Tesr e8 medium

Three hiPSC lines were used in this work. NESTIN-GFP knockin reporter (NES-GFP) and ND2-0 hiPSC were obtained from the NIH Center for Regenerative Medicine. hiPSC line USCK7 was generated in-house from human urine-derived cells by Cytotune (Life Technologies) reprogramming kit [21 (link)]. All hiPSCs were cultured on Matrigel-coated dishes in TeSR-E8 medium (Stemcell Technologies).

For the generation of iPSC-CFs a protocol developed by Zhang et al. was used^{13 (link)}. Briefly, human iPSCs were dissociated with 1 mL/well 0.5 µM EDTA solution (Invitrogen) at RT for 5 min and seeded on Vitronectin XF (StemCell Technologies) coated 6-well plates at a density of 15.000–30.000 cells/cm² in TeSR-E8 medium (StemCell Technologies) supplemented with 5 μM ROCK inhibitor (Y-27632) (Tocris) for 24 h. Cells were cultured for 6–7 days in TeSR-E8 medium with medium changes every other day until they reached 100% confluency and differentiation started (day 0). At day 0, the medium was changed to 2.5 mL/well RPMI + B27 without insulin (Gibco) and supplemented with 12 µM CHIR99021 (Tocris) for 24 h (day 1). After day 1, the medium was changed to 2.5 mL RPMI + B27 without insulin for 24 h (day 2). Afterwards, the medium was changed to 2.5 mL/well of the CFBM medium (Table S1) supplemented with 75 ng/mL bFGF (StemCell Technologies). Cells were refreshed with 2 mL/well CFBM supplemented with 75 ng/mL bFGF every other day until day 20 when RNA was collected, and cells were dissociated using TrypLE Select (10x) (Thermo Fisher) for 10 min at 37 °C. After dissociation, cells were cultured in DMEM + 10% Fetal bovine serum. For the first two passages, 5 μM ROCK inhibitor was added for 24 h to help cell attachment. Cells between passage 3–6 were used for experiments.

Human iPSCs were maintained in StemMACS human iPSC Brew medium (Miltenyi) or TeSR-E8 medium (Stemcell Technologies). The differentiation of iPSCs into neuronal precursor cells (NPCs) was promoted as previously described^{69 (link)}. Subsequent differentiation into astrocytes was performed as detailed elsewhere^{69 (link)}. Differentiation of NPCs into neurons was conducted by neurogenin 2 overexpression (NGN2). Briefly, NGN2 coding sequence was cloned into the pCW57.1 backbone (gift from David Root (Addgene plasmid # 41393). Human iPSC-derived NPCs were transduced with a VSV-G pseudotyped lentivirus carrying the LV-NGN2 transgene and amplified as previously described^{69 (link)} in the presence of puromycin (2 μg/ml). Cells were then plated on plates coated with poly-L-ornithine and Laminin in DMEM/F-12 medium supplemented with N-2 and B-27 supplements (1x) (Thermofisher), doxycycline (2 μg/ml) and Laminin (2 μg/ml). Medium was changed every two to three days. At day 8, medium was supplemented with BDNF (10 μg/ml) and GDNF (10 μg/ml). Neurons were used for the experiments after 14 days of differentiation.