Thiazovivin

iPS lines were maintained on MEF prior to EB-based differentiation, in serum-free hES medium supplemented with 8ng/mL hbFGF. Prior to monolayer-based iCD34 differentiation, iPS lines were cultured on Matrigel in hES media containing 0.4μM PD032590, 1uM CHIR99021, 5μM Thiazovivin, 2μM SB431542 (all Biovision), 10μM ROCK-inhibitor (Ascent) and 10ng/mL hbFGF (R&D Systems) as previously described^{63 (link)}.
Fresh media was provided every day and cells were passaged every 3–4 days as previously described^{12 (link), 51 (link)}. iPS lines were tested for mycoplasma contamination every 2 months.

Ngn2-iN and AD-iN cells were generated as described (Yang et al., 2017 (link); Zhang et al., 2013 (link)). To generate AM-iN cells, ES cells were treated with Accutase (Innovative Cell Technologies) and plated as dissociated cells in six-well plates (H1 cells: ~5 × 10⁴ cells/well) on day 0. Cells were plated on plates coated with Matrigel™ and mTeSR1 containing 2μM Thiazovivin (Bio Vision). At the same time of plating, lentivirus prepared as described above (1.5 μl/well of six-well plate) was added. On day 1, the culture medium was replaced with N2/DMEM/F12/NEAA (Invitrogen) containing doxycycline (2 μg/ml, Clontech) to induce TetO gene expression, and the culture was retained in the medium for ~2 weeks. On day 2 and 3 puromycin was used to selected transduced cells. From day 5 to day 7 AraC (4μM) was added to the media to selected for not dividing cells. On day 6, mouse glial cells were plated on matrigel-coated coverslips (~5 × 10⁴ cells/well of 24-well plate). On day 7, iN cells were dissociated using Accutase and plated on glial cells (~3 × 10⁵ cells/well of 24-well plate). Cultures were analyzed 5 weeks after induction of the transgenes. Cocultures of excitatory and inhibitory neurons (Ngn2 iN cells and AD iN) were generated as described (Yang et al., 2017 (link)).