Glutamax

Briefly, confluent hPSCs were washed once with PBS and differentiation was started by adding 500 μL of medium MN (KO DMEM/F12 (Gibco)), containing 1× N2-A Supplement (Stem Cell Technologies), 1× SM1 (Stem Cell Technologies), 1× Glutamax (Gibco), 50 mM of ascorbic acid (Sigma), 1 μM of Compound C, 1 μM of CHIR, 10 μM of Rock Inhibitor, and 1 μM SB431542. The medium was changed every 24 h for a total of 6 days, after which, the hPSC-derived ectodermal cells were collected for downstream analyses.

Human neural stem cells (hNSC) obtained from ThermoFisher Scientific (Schwerte, Germany) were cultivated according to the manufacturer's recommendations. Cells were maintained in an NSC medium, consisting of KnockOut D‐MEM/F12 (Gibco‐ThermoFisher Scientific) supplemented with 2 mM Glutamax (Gibco‐ThermoFisher Scientific), 2% StemPro Neural Supplement (Gibco‐ThermoFisher Scientific), 1% Penicillin–Streptomycin (Gibco‐ThermoFisher Scientific), 20 ng/μL bFGF (Peprotech, Hamburg, Germany), and 20 ng/μL EGF (Peprotech) in a humidified incubator at 37°C and with 5% CO₂. Differentiation of NSC to astrocytes was performed according to a protocol established in our lab (Alisch et al. 2021 (link); Kerkering et al. 2023 (link)). In short, for the differentiation of hNSCs to astrocytes, hNSCs were plated on Geltrex‐coated (ThermoFisher Scientific) 6‐well plates and cultivated in astrocyte differentiation medium (ADM), which contains D‐MEM (Gibco‐Thermo Fisher Scientific) supplemented with 2 mM Glutamax, 1% N‐2 (Thermo Fisher Scientific), 1% Penicillin–Streptomycin, 1% FCS (Sigma‐Aldrich‐Merck, Darmstadt, Germany), and 20 ng/mL CNTF (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were cultivated for 4 weeks, and medium was changed every 3–4 days. For experiments, astrocytes were plated on Geltrex‐coated 16‐well chamber slides in a density of 30,000 cells/0.5 cm³ and maintained in astrocyte cultivation medium (ACM) containing ADM without FCS for 5 days before starting experiments.