Introduction 23 1 role for neuronal dysfunction in 4H, expanding the focus beyond traditional glial-centric paradigms. Chapter 4: To study changes in neuron-glia interactions in 4H leukodystrophy and leukodystrophies in general, we developed an EU platform based on myelinating spheroids. As no existing standardized platform to investigate leukodystrophies was in place, we distributed iPSCs for the centralized generation of myelinating spheroids of GLD, CD, 4H and controls. We explored whether these 3D spheroids generate the cellular composition required for the investigation of leukodystrophies, including neurons, oligodendrocytes, and astrocytes. We confirmed relevance for investigating leukodystrophies and report differential expressed genes and significant gene sets that can guide future research directions. In conclusion, this chapter validated the potential of 3D spheroid models for studying leukodystrophies by mimicking disease-relevant cellular interactions. It emphasizes their utility in identifying cell-type-specific vulnerabilities and novel molecular targets, addressing the gap in comprehensive in vitro models. Chapter 5: In the myelinating spheroids, one cell type was not included, the microglia. Hence, we generated a platform for the co-culture of iPSC-derived microglia-like cells and analysed the effect of the microglia on the neuronal cultures using cellomics and multielectrode arrays (MEA). The addition of microglia significantly impacted the cultures. ALD microglia seemed to affect axons different compared to control microglia, while 4H microglia seemed to function fine. Chapter 6: To explore how patient-specific genetic variants affect POLR3 gene and protein expression during neuronal lineage differentiation in 4H leukodystrophy, we examined POLR3 expression levels, protein localization, and developmental dynamics across iPSCs, neural epithelial cells and neurons. We also evaluated whether the genetic background contributes to expression differences. This chapter integrates patient-specific data with iPSC models to elucidate disease mechanisms. By addressing genetic variability, this approach paves the way for precision medicine approaches in 4H leukodystrophy research. Chapter 7: In this concluding chapter, we evaluate the model systems used throughout the thesis, discussing their advantages, limitations, and suitability for future leukodystrophy research. We provide an overarching synthesis of the knowledge gained from each chapter and how these findings interconnect. Additionally, we present forward-looking perspectives
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