Revealing the Arabidopsis AtGRP7 mRNA binding proteome by specific enhanced RNA interactome capture.

BACKGROUND: The interaction of proteins with RNA in the cell is crucial to orchestrate all steps of RNA processing. RNA interactome capture (RIC) techniques have been implemented to catalogue RNA- binding proteins in the cell. In RIC, RNA-protein complexes are stabilized by UV crosslinking in vivo. Polyadenylated RNAs and associated proteins are pulled down from cell lysates using oligo(dT) beads and the RNA-binding proteome is identified by quantitative mass spectrometry. However, insights into the RNA-binding proteome of a single RNA that would yield mechanistic information on how RNA expression patterns are orchestrated, are scarce. RESULTS: Here, we explored RIC in Arabidopsis to identify proteins interacting with a single mRNA, using the circadian clock-regulated Arabidopsis thaliana GLYCINE-RICH RNA-BINDING PROTEIN 7 (AtGRP7) transcript, one of the most abundant transcripts in Arabidopsis, as a showcase. Seedlings were treated with UV light to covalently crosslink RNA and proteins. The AtGRP7 transcript was captured from cell lysates with antisense oligonucleotides directed against the 5'untranslated region (UTR). The efficiency of RNA capture was greatly improved by using locked nucleic acid (LNA)/DNA oligonucleotides, as done in the enhanced RIC protocol. Furthermore, performing a tandem capture with two rounds of pulldown with the 5'UTR oligonucleotide increased the yield. In total, we identified 356 proteins enriched relative to a pulldown from atgrp7 mutant plants. These were benchmarked against proteins pulled down from nuclear lysates by AtGRP7 in vitro transcripts immobilized on beads. Among the proteins validated by in vitro interaction we found the family of Acetylation Lowers Binding Affinity (ALBA) proteins. Interaction of ALBA4 with the AtGRP7 RNA was independently validated via individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP). The expression of the AtGRP7 transcript in an alba loss-of-function mutant was slightly changed compared to wild-type, demonstrating the functional relevance of the interaction. CONCLUSION: We adapted specific RNA interactome capture with LNA/DNA oligonucleotides for use in plants using AtGRP7 as a showcase. We anticipate that with further optimization and up scaling the protocol should be applicable for less abundant transcripts.

Trifunctional fatty acid derivatives: the impact of diazirine placement.

Functionalized lipid probes are a critical new tool to interrogate the function of individual lipid species, but the structural parameters that constrain their utility have not been thoroughly described. Here, we synthesize three palmitic acid derivatives with a diazirine at different positions on the acyl chain and examine their metabolism, subcellular localization, and protein interactions. We demonstrate that while they produce very similar metabolites and subcellular distributions, probes with the diazirine at either end pulldown distinct subsets of proteins after photo-crosslinking. This highlights the importance of thoughtful diazirine placement when developing probes based on biological molecules.

Interactomic exploration of LRRC8A in volume-regulated anion channels.

Ion channels are critical in enabling ion movement into and within cells and are important targets for pharmacological interventions in different human diseases. In addition to their ion transport abilities, ion channels interact with signalling and scaffolding proteins, which affects their function, cellular positioning, and links to intracellular signalling pathways. The study of "channelosomes" within cells has the potential to uncover their involvement in human diseases, although this field of research is still emerging. LRRC8A is the gene that encodes a crucial protein involved in the formation of volume-regulated anion channels (VRACs). Some studies suggest that LRRC8A could be a valuable prognostic tool in different types of cancer, serving as a biomarker for predicting patients' outcomes. LRRC8A expression levels might be linked to tumour progression, metastasis, and treatment response, although its implications in different cancer types can be varied. Here, publicly accessible databases of cancer patients were systematically analysed to determine if a correlation between VRAC channel expression and survival rate exists across distinct cancer types. Moreover, we re-evaluated the impact of LRRC8A on cellular proliferation and migration in colon cancer via HCT116 LRRC8A-KO cells, which is a current topic of debate in the literature. In addition, to investigate the role of LRRC8A in cellular signalling, we conducted biotin proximity-dependent identification (BioID) analysis, revealing a correlation between VRAC channels and cell-cell junctions, mechanisms that govern cellular calcium homeostasis, kinases, and GTPase signalling. Overall, this dataset improves our understanding of LRRC8A/VRAC and explores new research avenues while identifying promising therapeutic targets and promoting inventive methods for disease treatment.

