Glutamatergic differentiation protocol

Glutamatergic differentiation was performed as described by Ho et al.105 Briefly, cells were seeded on laminin-coated plates 24 h before infection. Cells were double-infected with TetO-hNGN2-P2A-EGFP-T2A-PuroR and CMV-rtTA as described above. 48 h after infection, doxycycline (1 μg/mL) was administered to the cells, followed by puromycin (0.5 μg/mL) the next day. After 24 h of puromycin selection, EGF and FGF-2 were withdrawn from the medium, and cells were allowed to differentiate for 21 days. During the first week of differentiation, half of the medium was changed every day, maintaining doxycycline concentration. In the second week of differentiation, the medium was changed every other day, reducing doxycycline concentration to half with each change until complete removal by the last week of differentiation. qRT-PCR was performed to confirmed overexpression of glutamatergic marker VGlut1.