Chapter 5 158 In this study, we describe the development of a novel in vitro co-culture system that combines hPSC-derived microglia-like cells with cortical neurons. Utilizing high-content morphological analysis and multi-electrode arrays (MEAs), we assess the influence of microglia-like cells on neuronal morphology and network activity. Our results demonstrate that the presence of microglia-like cells in neuronal cultures reduces cellular debris, reduces synaptic density, and enhances neuronal network activity, suggesting that these hPSCderived microglia-like cells actively shape the neuronal circuits in vitro. Additionally, we show that the morphology of microglia-like cells is influenced by the neuronal background, underscoring the bidirectional interactions between these cell types. We continued to use this model for the investigation of microglia-like cells from two leukodystrophies and identified that both ALD and 4H microglia-like cells in co-culture had no sign of intrinsic defects, as measured by numbers and roundness. However, co-cultures with ALD microglialike cells did display changes in axon morphology. This co-culture model represents an important advancement towards the creation of more physiologically relevant in vitro systems to study both normal brain function and the role of microglia in neurodegenerative and neurodevelopmental diseases. RESULTS Microglia-like cells dynamically interact with cortical neurons in vitro To develop a human-based in vitro assay suitable to study the modulating effects of microglia on neuronal network development, we created microglia-neuron co-cultures based on earlier established hPSC-derived microglia (Haenseler et al., 2017) and human cortical neuron (Dooves et al., 2023; Nadadhur et al., 2017) derivation protocols. Microglialike cells were generated from a human GFP-expressing embryonic stem cell line (H09 GFP+) and added to 4 different hPSC-derived mixtures of glutamatergic and GABAergic cortical neuron cultures at week 6 of neuron maturation. To the control group microglia-like cells were not added, but this group did get the same media regime. The cultures were maintained for an additional 6 weeks, during which we assessed integration, survival, and interaction of microglia-like cells with neurons using immunofluorescence and multielectrode assays (MEA) (Fig. 1A). We confirmed that microglia-like cells survived for the entire duration of the co-culture on both cellomics (Fig. 1B-E) and MEA plates (Supplementary Fig. 1A-H) across all neuronal backgrounds. In an independent experiment, we confirmed that the hPSC-derived microglia-like cells express Iba1 (Supplementary Fig. 2A-D). Additionally, using microglia-like cell mono-cultures we displayed that all of the plated cells went on to express TMEM119 as indicated by immunocytochemistry, displaying
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