Microglia morphological response to mesenchymal stromal cell extracellular vesicles demonstrates EV therapeutic potential for modulating neuroinflammation
Year: 2024
Type: article
Abstract: BACKGROUND: Mesenchymal stromal cell derived extracellular vesicles (MSC-EVs) are a promising therapeutic for neuroinflammation. MSC-EVs can interact with microglia, the resident immune cells of the brain, to exert their immunomodulatory effects. In response to inflammatory cues, such as cytokines, microglia undergo phenotypic changes indicative of their function e.g. morphology and secretion. However, these changes in response to MSC-EVs are not well understood. Additionally, no disease-relevant screening tools to assess MSC-EV bioactivity exist, which has further impeded clinical translation. Here, we developed a quantitative, high throughput morphological profiling approach to assess the response of microglia to neuroinflammation- relevant signals and whether this morphological response can be used to indicate the bioactivity of MSC-EVs. RESULTS: Using an immortalized human microglia cell-line, we observed increased size (perimeter, major axis length) and complexity (form factor) upon stimulation with interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Upon treatment with MSC-EVs, the overall morphological score (determined using principal component analysis) shifted towards the unstimulated morphology, indicating that MSC-EVs are bioactive and modulate microglia. The morphological effects of MSC-EVs in TNF-α /IFN-γ stimulated cells were concomitant with reduced secretion of 14 chemokines/cytokines (e.g. CXCL6, CXCL9) and increased secretion of 12 chemokines/cytokines (e.g. CXCL8, CXCL10). Proteomic analysis of cell lysates revealed significant increases in 192 proteins (e.g. HIBADH, MEAK7, LAMC1) and decreases in 257 proteins (e.g. PTEN, TOM1, MFF) with MSC-EV treatment. Of note, many of these proteins are involved in regulation of cell morphology and migration. Gene Set Variation Analysis revealed upregulation of pathways associated with immune response, such as regulation of cytokine production, immune cell infiltration (e.g. T cells, NK cells) and morphological changes (e.g. Semaphorin, RHO/Rac signaling). Additionally, changes in microglia mitochondrial morphology were measured suggesting that MSC-EV modulate mitochondrial metabolism. CONCLUSION: This study comprehensively demonstrates the effects of MSC-EVs on human microglial morphology, cytokine secretion, cellular proteome, and mitochondrial content. Our high-throughput, rapid, low-cost morphometric approach enables screening of MSC-EV batches and manufacturing conditions to enhance EV function and mitigate EV functional heterogeneity in a disease relevant manner. This approach is highly generalizable and can be further adapted and refined based on selection of the disease-relevant signal, target cell, and therapeutic product.
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,790
Funders: National Science Foundation, Achievement Rewards for College Scientists Foundation, Georgia Agricultural Experiment Stations, National Institutes of Health, National Institute on Aging, National Institute of General Medical Sciences, National Institute of Neurological Disorders and Stroke, Division of Chemical, Bioengineering, Environmental, and Transport Systems, Office of Research, University of Georgia
Awards: G1395737231, FMSG: 3D-printed, granular hydrogels for the controlled expansion of mesenchymal stromal cells, G3549828030, G4857410013, NSF Engineering Research Center for Cell Manufacturing Technologies (CMaT), Collaborative Research: Investigating the role and interplay of microenvironment, manufacturing, and metabolism on MSC production of extracellular vesicles, G6671297155, G7868097974, CAREER: Closing the Loop on Neuroinflammation, Role of Neuronal p38 MAPK After Repetitive Mild TBI, G9000106303, Neuron and microglia-specific proteomic signatures of ERK mediated mechanisms of Alzheimer's disease.
OpenAlex ID: W4403510787