Trifunctional fatty acid derivatives demonstrate the impact of diazirine placement.

Functionalized lipid probes are a critical new tool to interrogate the function of individual lipid species, but the structural parameters that constrain their utility have not been thoroughly described. Here, we synthesize three palmitic acid derivatives with a diazirine at different positions on the acyl chain and examine their metabolism, subcellular localization, and protein interactions. We demonstrate that while they produce very similar metabolites and subcellular distributions, probes with the diazirine at either end pulldown distinct subsets of proteins after photo-crosslinking. This highlights the importance of thoughtful diazirine placement when developing probes based on biological molecules.

Systematic analysis of RNA-binding proteins identifies targetable therapeutic vulnerabilities in osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumor with a strong tendency to metastasize, limiting the prognosis of affected patients. Genomic, epigenomic and transcriptomic analyses have demonstrated the exquisite molecular complexity of this tumor, but have not sufficiently defined the underlying mechanisms or identified promising therapeutic targets. To systematically explore RNA-protein interactions relevant to OS, we define the RNA interactomes together with the full proteome and the transcriptome of cells from five malignant bone tumors (four osteosarcomata and one malignant giant cell tumor of the bone) and from normal mesenchymal stem cells and osteoblasts. These analyses uncover both systematic changes of the RNA-binding activities of defined RNA-binding proteins common to all osteosarcomata and individual alterations that are observed in only a subset of tumors. Functional analyses reveal a particular vulnerability of these tumors to translation inhibition and a positive feedback loop involving the RBP IGF2BP3 and the transcription factor Myc which affects cellular translation and OS cell viability. Our results thus provide insight into potentially clinically relevant RNA-binding protein-dependent mechanisms of osteosarcoma.

MemPrep, a new technology for isolating organellar membranes provides fingerprints of lipid bilayer stress.

Biological membranes have a stunning ability to adapt their composition in response to physiological stress and metabolic challenges. Little is known how such perturbations affect individual organelles in eukaryotic cells. Pioneering work has provided insights into the subcellular distribution of lipids in the yeast Saccharomyces cerevisiae, but the composition of the endoplasmic reticulum (ER) membrane, which also crucially regulates lipid metabolism and the unfolded protein response, remains insufficiently characterized. Here, we describe a method for purifying organelle membranes from yeast, MemPrep. We demonstrate the purity of our ER membrane preparations by proteomics, and document the general utility of MemPrep by isolating vacuolar membranes. Quantitative lipidomics establishes the lipid composition of the ER and the vacuolar membrane. Our findings provide a baseline for studying membrane protein biogenesis and have important implications for understanding the role of lipids in regulating the unfolded protein response (UPR). The combined preparative and analytical MemPrep approach uncovers dynamic remodeling of ER membranes in stressed cells and establishes distinct molecular fingerprints of lipid bilayer stress.

Macromolecular crowding in human tenocyte and skin fibroblast cultures: A comparative analysis.

Although human tenocytes and dermal fibroblasts have shown promise in tendon engineering, no tissue engineered medicine has been developed due to the prolonged ex vivo time required to develop an implantable device. Considering that macromolecular crowding has the potential to substantially accelerate the development of functional tissue facsimiles, herein we compared human tenocyte and dermal fibroblast behaviour under standard and macromolecular crowding conditions to inform future studies in tendon engineering. Basic cell function analysis made apparent the innocuousness of macromolecular crowding for both cell types. Gene expression analysis of the without macromolecular crowding groups revealed expression of tendon related molecules in human dermal fibroblasts and tenocytes. Protein electrophoresis and immunocytochemistry analyses showed significantly increased and similar deposition of collagen fibres by macromolecular crowding in the two cell types. Proteomics analysis demonstrated great similarities between human tenocyte and dermal fibroblast cultures, as well as the induction of haemostatic, anti-microbial and tissue-protective proteins by macromolecular crowding in both cell populations. Collectively, these data rationalise the use of either human dermal fibroblasts or tenocytes in combination with macromolecular crowding in tendon engineering.

