Chapter 2 70 Differentially expressed gene analysis Raw count data was used for differentially expressed gene (DEG) analysis with DESeq2 package.(Love et al., 2014) For cell type comparison, we performed DESeq2 for a specific cell type against all other samples and tested all 21,542 genes. Control vs patient DEG was performed per cell type, and genes were further filtered on such with CPM > 1 in ≥ 50% of samples with the testing cell type. The number of tested genes for fibroblast, iPSC and product are 16,890, 18,855 and 19,032 genes, respectively. Genes with absolute log2 fold change (logFC) > 1 and Bonferroni corrected P-value < 0.05 were defined as DEG. Cortical neuron differentiation Neurons were generated from iPSCs according to previously published protocol (Nadadhur et al., 2017). Shortly, iPSCs were differentiated into neural epithelial stem cells (NES) using N2/B27 medium supplemented with Dorsomorphin and SB431. Within 1-2 weeks after plating neural rosettes appeared in the culture, which were selected by manual cutting. NES cells were maintained in N2/B27 medium supplemented with FGF2 and EGF for up to 4 passages. To differentiate NES cells into neurons, medium was changed to N2 medium supplemented with SHH for 4 days, followed by 3 days in Neurobasal/B27 medium with Valproic Acid. The day of the medium change to N2 medium with SHH was considered day 1 of neuronal differentiation. At day 8 of differentiation, cells were split into a new PLO/Laminin coated plate in Neurobasal/B27 medium supplemented with BDNF, GDNF, IGF1 and cAMP. At day 18, Neurons were passaged a final time and either frozen for later use or plated on rat astrocytes for further neuronal maturation. From now on, half of the medium was refreshed twice a week. At day 25, neurons were treated with AraC to remove proliferating progenitors. Neurons were kept in culture until day 56 (~5 weeks), at which point they resembled mature neurons in morphology and synaptic marker expression. For MEA and neuronal subtype proportion analysis, cultures were kept until 10 and 14 weeks post plating. Oligodendrocyte differentiation Oligodendrocyte progenitor cells (OPCs) were generated from iPSCs according to a previously published protocol (Dooves et al., 2019). Shortly, iPSCs were plated on an antiadherent plate in N2/B27 medium with EGF, FGF2 and T3 to induce embryoid body formation. From day 2 onwards, RA was additionally added to the medium. At day 10, cells were plated on geltrex coated plates in N2/B27 medium with EGF and T3. At day 18, medium was switched to N2/B27 medium without vitamin A. Throughout the differentiation, cells
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