Chapter 6 200 MATERIAL & METHODS iPSC generation and maintenance The Institutional Research Board of the VU University Medical Center approved the study, and written consent was obtained from all participants in accordance with the declaration of Helsinki. iPSCs were generated from fibroblasts of healthy donors and 4H patients according to the previously described procedure (Holmes & Heine, 2017) or via CytoTune™- iPS 2.0 Sendai Reprogramming Kit according to manufacturer protocol. Pluripotency was confirmed by immunocytochemistry, alkaline phosphatase assay, PCR, embryoid body formation and/or pluritest. Additionally karyotyping and/or confirmation of disease variants was carried out. The human embryonic stem cell lines H01 and H09 were obtained from WiCell. Pluripotent stem cells were maintained in TeSR™-E8™ (STEMCELL Technologies, 05990) on Vitronectin XF™ coated plates (STEMCELL Technologies, 07180). Media was replaced daily or using double feedings over the weekend. Confluent wells were split with a ratio of 1:10 to 1:50 into a new well for further maintenance. An overview of the used stem cell lines can be found in Supplementary Table I. Differentiation into neuronal lineage Cortical neurons were generated using the previously published protocol (Nadadhur et al., 2017). In short, high-density iPSCs 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, 21103049), 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, Sigma-Aldrich, 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).
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