Culture shock: microglial heterogeneity, activation, and disrupted single-cell microglial networks in vitro
Authors: Cadiz Mika P. , Jensen Tanner D. , Sens Jonathon P. , Zhu Kuixi , Song Won-Min , Zhang Bin , Ebbert Mark , Chang Rui , Fryer John D. 
Published: March 2022

AbstractBackgroundMicroglia, the resident immune cells of the brain, play a critical role in numerous diseases, but are a minority cell type and difficult to genetically manipulate in vivo with viral vectors and other approaches. Primary cultures allow a more controlled setting to investigate these cells, but morphological and transcriptional changes upon removal from their normal brain environment raise many caveats from in vitro studies.MethodsTo investigate whether cultured microglia recapitulate in vivo microglial signatures, we used single-cell RNA sequencing (scRNAseq) to compare microglia freshly isolated from the brain to primary microglial cultures. We performed cell population discovery, differential expression analysis, and gene co-expression module analysis to compare signatures between in vitro and in vivo microglia. We constructed causal predictive network models of transcriptional regulators from the scRNAseq data and identified a set of potential key drivers of the cultured phenotype. To validate this network analysis, we knocked down two of these key drivers,C1qcandPrdx1,in primary cultured microglia and quantified changes in microglial activation markers.ResultsWe found that, although often assumed to be a relatively homogenous population of cells in culture, in vitro microglia are a highly heterogeneous population consisting of distinct subpopulations of cells with transcriptional profiles reminiscent of macrophages and monocytes, and are marked by transcriptional programs active in neurodegeneration and other disease states. We found that microglia in vitro presented transcriptional activation of a set of “culture shock genes” not found in freshly isolated microglia, characterized by strong upregulation of disease-associated genes includingApoe,Lyz2,andSpp1, and downregulation of homeostatic microglial markers, includingCx3cr1,P2ry12, andTmem119. Finally, we found that cultured microglia prominently alter their transcriptional machinery modulated by key drivers from the homeostatic to activated phenotype. Knockdown of one of these drivers,C1qc, resulted in downregulation of microglial activation genesLpl, Lyz2, and Ccl4.ConclusionsOverall, our data suggest that when removed from their in vivo home environment, microglia suffer a severe case of “culture shock”, drastically modulating their transcriptional regulatory network state from homeostatic to activated through upregulation of modules of culture-specific genes. Consequently, cultured microglia behave as a disparate cell type that does not recapitulate the homeostatic signatures of microglia in vivo. Finally, our predictive network model discovered potential key drivers that may convert activated microglia back to their homeostatic state, allowing for more accurate representation of in vivo states in culture. Knockdown of key driverC1qcpartially attenuated microglial activation in vitro, despiteC1qcbeing only weakly upregulated in culture. This suggests that even genes that are not strongly differentially expressed across treatments or preparations may drive downstream transcriptional changes in culture.