Cortical interneuron development is affected in 4H leukodystrophy 39 2 Differentiation in neural cells Differentiation into cerebellar granule cell neurons was done using a previously reported protocol, involving minimal factors in an N2+B27 neural maintenance medium (NMM), modified for a two-dimensional monolayer culture (Holmes & Heine, 2017a, 2017b). Cortical neuron differentiation was performed as previously described (Nadadhur et al., 2017) using dual SMAD inhibition and consequent maturation of cells into glutamatergic and GABAergic neurons. For myelinating cultures, human iPSCs were differentiated into glial progenitor cells as previously described (Dooves et al., 2019). To start a co-culture, day 18 cortical neurons were plated on a monolayer of rat astrocytes and matured for ~2 weeks, after which iPSC-derived glial progenitor cells were added to the culture. See supplementary method section for more details. RNA sequencing Total RNA was isolated from cells using TRIzol and chloroform-isopropanol extraction. After quality control samples were run on an Illumina HiSeq2500 according to manufacturer’s protocol. Sequencing reads were aligned to Humane Genome hg38. Similarity between fibroblast, iPSC and cerebellar cells was analysed, and the data of iPSC and cerebellar cells were compared to previously published gene expression profiles of 2 iPS cells and 2 granule cells. Differentially expressed genes (DEG) between 4H and control were determined with DESeq2 package in R. See supplementary methods for more details. qPCR For quantitative PCR (qPCR) analysis RNA was isolated from neurons using TRIzolchloroform isolation. After production of cDNA, a qPCR was performed using SYBR green according to manufacturer’s protocol. For DEG analysis, data was normalized for housekeeping gene EIF4G2. For interneuron subtype analysis, data was normalized for housekeeping gene EIF4G2 and general neuronal marker NEUN. See Supplementary Table 2 for an overview of the primers used. From the results the fold change 2-∆∆CT values were calculated, and the log fold change was used for statistical analysis. Data is shown as relative expression compared to day 30 control neurons.
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