All cells described in this work were incubated at 37°C, 5% CO2, and 90% humidity unless otherwise stated. 409B2 (RIKEN BRC Cell Bank), Sc102a1 (System Biosciences) stem cells, and corresponding rtTA/NGN2-derivatives were cultured in standard feeder-free conditions in mTeSR1 (STEMCELL Technologies) on plates coated with Matrigel (Corning). Primary cortical rat astrocytes (Gibco) were cultured in high-glucose DMEM containing 10% fetal calf serum and 1% pen/strep on plates coated with poly-D-lysine (Sigma-Aldrich). Fresh medium was added to the astrocytes every 4–5 days and passaged once a week with trypsin-EDTA digestion at a standard ratio of 1:2. Astrocytes were used up to passage 10, with passage 0 being the culture of initial isolation. rtTA/NGN2 double-positive stem cell lines were generated and differentiated into NGN2-iNs as described previously (Frega et al., 2017).
