Chapter 7 218 shown, neurons in NB+ medium did not develop axonal transport, indicating reduced maturation, without the presence of astrocytes. Hence, the use of more costly commercial media, STEMdiff Forebrain Neuron Maturation Medium, was required. These factors highlight the need to carefully consider mono-culture limitations in the context of specific research objectives. Co-cultures, which in this thesis involved iPSC-derived neurons on rat astrocytes with or without hPSC-derived microglia (Chapter 2, 3 and 5), improved functionality, intercellular crosstalk, and a more accurately represented in vivo situation. The presence of multiple cell types influenced the observed responses, underscoring the potential advantages of more complex models when evaluating compounds or studying pathological mechanisms. For example, co-cultures have been shown to alter cellular reactions in ways that monocultures cannot (De Simone et al., 2017), suggesting they may be more suitable for assessing therapies or exploring disease interactions. Spheroid systems represent another layer of complexity, incorporating multiple cell types to mimic in vivo interactions more closely. These models allow for unbiased exploration of affected cell types, potential therapeutic targets, and their response to therapies. Though we have only used one spheroid system, the models are rapidly evolving including bioreactor culture, organ-on-a-chip and/or biomaterials (Gong et al., 2021). Their structural similarity to the brain is argued to make them better for testing interventions at both the cellular and system levels. While those complex models are promising, in our research we have shown unexpected results. For example, we hypothesized that when using a spheroid system with myelination we capture the hypomyelinating phenotype. Nevertheless, there were no differences in OPC and oligodendrocyte abundance and compacted myelin was identified in all spheroids (Chapter 4). This raises critical questions: does hypomyelination occur only within a specific time window, or are factors like growth medium supplementation masking the phenotype? Such advanced models are also costly, laborintensive, and prone to batch effects, highlighting the need for careful protocol optimization. Another important factor in in vitro modelling is the use of all-human component vs xenoassisted systems. In general, the field of in vitro modelling is moving towards all-human, xeno-free systems, which aligns with eliminating animal-derived components to better replicate human biology and safety when used for cell replacement therapy. In our
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