Molecular profiling of sponge deflation reveals an ancient relaxant-inflammatory response.

A hallmark of animals is the coordination of whole-body movement. Neurons and muscles are central to this, yet coordinated movements also exist in sponges that lack these cell types. Sponges are sessile animals with a complex canal system for filter-feeding. They undergo whole-body movements resembling "contractions" that lead to canal closure and water expulsion. Here, we combine live 3D optical coherence microscopy, pharmacology, and functional proteomics to elucidate the sequence and detail of shape changes, the tissues and molecular physiology involved, and the control of these movements. Morphometric analysis and targeted perturbation suggest that the movement is driven by the relaxation of actomyosin stress fibers in epithelial canal cells, which leads to whole-body deflation via collapse of the incurrent and expansion of the excurrent canal system. Thermal proteome profiling and quantitative phosphoproteomics confirm the control of cellular relaxation by an Akt/NO/PKG/PKA pathway. Agitation-induced deflation leads to differential phosphorylation of proteins forming epithelial cell junctions, implying their mechanosensitive role. Unexpectedly, untargeted metabolomics detect a concomitant decrease in antioxidant molecules during deflation, reflecting an increase in reactive oxygen species. Together with the secretion of proteinases, cytokines, and granulin, this indicates an inflammation-like state of the deflating sponge reminiscent of vascular endothelial cells experiencing oscillatory shear stress. These results suggest the conservation of an ancient relaxant-inflammatory response of perturbed fluid-carrying systems in animals and offer a possible mechanism for whole-body coordination through diffusible paracrine signals and mechanotransduction.

Trifunctional Sphinganine: A New Tool to Dissect Sphingolipid Function.

Functions and cell biology of the sphingolipids sphingosine and sphinganine in cells are not well understood. While some signaling roles for sphingosine have been elucidated, the closely related sphinganine has been described only insofar as it does not elicit many of the same signaling responses. Here, we prepared multifunctionalized derivatives of the two lipid species that differ only in a single double bond of the carbon backbone. Using these novel probes, we were able to define their spatiotemporal distributions within cells. Furthermore, we used these tools to systematically map the protein interactomes of both lipids. The lipid-protein conjugates, prepared through photo-crosslinking in live cells and extraction via click chemistry to azide beads, revealed significant differences in the captured proteins, highlighting their distinct roles in various cellular processes. This work elucidates mechanistic differences between these critical lipids and sets the foundation for further studies of the cellular functions of sphingosine and sphinganine.

Global Hfq-mediated RNA interactome of nitrogen starved Escherichia coli uncovers a conserved post-transcriptional regulatory axis required for optimal growth recovery.

The RNA binding protein Hfq has a central role in the post-transcription control of gene expression in many bacteria. Numerous studies have mapped the transcriptome-wide Hfq-mediated RNA-RNA interactions in growing bacteria or bacteria that have entered short-term growth-arrest. To what extent post-transcriptional regulation underpins gene expression in growth-arrested bacteria remains unknown. Here, we used nitrogen (N) starvation as a model to study the Hfq-mediated RNA interactome as Escherichia coli enter, experience, and exit long-term growth arrest. We observe that the Hfq-mediated RNA interactome undergoes extensive changes during N starvation, with the conserved SdsR sRNA making the most interactions with different mRNA targets exclusively in long-term N-starved E. coli. Taking a proteomics approach, we reveal that in growth-arrested cells SdsR influences gene expression far beyond its direct mRNA targets. We demonstrate that the absence of SdsR significantly compromises the ability of the mutant bacteria to recover growth competitively from the long-term N-starved state and uncover a conserved post-transcriptional regulatory axis which underpins this process.

