Discussion 217 7 2. Human iPSC based disease modelling We successfully applied several in vitro systems to investigate 4H leukodystrophy relevant cell types and tissue. These models provided us, in absence of scarce 4H relevant human tissue, to identify pathological changes such as altered inhibitory neuron populations (Chapter 2) and transcriptomic differences (Chapter 3 and Chapter 4). We also demonstrated that the selection of model systems greatly influences the research outcomes (Chapter 3). To exemplify, we showed that both iPSC-derived neuron rat astrocyte cocultures and neuron mono-cultures in maturation medium lacked typical psychosine buildup in GLD lines. This accumulation however was shown in our early neuron mono-cultures and spheroids (data not shown), highlighting that certain model systems may obscure key pathological features. As familiarizing yourself with in vitro techniques is a long, labour intensive and expensive process, it is crucial to select appropriate models. But, how to select the most promising model? Here I will discuss some of my takes on iPSC model selection and reflect on the models we used. In hindsight were they the appropriate models? Subsequently I will pay some attention to how I would continue by discussing the selection of samples and read outs. 2.1 iPSC model selection We have used various iPSC models such as hiPSC-derived neuron mono-cultures, cocultures of those neurons with rat astrocytes and hPSC-derived microglia as well as spheroids. These models were all based on classical differentiation using patterning factors, a method we choose because it resembles more closely the in vivo developmental stages (Nadadhur et al., 2018), during which leukodystrophies often appear. While other iPSCderived models and other disease modelling approaches exist, we will discuss here what we have learned about selecting iPSC-based strategies to model leukodystrophies. Mono-cultures (Chapter 3) i.e., no astrocytes present, revealed transcriptional changes in 4H neurons but lacked clear hallmark phenotypes in GLD. This model was applied because of previous shown neuronal deficits in 4H and GLD, and because co-cultures were void of psychosine build up, a GLD hallmark. The benefit of these cultures is that the limited complexity of these models allow for the investigation of one cell type in great detail, with usually less lengthy and less difficult protocols. Additionally, these cultures have been optimized for years, providing detailed knowledge on the cells created. However, monocultures also have notable limitations. Neurons and astrocytes benefit from the presence of each other, they become more mature (Supakul et al., 2024). Although results are not
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