Towards a 3D spheroid system for modelling leukodystrophies 133 4 could still be a shared pathological mechanism in leukodystrophies, we do not have other evidence to support those observations. Similarly, oxidative phosphorylation was found to be highly significant in all leukodystrophies, but also this pathways was down and upregulation changed per cluster and per timepoint. Since oxidative stress is linked to impaired OPC differentiation in neurological disorders, it is an interesting topic for future research, but no conclusions can be drawn yet (Spaas et al., 2021). This emphasizes the necessity for this data set to become publicly available for other researchers to use as validation and discovery data set. Simultaneously it shows that when designing spheroid studies it is important to consider the inclusion of more than one disease, as this prevents overinterpretation of the data. Methodological Considerations While our data provides robust evidence for cellular and molecular changes, several methodological limitations should be considered. First, the annotation of cell clusters into predefined categories potentially introduces biases, particularly when clusters may not correspond to well-defined in vivo populations. Although the annotation is done carefully, more standardized clustering protocols throughout the field could avoid potential bias. Including isogenic controls can improve the precision of disease-specific findings by minimizing background noise, but the largest changes in gene expression in GLD were observed in comparison to both healthy donors and isogenic controls. Conversely, the lack of population-level genetic diversity in isogenic models may also be a limitation, as it does not capture the full range of human genetic variability relevant to disease expression. CONCLUSION To conclude, this study provides a comprehensive overview of cell types and gene expression changes involved in leukodystrophies GLD, CD and 4H. The 3D spheroids offer a promising platform for studying leukodystrophies by recapitulating key aspects of white matter development and pathology. By studying 3 different leukodystrophies in one experiment, we were able to identify a number of shared mechanisms. This commonality raises the possibility that therapeutic strategies developed for one leukodystrophy could be repurposed for others, potentially broadening the clinical applicability of future treatments. Additionally, disease-specific findings were observed and provide interesting candidate DEGs and pathways for follow-up studies. The generated dataset will be freely available for further analysis by the broader research community, allowing for more in-depth analysis of specific cell types and/or disorders and novel hypothesis generation.
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