Chapter 1 20 Combining Cell Types: Towards More Physiological Models To better reflect the complex, interconnected nature of the human brain, differentiated cell types can be combined into co-culture systems. This approach allows for the study of cellcell interactions relevant to myelination and neuronal network formation. For instance, neurons and glia can be differentiated separately and later co-cultured at specific maturation stages, enabling the investigation of glial support or impairment to neuron development in 4H (Dooves et al., 2019). This strategy provides an opportunity to model disease-specific cellular interactions while maintaining experimental flexibility. Alternatively, a more integrated approach involves generating three-dimensional (3D) brain-like structures known as organoids. These self-organizing cultures emerge from pluripotent stem cells when exposed to patterning cues that guide them toward specific brain regions (Lancaster et al., 2013). Organoids offer a more comprehensive representation of brain development, enabling multi-lineage differentiation and spatial organization. Offering a unique platform for studying complex, multi-cellular interactions in 4H leukodystrophy. Given the importance of replicating early brain development in studying 4H leukodystrophy, this thesis employs a combination of classical patterning-based monocultures, co-cultures, and brain organoids. By leveraging these models, we aim to uncover how key brain cell types contribute to the disease's pathophysiology, including hypomyelination and related neurological deficits. This multi-faceted approach provides a platform not only to investigate the molecular and cellular basis but hopefully also helps to pinpoint potential therapeutic targets. Insights gained from these models could inform future treatment strategies, including gene and cell replacement therapies, and the models could be used for high-throughput drug screening for disease-modifying compounds. BRIDGING RESEARCH TO TREATMENT DEVELOPMENT While restorative treatments have been developed for some leukodystrophies (Krivit, 2004; Krivit et al., 1999; van den Broek et al., 2018), no such therapies currently exist for 4H. Possibly gene replacement or correction of endogenous mutations could restore Pol III function. Similarly, cell replacement therapy, involving donor cells or ex vivo gene-corrected cells, could replenish damaged cell populations. However, developing these therapies requires a deep understanding of which cell types are primarily affected and need to be replaced or corrected.
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