In brief, the differentiation was carried out by adding doxycycline hyclate (2 μg/mL) to N2 supplemented media (Thermo Fisher, 17502048) with patterning factors SB431542 (Tocris, 1614, 10 μM), XAV939 (Stemgent, 04–00046, 2 μM) and LDN-193189 (Stemgent, 04–0074, 100 nM), as described previously^{92 (link)–94 (link)}. Puromycin selection was used (5μg/μL), from days 2 to 6 to remove non-transduced cells. At 4 days post induction, neuronal cells were resuspended into Neurobasal media (Gibco, 21103049) that was supplemented with B27 (Gibco, 17504044, 50X), doxycycline (2 μg/mL), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CTNF), and glial cell-derived neurotrophic factor (GDNF) (R&D Systems 248-BD/CF, 257–33 NT/CF, and 212-GD/CF at 10 ng/mL each). From this point onwards the neurons were either co-cultured with murine glial cells that were derived from early postnatal (P1-P3) mouse brains as described previously^{95 (link)} or were left to grow as monocultures (mouse strain https://www.jax.org/strain/100012; animal ethical committee approval by Harvard University: Animal Experimentation Protocol (AEP) # 93–15).