Towards a 3D spheroid system for modelling leukodystrophies 111 4 INTRODUCTION Leukodystrophies (LDs) are a heterogenous group of rare genetic brain disorders characterized by progressive degradation of white matter (WM), typically diagnosed in early childhood and often reducing life expectancy. Due to their progressive nature, there is an urgent need for curative treatments. However, the development of such therapies is challenged by limited knowledge on cell types and cellular mechanisms primarily affected in disease aetiology. Although the genetic basis of LDs often lies in genes that are expressed ubiquitously, due to the prominent white matter abnormalities these disorders have traditionally been attributed to glial dysfunction. Recent studies, however, point to significant neuronal involvement in the WM pathology of LDs, suggesting that not just myelin but all white matter constituents are essential for maintaining WM integrity and function. These new insights have significant impact on how we need to approach therapy development for LDs and emphasize the need to expand our knowledge on neurons, glia and their interactions in WM pathology. Given that LDs arise during critical stages of brain development, it is essential to study WMpathology in tissue that mimics these stages of brain development. Post-mortem tissue is not suitable as it reflects the end stage of disease and the rarity of LDs makes post-mortem tissue scarce. Studying LDs in animal models would enable the insight in developmental stages, however the translation from findings in animal models to human disease is complicated by species differences. To overcome these challenges, there is a growing need for in vitro models that recapitulate later stages of brain development. Advances in human in vitro models, particularly spheroids, offer a promising solution. Spheroids, derived from human stem cells, can recapitulate the cellular diversity of brain tissue, enabling the study of complex cell interactions during development. When combined with recent advances in analytical techniques, spheroids can be useful for studying neurodevelopmental disorders, including LDs. However, certain concerns about how brain-like spheroids are and which developmental stages can be reached using spheroids are often not addressed in depth. This study investigates whether 3D spheroid models can effectively replicate the cellular composition and interactions relevant to white matter (WM) pathology in leukodystrophies. To address this, we characterized the spheroids based on size, immunofluorescence and electron microscopy. We showed that all cells of interest; neurons, oligodendrocytes and
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