Chapter 3 94 Taken together, the detected transcriptomic changes indicate that this model does capture certain aspects of 4H neuronal phenotypes. However, the changes are subtle, and more work needs to be done to confirm the involvement of signalling pathways and/or biological processes in 4H neuronal pathology. To validate the relevance of these findings, future research could focus on the relation of these pathways and Pol III using possible conditional Pol III knock-outs or knock-downs using techniques such as siRNAs. Such investigations should also consider extended culture periods, as models that have shown neuronal phenotypes in 4H involved more mature stages (Dooves et al., 2023). Conclusion and Future Recommendations This study revealed limited morphological abnormalities in 4H and GLD neuronal models, with only moderate psychosine accumulation observed in GLD monocultures. While transcriptomic analysis identified molecular changes in 4H neurons, that direct future research opportunities, no conclusive phenotypes were found in GLD models. More appropriate in vitro models to address neuronal defects in leukodystrophies are required, especially for GLD. The optimization and identification of such models could start by exploring extended timepoints, psychosine levels over time and lipidomic profiling. MATERIALS & METHODS iPSC culture iPSCs were generated and maintained according to the protocols of the origin institutes (Dooves et al., 2023; Mangiameli et al., 2021). Written consent was obtained from all participants in accordance with the declaration of Helsinki. Neural Differentiation Cortical neurons were generated using the previously published protocol using dual SMAD inhibition and consequent maturation of cells into glutamatergic and GABAergic neurons (Nadadhur et al., 2017). Neurons were frozen at day 18 of differentiation in 10% DMSO in KSR. Neural Maturation Co-Culture Rat astrocytes were obtained by papaïn dissociation of cortex of postnatal day 1 pups. To minimize contamination of other cells rat astrocytes were vigorously tapped before each medium change. Medium consisted of DMEM/F12 medium + Glutamax + 10% FBS + 1x NEAA
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