Culture shock: microglial heterogeneity, activation, and disrupted single-cell microglial networks in vitro.
Culture shock: microglial heterogeneity, activation, and disrupted single-cell microglial networks in vitro.
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DOI：
10.1186/s13024-022-00531-1
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发表时间：
2022-03-28
影响因子：
15.1
通讯作者：
Fryer JD
中科院分区：
文献类型：
--
作者：
Cadiz MP;Jensen TD;Sens JP;Zhu K;Song WM;Zhang B;Ebbert M;Chang R;Fryer JD
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，难以用病毒载体和其他方法在体内进行遗传操作。原代培养允许更受控的设置来研究这些细胞，但从正常脑环境中取出后的形态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现体内小胶质细胞的特征，我们使用单细胞RNA测序（scRNAseq）来比较从大脑新鲜分离的小胶质细胞与原代小胶质细胞培养物。我们进行了细胞群体发现，差异表达分析，和基因共表达模块分析，以比较在体外和体内小胶质细胞之间的签名。我们从scRNAseq数据构建了转录调控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。为了验证这种网络分析，我们在原代培养的小胶质细胞中敲除了其中两个关键驱动因素C1 qc和Prdx 1，并量化了小胶质细胞活化标志物的变化。我们发现，虽然通常被认为是一个相对同质的细胞群体在文化中，在体外小胶质细胞是一个高度异质性的人口组成的不同亚群的细胞与转录谱联想到巨噬细胞和单核细胞，并标记的转录程序活跃在神经退行性疾病和其他疾病状态。我们发现，小胶质细胞在体外转录激活的一组“文化休克基因”没有发现在新鲜分离的小胶质细胞，其特征是强烈上调的疾病相关基因，包括载脂蛋白，Lyz 2和Spp 1，和下调的稳态小胶质细胞标志物，包括Cx 3cr 1，P2 ry 12和Tmem 119。最后，我们发现，培养的小胶质细胞显着改变其转录机制调制的关键驱动程序从稳态激活表型。敲低这些驱动因子之一C1 qc，导致小胶质细胞激活基因Lpl、Lyz 2和Ccl 4下调。总的来说，我们的数据表明，当从它们的体内家庭环境中取出时，小胶质细胞遭受严重的“文化休克”，通过上调培养特异性基因的模块，将它们的转录调控网络状态从稳态急剧调节到激活。因此，培养的小胶质细胞表现为一种不同的细胞类型，不重演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，可以将激活的小胶质细胞转换回其稳态，从而更准确地表示培养物中的体内状态。敲除的关键驱动程序C1 qc部分衰减小胶质细胞的激活在体外，尽管C1 qc仅微弱上调文化。这表明，即使在处理或制备过程中没有强烈差异表达的基因也可能驱动培养物中的下游转录变化。在线版本包含补充材料，可通过10.1186/s13024-022-00531-1获得。
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. The online version contains supplementary material available at 10.1186/s13024-022-00531-1.
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影响因子：
8.8
作者：
Mathys H;Adaikkan C;Gao F;Young JZ;Manet E;Hemberg M;De Jager PL;Ransohoff RM;Regev A;Tsai LH
通讯作者：
Tsai LH
神经胶质瘤相关的小胶质细胞/巨噬细胞表现出与 M1 和 M2 极化不同的表达谱，并高度表达 Gpnmb 和 Spp1。
影响因子：
3.7
作者：
Szulzewsky F;Pelz A;Feng X;Synowitz M;Markovic D;Langmann T;Holtman IR;Wang X;Eggen BJ;Boddeke HW;Hambardzumyan D;Wolf SA;Kettenmann H
通讯作者：
Kettenmann H
影响因子：
32.4
作者：
Hammond, Timothy R.;Dufort, Connor;Stevens, Beth
通讯作者：
Stevens, Beth
影响因子：
25
作者：
Van Hove, Hannah;Martens, Liesbet;Movahedi, Kiavash
通讯作者：
Movahedi, Kiavash
DOI：
10.1093/brain/awy215
发表时间：
2018-09-01
期刊：
Brain : a journal of neurology
影响因子：
--
作者：
Petyuk VA;Chang R;Ramirez-Restrepo M;Beckmann ND;Henrion MYR;Piehowski PD;Zhu K;Wang S;Clarke J;Huentelman MJ;Xie F;Andreev V;Engel A;Guettoche T;Navarro L;De Jager P;Schneider JA;Morris CM;McKeith IG;Perry RH;Lovestone S;Woltjer RL;Beach TG;Sue LI;Serrano GE;Lieberman AP;Albin RL;Ferrer I;Mash DC;Hulette CM;Ervin JF;Reiman EM;Hardy JA;Bennett DA;Schadt E;Smith RD;Myers AJ
通讯作者：
Myers AJ