hPSC-derived microglia shape neuronal morphology and enhance network activity in vitro 169 5 Further research is required to confirm this hypothesis and to explore the mechanisms underlying these changes. In the context of ALD, microglia-like cells appear to impair neuronal morphology, as indicated by changes in axonal segments. While ALD microglia-like cells themselves do not seem to exhibit increased stress, the alterations in axonal morphology raise intriguing questions about disease-specific microglial dysfunction. Interestingly, axonal abnormalities are a characteristic feature in ALD (Bergner et al., 2021). This study suggests that ALD microglia may contribute to the neuronal phenotype in ALD. However, further research is necessary as the observation of shorter axonal segments observed in ALD co-cultures could also reflect change in neuronal subtypes or subtype-specific vulnerabilities. Additionally, the engulfing of debris from healthy cells, potentially affects the disease presentation of microglia. It would be very interesting to make co-cultures with ALD neurons and/or ALD astrocytes. Nevertheless, we do show that this in vitro model can be used to identify microglia induced alterations to neuronal networks. In conclusion, we have established an in vitro co-culture model that successfully incorporates hPSC-derived microglia with neuronal networks, offering a more physiologically relevant system for studying neuron-glia interactions. This model presents a promising platform for future research into the complex roles of microglia and their connections to both normal brain function and disease. By leveraging this system, researchers may deepen their understanding of the cellular mechanisms underlying neurodegenerative and neurodevelopmental diseases, potentially paving the way for novel therapeutic approaches. MATERIALS & METHODS hPSC culture iPSC were generated from fibroblasts of healthy donors according to the previously described procedure (Holmes & Heine, 2017) or via CytoTune™-iPS 2.0 Sendai Reprogramming Kit according to manufacturer protocol. Pluripotency was confirmed by immunocytochemistry, alkaline phosphatase assay, PCR, embryoid body formation, pluritest and/or karyotyping. The human embryonic stem cell lines H01 and H09 GFP were obtained from WiCell. Pluripotent stem cells were maintained in TeSR™-E8™ (STEMCELL Technologies, 05990) on Vitronectin XF™ coated plates (STEMCELL Technologies, 07180). Media was replaced daily, or using double feedings over the weekend. Confluent wells were
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