Chapter 5 170 split with a ratio of 1:10 - 1:50 into a new well for further maintenance. All subjects have given informed consent in accordance with the declaration of Helsinki. An overview of the used stem cell lines can be found in Supplementary Table I. Neural differentiation Human cortical neurons were generated using the previously published protocol (Nadadhur et al., 2017). In short, high-density human PSCs on GeltrexTM coated plates (±150 µg/ml, Gibco, A1413302) were induced into neurons by dual inhibition of the BMP and Activin/Nodal/TGF-β signaling pathways using 1 µM Dorsomorphin (Selleckchem, S7306) and 10 µM SB431542 (Selleckchem, S1067) in Neural Maintenance Medium (NMM) which consisted of 50% DMEM/F-12 with GlutaMAXTM (Gibco, 31331028), 50% NeurobasalTM medium (Gibco, 21103-049), 0.5x N2 supplement (Gibco, 17502001), 0.5x B27 supplement (Gibco, 17504-044), 2.5 µg/ml Insulin (Merck/Sigma-Aldrich, I9278), 1 mM L-Glutamine (Gibco, 25030081), 50 µM Minimum Essential Medium (MEM) Non-Essential Amino Acids (Gibco, 11140050), 50 µM Beta-Mercaptoethanol (Gibco, 21985023) and 1x P/S (Penicillin/Streptomycin, Sigma, P0781). Neural rosettes formed between 8 and 12 days after neural induction and were manually picked for transfer to PLO (20 µg/ml, SigmaAldrich, P3655)/ mouse laminin (20 µg/ml, Sigma-Aldrich, L2020) coated wells containing NMM supplemented with 20 ng/ml FGF2 (PeproTech, 100-18B) and 20 ng/ml EGF (PeproTech, AF-100-15). When confluent, neural rosettes were split 1:2 to 1:3 using TrypLETM (Gibco, 12563-011) and defined trypsin inhibitor (DTI, Gibco, R007100) or frozen in KnockOutTM Serum Replacement (KSR, Gibco, 10828028) with 10% dimethyl sulfoxide (DMSO, Sigma-Aldrich, D2650). Passage 1 – 3 of NES were used for differentiation into neurons using N2 medium, which consisted of DMEM/F-12 (without L-Glutamine, Gibco, 21331020), 1x N2 supplement, 0.1 mM MEM-NEAA, 2 mM L-Glutamine, 2 µg/ml Heparin (Sigma-Aldrich, H3393) and 1x P/S, supplemented with 400 ng/ml Human Sonic Hedgehog (Shh, PeproTech, 100-45) for 4 days. This was followed by 3 days of 10 µM Valproic acid (VPA, Sigma-Aldrich, P4543) in NB+ medium which consisted of NeurobasalTM medium (Gibco, 21103-049) with 1x B27 (Gibco, 17504-044), 18 mM HEPES (Gibco, 15630-056), 0.25x GlutaMAXTM (Gibco, 35050-038) and 1x P/S. On day 8, cells are passaged 1:2 – 1:4 (depending on density) using accutase and maintained in NB+ medium supplemented with 20 ng/ml BDNF (PeproTech, 450-02), 10 ng/ml GDNF (PeproTech, 450-10), 10 ng/ml IGF (PeproTech, 100-11) and 1 µM cyclic-AMP (Sigma-Aldrich, D0260) until day 18. At day 18 the cells were frozen in KSR with 10% DMSO.
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