The H9 hESCs (WA09, purchased from the WiCell research Institute, female, RRID: CVCL_9773) colonies were maintained on matrigel‐coated 6‐well plates (Corning) in E8 flex medium [E8 basal medium (Gibco) supplemented with E8 supplement Flex and 5 U ml−1 Penicillin−Streptomycin]. The colonies were passaged twice a week (in 1:3 split ratio) using 0.5 mM EDTA (Gibco). To induce NPC, the hESCs were harvested into single‐cell suspensions by accutase (Sigma) and seeded at 2.5 million cells per well of 6‐well plate in the neural induction medium (NIM) consisting of neural maintenance medium (NMM) supplemented with the dual‐SMAD inhibitors SB431542 (10 µM, Tocris) and LDN193189 (1 µM, Miltenyi)[34 (link)
]. (For NMM preparation, see Supporting Information: Extended Description of Methods.) At day 10, the neuroepithelial cells were passaged three times with Dispase II (Sigma) in the NMM up to 34 days to further purify the NPC. The generated DIV (Day In Vitro) 34 NPC were stored in liquid nitrogen cell bank for subsequent experiments. To enable cell viability tracking, a genome‐engineered H9 hESCs line bearing the constitutive tdTomato reporter expression cassette in the Adeno‐Associated Virus Integration Site 1 (AAVS1) locus were used to generate NPC as described above. The use of hESCs was approved by the Human Ethics Committee at the University Hospital, Gasthuisberg, KU Leuven, Belgium.
]. (For NMM preparation, see Supporting Information: Extended Description of Methods.) At day 10, the neuroepithelial cells were passaged three times with Dispase II (Sigma) in the NMM up to 34 days to further purify the NPC. The generated DIV (Day In Vitro) 34 NPC were stored in liquid nitrogen cell bank for subsequent experiments. To enable cell viability tracking, a genome‐engineered H9 hESCs line bearing the constitutive tdTomato reporter expression cassette in the Adeno‐Associated Virus Integration Site 1 (AAVS1) locus were used to generate NPC as described above. The use of hESCs was approved by the Human Ethics Committee at the University Hospital, Gasthuisberg, KU Leuven, Belgium.
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