Towards a 3D spheroid system for modelling leukodystrophies 131 4 DISCUSSION Our study demonstrates that 3D spheroid models provide valuable access to leukodystrophy-relevant cell types and molecular pathways, enabling the exploration of both known and potentially novel disease mechanisms. With immunofluorescence, we confirmed the presence of key white matter cell types, including oligodendrocytes, astrocytes, and neurons. Using single cell RNA-seq we showed more detailed cellular diversity. The generation of spheroids in a single centralized facility enabled meaningful disease-specific comparisons. This was particularly evident in the replication of differentially expressed genes (DEGs) identified through both cell cluster-based analysis and isogenic line comparisons. These findings underline the model's robustness in detecting disease-driven molecular signatures. Beyond confirming previously implicated pathways, this model serves as a dynamic platform for generating novel hypotheses in leukodystrophy research. The integration of high-throughput sequencing with advanced bioinformatics pipelines supports comprehensive data exploration, allowing for the identification of new molecular targets and potential therapeutic pathways. Although we have shown the potential use of this data set, results as described in this thesis are preliminary, hence, we point out future directions of research that should be explored before publication. Disease-specific alterations of cellular composition We identified cellular, molecular and pathway differences in clusters from the oligodendrocyte, astrocyte, and neuronal lineage in spheroids of several leukodystrophies. Particularly interesting is the underrepresentation of some clusters in LDs such as the clusters cycling radial glia and outer radial glia. Radial glial cells develop early in development from neuroepithelial stem cells that line the ventricular zone. Radial glial cells both provide a scaffold for neuronal migration and give rise to intermediate progenitors for neurogenesis and later gliogenesis. Outer radial glia are a type of radial glia specifically present in primate brains, which are involved in cortical expansion and also give rise to specific oligodendrocyte progenitor cell populations (Pollen et al., 2015; Zhou et al., 2024). It would be interesting to study whether radial glial cells show a normal abundance in early spheroids, and whether this would be correlated to a normal neurogenesis but possible disrupted gliogenesis, which only starts in later developmental stages. Possibly, pseudotime analysis of the existing data can be applied to see from which clusters those underrepresented clusters originate and if other clusters are derived from those clusters. Next, is to wonder whether those cells are completely absent in the model, or severely affected by the pathology and hence mislabelled or not sequenced in the single cell
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