Chapter 4 134 MATERIALS & METHODS iPSC culture iPSCs were generated by each institute according to previously described protocols (Dooves et al., 2023; Holmes & Heine, 2017; Mangiameli et al., 2021) All subjects have given informed consent in accordance with the declaration of Helsinki. Briefly, fibroblasts were reprogrammed into iPSCs using either the CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Invitrogen, A16517), polycistronic construct with OCT4, SOX2, KLF4, and C-MYC within a lentiviral vector or StemMACS mRNA Reprogramming Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Pluripotency was confirmed by immunocytochemistry, alkaline phosphatase assay, PCR, embryoid body formation and/or pluritest. Karyotype analysis was also performed to exclude chromosomal abnormalities. GALC KO and KI iPSC were produced by CRISPR/Cas9 mediated gene editing of the HD2 iPSC line: in particular GALC KO iPSC were obtained using the Gene Knockout Kit v2 (Synthego), designed to target the first exon of the human GALC gene, according to the manufacturer directions; while the production of GALC KI iPSC was outsourced to Synthego and was obtained by inserting the pathogenic G553R mutations (GALC, c.1657G>A ) in both alleles. After generation and characterization, all cell culture procedures were performed centralized in the IRCCs San Raffaele Scientific Institute. iPSCs from IRCCs San Raffaele Scientific Institute and the Institute of Reconstructive Neurobiology, were thawed and maintained in matrigel-coated (Merck Millipore, CLS354230) plates with StemMACS iPSBrew XF medium (iPS-Brew, Miltenyi Biotech, 130-104-368). iPSCs were refreshed daily and incubated at 37°C in controlled humidified atmosphere (5% CO2, 5% O2). When confluent, cells were split in a 1:3 to 1:10 ratio using 0.5 mM EDTA (ThermoFisher, 15575020) diluted in PBS, for further maintenance. iPSCs of the Vrije Universiteit were thawed in the culture condition of the origin institute, TeSR™-E8™ (STEMCELL Technologies, 05990) supplemented with 10 µM ROCK inhibitor Y-27632 (Selleckchem, S1049) on Vitronectin XF™ coating (STEMCELL Technologies, 07180). The following passage, cells were plated on Matrigel-coated plates and were adapted to iPS-Brew medium. An overview of the used stem cell lines can be found in Supplementary Table I. Spheroid culture Spheroids from a total of five control, three Globoid Leukodystrophy, three Canavan disease and two 4H leukodystrophy iPSC lines (See Supplementary Table I) were generate according
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