Enzymatic Dissociation Induces Transcriptional and Proteotype Bias in Brain Cell Populations

Mattei, Daniele; Ivanov, Andranik; Oostrum, Marc van; Pantelyushin, Stanislav; Richetto, Juliet; Mueller, F; Beffinger, Michal; Schellhammer, Linda; Berg, Johannes vom; Wollscheid, Bernd; Beule, Dieter; Paolicelli, Rosa Chiara; Meyer, Urs

doi:10.3390/ijms21217944

Cited by 97 publications

(96 citation statements)

References 35 publications

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“…In addition, we used another independent statistical approach for signature enrichment in single cell data based on the recently published CERNO algorithm 77 , 78 . Briefly, we used the CERNO method to recalculate the enrichment for a specific gene signature per cell.…”

Section: Methodsmentioning

confidence: 99%

P-selectin axis plays a key role in microglia immunophenotype and glioblastoma progression

et al. 2021

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Glioblastoma (GB) is a highly invasive type of brain cancer exhibiting poor prognosis. As such, its microenvironment plays a crucial role in its progression. Among the brain stromal cells, the microglia were shown to facilitate GB invasion and immunosuppression. However, the reciprocal mechanisms by which GB cells alter microglia/macrophages behavior are not fully understood. We propose that these mechanisms involve adhesion molecules such as the Selectins family. These proteins are involved in immune modulation and cancer immunity. We show that P-selectin mediates microglia-enhanced GB proliferation and invasion by altering microglia/macrophages activation state. We demonstrate these findings by pharmacological and molecular inhibition of P-selectin which leads to reduced tumor growth and increased survival in GB mouse models. Our work sheds light on tumor-associated microglia/macrophage function and the mechanisms by which GB cells suppress the immune system and invade the brain, paving the way to exploit P-selectin as a target for GB therapy.

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Section: Methodsmentioning

confidence: 99%

P-selectin axis plays a key role in microglia immunophenotype and glioblastoma progression

et al. 2021

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“…We and the manufacturer (Miltenyi Biotec) have experimentally verified that the Multi-Tissue Dissociation Kit I preserves many common immune cell surface epitopes, including all listed in the Key Resources Table. CRITICAL: Enzymatic tissue dissociation methods have been shown to reduce the astrocyte and neuron viability and activate and/or alter the transcriptome of microglia and other cell types ( Mattei et al., 2020 ). Users should evaluate their cells to examine whether the digestion has artificially activated or changed their transcriptional status.…”

Section: Before You Beginmentioning

confidence: 99%

“… CRITICAL: Enzymatic tissue dissociation methods have been shown to reduce the astrocyte and neuron viability and activate and/or alter the transcriptome of microglia and other cell types ( Mattei et al., 2020 ). Users should evaluate their cells to examine whether the digestion has artificially activated or changed their transcriptional status.…”

Section: Before You Beginmentioning

confidence: 99%

Isolation of mouse brain-infiltrating leukocytes for single cell profiling of epitopes and transcriptomes

et al. 2021

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Summary High dimensional compositional and transcriptional profiling of heterogeneous brain-infiltrating leukocytes can lead to novel biological and therapeutic discoveries. High-quality single-cell leukocyte preparations are a prerequisite for optimal single cell profiling. Here, we describe a protocol for epitope and RNA-preserving dissociation of adult mouse brains and subsequent leukocyte purification and staining, which is adaptable to homeostatic and pathogenic brains. Leukocyte preparation following this protocol permits exquisite single-cell surface protein and RNA profiling in applications including CyTOF and CITE-seq. For complete details on the use and execution of this protocol, please refer to Guldner et al. (2020) and Golomb et al. (2020) .

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“…Given new evidence showing that partial CX3CR1 deficiency may alter microglial functions in a transgenic AD mouse model (Hickman, Allison, Coleman, Kingery-Gallagher, & El Khoury, 2019), whether partial CX3CR1 deficiency and tamoxifen affect understanding sex-based differences in microglial transcriptomes remains to be investigated. Third, a recent study reports that standard enzymatic digestion of brain tissue at 37 C induces profound and consistent alterations in the transcriptome and proteotype of neuronal and glial cells, as compared to an optimized mechanical dissociation protocol at 4 C (Mattei et al, 2020). Combined single-cell and spatial transcriptomics may be better approaches to dissect molecular architecture in TBI.…”

Section: Discussionmentioning

confidence: 99%

Transcriptional profiling of microglia in the injured brain reveals distinct molecular features underlying neurodegeneration

Cong

Dai

Shi

et al. 2021

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Neurotrauma has been recognized as a risk factor for neurodegenerative diseases, and sex difference of the incidence and outcome of neurodegenerative diseases has long been recognized. Past studies suggest that microglia could play a versatile role in both health and disease. So far, the microglial mechanisms underlying neurodegeneration and potentially lead to sex‐specific therapies are still very open. Here we applied whole transcriptome analysis of microglia acutely isolated at different timepoints after a cortical stab wound injury to gain insight into genes that might be dysregulated and transcriptionally different between males and females after cortical injury. We found that microglia displayed distinct temporal and sexual molecular signatures of transcriptome after cortical injury. Hypotheses and gene candidates that we presented in the present study could be worthy to be examined to explore the roles of microglia in neurotrauma and in sex‐biased neurodegenerative diseases.

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Enzymatic Dissociation Induces Transcriptional and Proteotype Bias in Brain Cell Populations

Cited by 97 publications

References 35 publications

P-selectin axis plays a key role in microglia immunophenotype and glioblastoma progression

P-selectin axis plays a key role in microglia immunophenotype and glioblastoma progression

Isolation of mouse brain-infiltrating leukocytes for single cell profiling of epitopes and transcriptomes

Transcriptional profiling of microglia in the injured brain reveals distinct molecular features underlying neurodegeneration

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