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Minimizing the ex vivo confounds of cell-isolation techniques on transcriptomic profiles of purified microglia

ABSTRACT: Modern molecular neuroscience studies require analysis of specific cellular populations derived from brain tissue samples to disambiguate cell-type specific events. This is particularly true in glial cell types, such as microglia, which as minority cell populations in the brain, may be obscured in whole tissue analyses. Microglia have central functions in development, aging, and neurodegeneration and are a current focus of neuroscience research. A long-standing concern for glial biologists using in vivo models is whether cell isolation from CNS tissue could introduce ex vivo artifacts in microglia, which respond quickly to changes in the environment. Mouse microglia were purified by antibody-conjugated magnetic bead, and cytometer- and cartridge-based fluorescence-activated cell sorting approaches to compare and contrast performance. The Cx3cr1-NuTRAP (nuclear tagging and translating ribosome affinity purification) model was used to provide an endogenous fluorescent microglial marker and a microglial-specific translatome profile lacking cell isolation artifacts. All methods performed similarly for microgial purity with main differences being in cell yield and time of isolation. Ex vivo activation signatures occurred principally during the initial tissue dissociation and cell preparation and not the microglial cell sorting. Utilizing transcriptional and translational inhibitors during the cell preparation prevented the activational phenotype. These data demonstrate that a variety of microglial isolation approaches can be used, depending on experimental needs, and that inhibitor cocktails are effective at reducing cell preparation artifacts.

ORGANISM(S): Mus musculus

PROVIDER: GSE179721 | GEO | 2021/09/30

REPOSITORIES: GEO

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Project description:Expression profiling of distinct central nervous system (CNS) cell populations has been employed to facilitate disease classification and to provide insights into the molecular basis of brain pathology. One important cell type implicated in a wide variety of CNS disease states is the resident brain macrophage (microglia). In these studies, microglia are often isolated from dissociated brain tissue by flow sorting procedures [fluorescence-activated cell sorting (FACS)] or from postnatal glial cultures by mechanic isolation. Given the highly dynamic and state-dependent functions of these cells, the use of FACS or short-term culture methods may not accurately capture the biology of brain microglia. In the current study, we performed RNA-sequencing using Cx3cr1+/GFP labeled microglia isolated from the brainstem of 6-week-old mice to compare the transcriptomes of FACS-sorted versus laser capture microdissection (LCM). While both isolation techniques resulted in a large number of shared (common) transcripts, we identified transcripts unique to FACS-isolated and LCM-captured microglia. In particular, M-bM-^HM-<50% of these LCM-isolated microglial transcripts represented genes typically associated with neurons and glia. While these transcripts clearly localized to microglia using complementary methods, they were not translated into protein. Following the induction of murine experimental autoimmune encephalomyelitis, increased oligodendrocyte and neuronal transcripts were detected in microglia, while only the myelin basic protein oligodendrocyte transcript was increased in microglia after traumatic brain injury. Collectively, these findings have implications for the design and interpretation of microglia transcriptome-based investigations. Wildtype and GFP expressing microglia from mouse brainstems were flow sorted or captured by laser microdissection. Differences between the two isolation methods were verified and further examined in neurodegenerative disease models.

Project description:Background: Microglial isolation and culturing methods continue to be explored to maximize cellular yield, purity, responsiveness to stimulation and similarity to in vivo microglia. This study aims to evaluate different microglia isolation methods — 3 variants of microglia isolation from neonatal mice and 2 variants of microglia isolation from adult mice — on transcriptional profile and response to HMGB1. Methods: Microglia from neonatal mice, age 0-3 days (P0-P3) were isolated from mixed glial cultures (MGC). We included three variations of this protocol that differed by use of GM-CSF in culture (No GM-CSF or 500 pg/mL GM-CSF), and days of culture in MGC before microglial separation (10 or 21). Protocols for studying microglia from adult mice age 6-8 weeks included isolation by adherence properties followed by 7 days of culture with 100 ng/mL GM-CSF and 100 ng/mL M-CSF (Vijaya et al., 2023), or acute isolation using CD11b beads (Bordt et al., 2020). Purity, yield, and RNA quality of the isolated microglia were assessed by flow cytometry, hemocytometer counting, Nanodrop, and Bioanalyzer, respectively. Microglial responsiveness to an inflammatory stimulus, HMGB1, was evaluated by measuring TNFα, IL1β, and IFNβ secretion by ELISA and assessing gene expression patterns using bulk RNA sequencing. Results: All five methods demonstrated greater than 90% purity. Microglia from all cultures increased transcription of or secreted TNFα, IL1β, and IFNβ in response to HMGB1. RNA sequencing showed a larger number of differentially expressed genes in response to HMGB1 treatment in microglia cultured from pups than from adult mice, with sparse changes among the 3 MGC culturing conditions. Conclusion: These findings suggest that while all methods provided high purity, the choice of protocol may significantly influence yield, RNA quality, baseline transcriptional profile and response to stimulation. This comparative study provides valuable insights to inform the choice of microglial isolation and culture method.

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Minimizing the ex vivo confounds of cell-isolation techniques on transcriptomic profiles of purified microglia

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