modeling to uncover cues involved in establishing microglia identity: TGF-β3 and laminin can drive microglia signature gene expression.

Microglia are the resident macrophages of the central nervous system (CNS) and play a key role in CNS development, homeostasis, and disease. Good models are indispensable to study their cellular biology, and although much progress has been made, cultures of primary microglia still only partially recapitulate the transcriptome of microglia. In this study, we explored a combination of and methodologies to gain insight into cues that are involved in the induction or maintenance of the microglia reference transcriptome. First, we used the tool NicheNet to investigate which (CNS-derived) cues could underlie the differences between the transcriptomes of and microglia. Modeling on basis of gene products that were found to be upregulated , predicted that high mobility group box 2 (HMGB2)- and interleukin (IL)-1β-associated signaling pathways were driving their expression. Modeling on basis of gene products that were found to be downregulated , did not lead to predictions on the involvement of specific signaling pathways. This is consistent with the idea that microenvironmental cues that determine microglial identity are for most part of inhibitory nature. In a second approach, primary microglia were exposed to conditioned medium from different CNS cell types. Conditioned medium from spheres composed of microglia, oligodendrocytes, and radial glia, increased the mRNA expression levels of the microglia signature gene NicheNet analyses of ligands expressed by oligodendrocytes and radial glia predicted transforming growth factor beta 3 (TGF-β3) and LAMA2 as drivers of microglia signature gene expression. In a third approach, we exposed microglia to TGF-β3 and laminin. exposure to TGF-β3 increased the mRNA expression levels of the microglia signature gene . Microglia cultured on laminin-coated substrates were characterized by reduced mRNA expression levels of extracellular matrix-associated genes and , and by increased mRNA expression levels of the microglia signature genes and . Together, our results suggest to explore inhibition of HMGB2- and IL-1β-associated pathways in microglia. In addition, exposure to TGF-β3 and cultivation on laminin-coated substrates are suggested as potential improvements to current microglia culture protocols.