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Culture shock: microglial heterogeneity, activation, and disrupted single-cell microglial networks in vitro
Molecular Neurodegeneration ( IF 15.1 ) Pub Date : 2022-03-28 , DOI: 10.1186/s13024-022-00531-1
Mika P Cadiz 1, 2 , Tanner D Jensen 1 , Jonathon P Sens 1, 2 , Kuixi Zhu 3 , Won-Min Song 4 , Bin Zhang 4 , Mark Ebbert 5 , Rui Chang 3 , John D Fryer 1, 2
Affiliation  

Microglia, 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. To 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, C1qc and Prdx1, in primary cultured microglia and quantified changes in microglial activation markers. We 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 including Apoe, Lyz2, and Spp1, and downregulation of homeostatic microglial markers, including Cx3cr1, P2ry12, and Tmem119. 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 genes Lpl, Lyz2, and Ccl4. Overall, 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 driver C1qc partially attenuated microglial activation in vitro, despite C1qc being 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.

中文翻译:

文化冲击:体外小胶质细胞异质性、激活和破坏的单细胞小胶质细胞网络

小胶质细胞是大脑的常驻免疫细胞,在许多疾病中起着关键作用,但它是少数细胞类型,难以通过病毒载体和其他方法在体内进行基因操作。原代培养允许更受控制的环境来研究这些细胞,但从正常大脑环境中移除后的形态学和转录变化引起了体外研究的许多警告。为了研究培养的小胶质细胞是否重演体内小胶质细胞特征,我们使用单细胞 RNA 测序 (scRNAseq) 来比较从大脑中新鲜分离的小胶质细胞与原代小胶质细胞培养物。我们进行了细胞群发现、差异表达分析和基因共表达模块分析,以比较体外和体内小胶质细胞之间的特征。我们从 scRNAseq 数据构建了转录调节因子的因果预测网络模型,并确定了一组培养表型的潜在关键驱动因素。为了验证此网络分析,我们在原代培养的小胶质细胞中敲除其中两个关键驱动因素 C1qc 和 Prdx1,并量化了小胶质细胞激活标记物的变化。我们发现,虽然通常被认为是培养中相对同质的细胞群,但体外小胶质细胞是一个高度异质的群体,由不同的细胞亚群组成,其转录谱让人联想到巨噬细胞和单核细胞,并且以活跃于神经变性的转录程序为标志和其他疾病状态。我们发现体外小胶质细胞表现出一组在新鲜分离的小胶质细胞中未发现的“文化休克基因”的转录激活,其特征是疾病相关基因(包括 Apoe、Lyz2 和 Spp1)的强烈上调,以及稳态小胶质细胞标记物的下调,包括Cx3cr1、P2ry12 和 Tmem119。最后,我们发现培养的小胶质细胞显着改变了它们由关键驱动因素调节的转录机制,从稳态到激活的表型。这些驱动程序之一 C1qc 的敲低导致小胶质细胞激活基因 Lpl、Lyz2 和 Ccl4 的下调。总的来说,我们的数据表明,当小胶质细胞从它们体内的家庭环境中移除时,会遭受严重的“文化休克”,通过上调文化特异性基因的模块,大幅调节它们的转录调控网络状态,从稳态到激活。因此,培养的小胶质细胞表现为一种不同的细胞类型,不会概括体内小胶质细胞的稳态特征。最后,我们的预测网络模型发现了潜在的关键驱动因素,这些驱动因素可能会将激活的小胶质细胞转换回其稳态状态,从而更准确地表示培养物中的体内状态。关键驱动 C1qc 的敲低在体外部分减弱​​了小胶质细胞的激活,尽管 C1qc 在培养中仅微弱上调。这表明,即使是在处理或准备过程中没有强烈差异表达的基因也可能驱动下游文化的转录变化。
更新日期:2022-03-28
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