Discussion 219 7 laboratory, we have moved towards xeno-free hPSC cultures years ago, reflecting this paradigm shift. Though, some of our differentiation protocols continue to rely on products such as Matrigel, a mouse sarcoma extracellular matrix (ECM) product (Nadadhur et al., 2017). Similarly, bovine serum, a product not normally found in the brain, is used to generate astrocytes (Nadadhur et al., 2018) but this pushes them in a reactive state (Du et al., 2010). In our research, we not only used cultures with animal-derived products, we cocultured with rat astrocytes. Formerly, rat astrocytes were the gold standard for co-cultures. However, reliable, more physiological all human systems protocols now exist (Batenburg et al., 2023; Dooves et al., 2021; Lendemeijer et al., 2024; White et al., 2024). Nevertheless, we identified 4H neuronal deficits in the presence of rat astrocytes, but also show that their presence obscured GLD phenotypes. We hypothesize that this happened as they did not have GLD associated mutations. Since additional experiments (Chapter 3) showed that 4H neurons co-cultured with rat astrocytes, also exhibit pathological changes, co-cultures with rat astrocytes could still be a plausible option for modelling 4H leukodystrophy, but moving towards all human systems would be a next step to model 4H more physiological. The current thesis emphasizes that iPSC model selection depends on the disease and research question under investigation. To continue 4H research, a logic next step is to formulate new hypotheses based on the leukodystrophy spheroid dataset, which will become publicly available. These hypotheses can be tested in simpler mono- or co-culture settings before advancing to spheroid or brain-on-chip systems for preclinical validation. Additionally, we have been intrigued by the absence of psychosine build-up in GLD monocultures in maturation medium. Investigation of the spent medium could reveal whether certain factors in this medium mask the phenotype, or in the best case, could relief the phenotype. Lastly, the future of iPSC models likely lies in adopting all-human systems for increased relevance, though xeno (assisted) cultures will retain value where more rapid maturation or scalability is needed. 2.2 Sample selection The main focus has been to investigate 4H syndrome utilizing patient-derived iPSC lines. Additionally, we used KO and KI lines with isogenic controls to investigate GLD and CD. Isogenic controls are typically used to gain higher power than when using case-control designs only. However the ability to generalize findings from isogenic models can be limited (Brunner et al., 2023). This section evaluates sample selection decisions made during this work and proposes strategies for future research.
RkJQdWJsaXNoZXIy MTk4NDMw