Chapter 4 110 ABSTRACT Leukodystrophies (LDs) are a heterogeneous group of rare genetic disorders characterized by progressive white matter (WM) degeneration. Often, the genetic cause is known, but knowledge of underlying disease mechanisms remains poorly understood, limiting therapeutic development. Advances in in vitro disease models, particularly brain spheroids, might recapitulate the cellular diversity of brain tissue, enabling the study of complex cell interactions during development. In this study, we aimed to investigate whether 3D spheroids can be used to model and study several leukodystrophies. We applied a 3D spheroid model derived from human induced pluripotent stem cells (iPSCs) to investigate Globoid Leukodystrophy (GLD), 4H Leukodystrophy, and Canavan Disease (CD). We characterized spheroid growth, cellular composition, and myelination capacity using immunofluorescence and electron microscopy, confirming the presence of a diverse range of brain cell types, including neurons, oligodendrocytes, and astrocytes. Single-cell mRNA sequencing identified 27 cell clusters. Notably, the clusters of cycling radial glia and neural progenitor cells were significantly underrepresented in all LD spheroids. Furthermore, differential gene expression and gene set enrichment analysis identified numerous disease-specific changes. For example, the gene sets “translation” and “oxidative phosphorylation” were significant in many clusters across all leukodystrophies. However, the dynamics are intricate, as they are upregulated in some clusters and timepoints and downregulated in others. Additionally, we showed that including more than one disease model can help prevent overinterpretation based on biases in existing knowledge. To summarize, this work establishes 3D spheroids as a suitable model for studying leukodystrophies and provides interesting candidate DEGs and gene sets that can guide follow-up studies. Additionally, our dataset offers a resource for future investigations into leukodystrophy mechanisms and treatment strategies. Keywords: Leukodystrophies, spheroids, single-cell mRNA sequencing, in vitro disease modelling.
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