Chapter 2 38 and therefore provide a promising tool to study rare diseases like 4H leukodystrophy. To get insight into disease mechanisms and identify genes that are dysregulated by 4H mutations, we generated patient iPSC-derived cerebellar and cortical cell populations, including neuron-oligodendrocyte co-cultures and studied them by transcriptome, cellomics and multi-electrode array (MEA) analysis. In patient iPSC-derived cerebellar neurons we found a significantly lower expression of ARX, which is involved in early brain development and severe infantile-onset encephalopathies (Shoubridge et al., 2010). As ARX is associated with cortical interneuron development, we performed immunohistochemistry for GAD65/67 on patient material and confirmed alterations in inhibitory neurons in 4H. So, we decided to study the development of cortical neurons in more depth. Indeed, the number of GABAergic synapses was significantly decreased in 4H neuronal cultures, which correlated to an increased neuronal network activity. To further identify the specific subtypes that are affected in 4H, qPCR analysis for the major cortical interneuron populations was performed. Interestingly, the expression of ERBB4, important for the development of parvalbumin (PV) interneurons, was significantly increased in 4H neurons. Together, our results show that cortical interneuron development is affected in 4H patients, possibly due to pathway changes involving ARX that may affect development of parvalbumin interneurons. MATERIAL AND METHODS Patient consent 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. iPSC culture Fibroblasts from anonymous donors and 4H patients were reprogrammed into iPSC lines as described previously (Holmes & Heine, 2017a). Briefly, iPSCs were generated using an overexpression of OCT4, SOX2, KLF4, and C-MYC. iPSC lines were confirmed for pluripotency by ICC, PCR, alkaline phosphatase, embryoid body formation assay, karyotyping and/or a pluritest. Human embryonic stem cell line H01 was obtained from WiCELL and used as a control line for RNA sequencing experiments. See Supplementary Table 1 for an overview of the iPSC lines. Control and 4H iPSCs were maintained on a vitronectin coating in TeSR E8 medium. Medium was refreshed daily, and cells were passaged once a week using Gentle Cell Dissociation reagent (StemCell Technologies) according to manufacturer’s protocol. Cells were split 1:10-1:50 to a new well for further maintenance.
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