Chapter 7 222 Time points for sample collection Considering that the transcriptomic differences in 4H cortical neurons was observed after only one week of culture, we hypothesize that 4H neuronal defects occur over time, hence future in vitro work with similar methods should be done on 4H neurons at later time points. Mature neurons in the models for 4H are critical, considering that we aim to investigate interneurons, a neuronal subtype that arises later in development and takes time to mature (Fitzgerald et al., 2020). Likewise, our myelination models for 4H, using both spheroids (Chapter 4) and 2D co-cultures (Chapter 2), did not reveal significant impairments in myelination. These results suggest that these models may need further optimization to capture the relevant time window or environmental conditions for hypomyelination. Careful selection of sampling timelines is crucial for resolving such questions. So in conclusion, while decisions made during this work were justified, there is room for improvement: 1) Isogenic lines: We have seen in the neuron-mono cultures (Chapter 3) that changes can be subtle in 4H, for this isogenic experimental set-up that generally have increased power can be useful. We could make either stable genetically modified lines, or use techniques such as RNA interference to create POLR3A or B knock-downs; 2) Patientderived diversity: For generalizing findings to the entire patient population, and later patient-specific treatments, it is still important to use patient-derived lines with larger genetic background variations. In this thesis we have worked with a small patient cohort. Future aims should focus on including more patients to validate whether the findings are representative of the entire patient population. In chapter 5, we have shown that it is possible that there are patient-specific differences. Addressing these issues will be essential for advancing 4H research and improving the reliability of experimental findings; 3) Optimize time points: As subtle changes may emerge over time, longitudinal studies of neurons and spheroids at different maturation stages are essential for capturing the dynamic progression of 4H pathology. 2.3 Read out selection Selecting the appropriate read-outs is a fundamental aspect of any study. In this thesis, we have used a broad scale of read-outs such as high-throughput morphological analysis using cellomics, electrophysiology analysis using MEAs, classical approaches such as qPCR and western blot, and transcriptomics. Each research question justifies the use of specific techniques; however, I would like to elaborate on the growing prominence of transcriptomics in research.
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