Culture shock: microglial heterogeneity, activation, and disrupted single-cell microglial networks in vitro

Mika P. Cadiz, Tanner D. Jensen, Jonathon P. Sens, Kuixi Zhu, Won Min Song, Bin Zhang, Mark Ebbert, Rui Chang, John D. Fryer

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

Original languageEnglish
Article number26
JournalMolecular Neurodegeneration
Volume17
Issue number1
DOIs
StatePublished - Dec 2022

Bibliographical note

Funding Information:
JDF was supported by the Mayo Foundation, The Ben Dov Family Luminescence Foundation, the Ed and Ethel Moore Alzheimer’s Disease Research Program of Florida Department of Health (6AZ06), The Rotary Coins for Alzheimer’s Research Trust Fund, CureAlz Foundation, the Goodman Family Foundation, and NIH Grants NS084974, AG062556, AG062110, NS094137, AG057997, AG062077, NS110435, AG047327, AG049992, AG062677. RC was supported by NIH grants AG057457, AG062620, AG057931 and, AG059093. BZ was supported by NIH grants AG046170 and AG068030.

Funding Information:
RC is founder of INTelico Therapeutics LLC, AZ, 85718, USA, a company focused on drug target discovery and precision therapeutics based upon probabilistic models of disease. This study is not supported by any funding from INTelico Therapeutics LLC.

Publisher Copyright:
© 2022, The Author(s).

Keywords

  • Causal network analysis
  • In vitro culture
  • Microglia
  • Single cell RNA sequencing

ASJC Scopus subject areas

  • Molecular Biology
  • Clinical Neurology
  • Cellular and Molecular Neuroscience

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