Microglia Morphological Response to Mesenchymal Stromal Cell Extracellular Vesicles Demonstrates EV Therapeutic Potential for Modulating Neuroinflammation.

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-γ/IFN-α 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 morphological 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.

High throughput screening of mesenchymal stromal cell morphological response to inflammatory signals for bioreactor-based manufacturing of extracellular vesicles that modulate microglia.

Due to their immunomodulatory function, mesenchymal stromal cells (MSCs) are a promising therapeutic with the potential to treat neuroinflammation associated with neurodegenerative diseases. This function is mediated by secreted extracellular vesicles (MSC-EVs). Despite established safety, MSC clinical translation has been unsuccessful due to inconsistent clinical outcomes resulting from functional heterogeneity. Current approaches to mitigate functional heterogeneity include 'priming' MSCs with inflammatory signals to enhance function. However, comprehensive evaluation of priming and its effects on MSC-EV function has not been performed. Furthermore, clinical translation of MSC-EV therapies requires significant manufacturing scale-up, yet few studies have investigated the effects of priming in bioreactors. As MSC morphology has been shown to predict their immunomodulatory function, we screened MSC morphological response to an array of priming signals and evaluated MSC-EV identity and potency in response to priming in flasks and bioreactors. We identified unique priming conditions corresponding to distinct morphologies. These conditions demonstrated a range of MSC-EV preparation quality and lipidome, allowing us to discover a novel MSC-EV manufacturing condition, as well as gain insight into potential mechanisms of MSC-EV microglia modulation. Our novel screening approach and application of priming to MSC-EV bioreactor manufacturing informs refinement of larger-scale manufacturing and enhancement of MSC-EV function.

HILIC-IM-MS for Simultaneous Lipid and Metabolite Profiling of Bacteria.

Although MALDI-ToF platforms for microbial identifications have found great success in clinical microbiology, the sole use of protein fingerprints for the discrimination of closely related species, strain-level identifications, and detection of antimicrobial resistance remains a challenge for the technology. Several alternative mass spectrometry-based methods have been proposed to address the shortcomings of the protein-centric approach, including MALDI-ToF methods for fatty acid/lipid profiling and LC-MS profiling of metabolites. However, the molecular diversity of microbial pathogens suggests that no single "ome" will be sufficient for the accurate and sensitive identification of strain- and susceptibility-level profiling of bacteria. Here, we describe the development of an alternative approach to microorganism profiling that relies upon both metabolites and lipids rather than a single class of biomolecule. Single-phase extractions based on butanol, acetonitrile, and water (the BAW method) were evaluated for the recovery of lipids and metabolites from Gram-positive and -negative microorganisms. We found that BAW extraction solutions containing 45% butanol provided optimal recovery of both molecular classes in a single extraction. The single-phase extraction method was coupled to hydrophilic interaction liquid chromatography (HILIC) and ion mobility-mass spectrometry (IM-MS) to resolve similar-mass metabolites and lipids in three dimensions and provide multiple points of evidence for feature annotation in the absence of tandem mass spectrometry. We demonstrate that the combined use of metabolites and lipids can be used to differentiate microorganisms to the species- and strain-level for four of the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa) using data from a single ionization mode. These results present promising, early stage evidence for the use of multiomic signatures for the identification of microorganisms by liquid chromatography, ion mobility, and mass spectrometry that, upon further development, may improve upon the level of identification provided by current methods.

High throughput screening of mesenchymal stromal cell morphological response to inflammatory signals for bioreactor-based manufacturing of extracellular vesicles that modulate microglia.

Due to their immunomodulatory function, mesenchymal stromal cells (MSCs) are a promising therapeutic with the potential to treat neuroinflammation associated with neurodegenerative diseases. This function can be mediated by secreted extracellular vesicles (MSC-EVs). Despite established safety, MSC clinical translation has been unsuccessful due to inconsistent clinical outcomes resulting from functional heterogeneity. Current approaches to mitigate functional heterogeneity include 'priming' MSCs with inflammatory signals to enhance function. However, comprehensive evaluation of priming and its effects on MSC-EV function has not been performed. Clinical translation of MSC-EV therapies requires significant manufacturing scale-up, yet few studies have investigated the effects of priming in bioreactors. As MSC morphology has been shown to predict their immunomodulatory function, we screened MSC morphological response to an array of priming signals and evaluated MSC-EV identity and potency in response to priming in flasks and bioreactors. We identified unique priming conditions corresponding to distinct morphologies. These conditions demonstrated a range of MSC-EV preparation quality and lipidome, allowing us to discover a novel MSC-EV manufacturing condition, as well as gain insight into potential mechanisms of MSC-EV microglia modulation. Our novel screening approach and application of priming to MSC-EV bioreactor manufacturing informs refinement of larger-scale manufacturing and enhancement of MSC-EV function.

Revealing Fatty Acid Heterogeneity in Staphylococcal Lipids with Isotope Labeling and RPLC-IM-MS.

Up to 80% of the fatty acids in Staphylococcus aureus membrane lipids are branched, rather than straight-chain, fatty acids. The branched fatty acids (BCFAs) may have either an even or odd number of carbons, and the branch position may be at the penultimate carbon (iso) or the antepenultimate (anteiso) carbon of the tail. This results in two sets of isomeric fatty acid species with the same number of carbons that cannot be resolved by mass spectrometry. The isomer/isobar challenge is further complicated when the mixture of BCFAs and straight-chain fatty acids (SCFAs) are esterified into diacylated lipids such as the phosphatidylglycerol (PG) species of the S. aureus membrane. No conventional chromatographic method has been able to resolve diacylated lipids containing mixtures of SCFAs, anteiso-odd, iso-odd, and iso-even BCFAs. A major hurdle to method development in this area is the lack of relevant analytical standards for lipids containing BCFA isomers. The diversity of the S. aureus lipidome and its naturally high levels of BCFAs present an opportunity to explore the potential of resolving diacylated lipids containing BCFAs and SFCAs. Using our knowledge of lipid and fatty acid biosynthesis in S. aureus, we have used a stable-isotope-labeling strategy to develop and validate a 30 min C18 reversed-phase liquid chromatography method combined with traveling-wave ion mobility-mass spectrometry to provide resolution of diacylated lipids based on the number of BCFAs that they contain.