hPSC-derived microglia shape neuronal morphology and enhance network activity in vitro 159 5 that a pure pool of microglia-like cell precursors was plated in the co-cultures (Supplementary Fig. 2E). Also on RNA level, these cells show microglia-like expression of canonical microglia markers such as Iba1, P2RY12 and TMEM119 (Supplementary Fig. 2F)(Pettas et al., 2022). While all neuron cultures received the same microglia differentiation batch, the neuronal background had a significant influence on microglia quantities (Supplementary Table III, Fig. 1F). As in vivo microglia are highly dynamic and plastic cells that undergo morphology changes, particularly increased cell roundness is an important indicator of altered microglia function, we analyzed morphology (Paolicelli et al., 2022). Our analysis revealed that microglia-like cells in co-culture do not present roundness that is associated with microglia reactivity (mean roundness = 0.49, Fig. 1B-E). Though, neuronal background significantly influenced various aspects of their morphology, including roundness, size, length and width to length ratio (see Supplementary Table III, Fig. 1G-K), underscoring the dynamic interaction between the neurons and microglia-like cells. Additionally, microglia morphology significantly differed between timepoints (see Supplementary Table III, Fig 1G-K). Together, we showed the successful incorporation of hPSC-derived microglia-like cells and their interaction with human cortical neurons in vitro. Microglia support neuronal maturation in vitro In vivo microglia have important functions in brain development and maintaining brain homeostasis by functions such as clearing debris, pruning synapses and shaping neuronal circuits by phagocytosis of, amongst others, neural progenitors, neurons, axons and dendrites (Paolicelli & Ferretti, 2017; Riccomagno & Kolodkin, 2015). Therefore, we hypothesized that co-cultures with microglia-like cells have different morphological features compared to cultures without microglia. Specifically, we hypothesized that microglia co-cultures have decreased nuclear debris and alterations on neuron morphology that we aimed to address by investigating the number of mature neurons (NeuN+) and the number of dendrite and axonal protrusions. High content analysis was used to validate this hypothesis. Results were first tested separately and subsequently aggregated using Fisher’s method to avoid multiple testing and improve power. We confirmed that the addition of microglia-like cells had a statistical significant effect on the combination of these parameters (p < 0.0001, Fisher’s method, Supplementary Fig. 3A-D). Post hoc testing showed reduced nuclear debris (Fig. 2A-C, p < 0.0001), unchanged levels of mature neurons (Fig. 2D, p = 0.609) and reduced numbers of axonal and dendritic segments in the microglia cocultures (Fig. 2E, p = 0.044 and Fig. 2F, p = 0.0001).
RkJQdWJsaXNoZXIy MTk4NDMw