Flow in fetoplacental-like microvessels in vitro enhances perfusion, barrier function, and matrix stability.

Proper placental vascularization is vital for pregnancy outcomes, but assessing it with animal models and human explants has limitations. We introduce a 3D in vitro model of human placenta terminal villi including fetal mesenchyme and vascular endothelium. By coculturing HUVEC, placental fibroblasts, and pericytes in a macrofluidic chip with a flow reservoir, we generate fully perfusable fetal microvessels. Pressure-driven flow facilitates microvessel growth and remodeling, resulting in early formation of interconnected and lasting placental-like vascular networks. Computational fluid dynamics simulations predict shear forces, which increase microtissue stiffness, decrease diffusivity, and enhance barrier function as shear stress rises. Mass spectrometry analysis reveals enhanced protein expression with flow, including matrix stability regulators, proteins associated with actin dynamics, and cytoskeleton organization. Our model provides a powerful tool for deducing complex in vivo parameters, such as shear stress on developing vascularized placental tissue, and holds promise for unraveling gestational disorders related to the vasculature.

STPP-UP: An alternative method for drug target identification using protein thermal stability.

Thermal proteome profiling (TPP) has significantly advanced the field of drug discovery by facilitating proteome-wide identification of drug targets and off-targets. However, TPP has not been widely applied for high-throughput drug screenings, since the method is labor intensive and requires a lot of measurement time on a mass spectrometer. Here, we present Single-tube TPP with Uniform Progression (STPP-UP), which significantly reduces both the amount of required input material and measurement time, while retaining the ability to identify drug targets for compounds of interest. By using incremental heating of a single sample, changes in protein thermal stability across a range of temperatures can be assessed, while alleviating the need to measure multiple samples heated to different temperatures. We demonstrate that STPP-UP is able to identify the direct interactors for anticancer drugs in both human and mice cells. In summary, the STPP-UP methodology represents a useful tool to advance drug discovery and drug repurposing efforts.

Comparative analysis of wild-type and chloroplast MCU-deficient plants reveals multiple consequences of chloroplast calcium handling under drought stress.

Introduction: Chloroplast calcium homeostasis plays an important role in modulating the response of plants to abiotic and biotic stresses. One of the greatest challenges is to understand how chloroplast calcium-permeable pathways and sensors are regulated in a concerted manner to translate specific information into a calcium signature and to elucidate the downstream effects of specific chloroplast calcium dynamics. One of the six homologs of the mitochondrial calcium uniporter (MCU) was found to be located in chloroplasts in the leaves and to crucially contribute to drought- and oxidative stress-triggered uptake of calcium into this organelle. Methods: In the present study we integrated comparative proteomic analysis with biochemical, genetic, cellular, ionomic and hormone analysis in order to gain an insight into how chloroplast calcium channels are integrated into signaling circuits under watered condition and under drought stress. Results: Altogether, our results indicate for the first time a link between chloroplast calcium channels and hormone levels, showing an enhanced ABA level in the cmcu mutant already in well-watered condition. Furthermore, we show that the lack of cMCU results in an upregulation of the calcium sensor CAS and of enzymes of chlorophyll synthesis, which are also involved in retrograde signaling upon drought stress, in two independent KO lines generated in Col-0 and Col-4 ecotypes. Conclusions: These observations point to chloroplasts as important signaling hubs linked to their calcium dynamics. Our results obtained in the model plant Arabidopsis thaliana are discussed also in light of our limited knowledge regarding organellar calcium signaling in crops and raise the possibility of an involvement of such signaling in response to drought stress also in crops.

RNA-Binding Proteins Regulate Post-Transcriptional Responses to TGF-ß to Coordinate Function and Mesenchymal Activation of Murine Endothelial Cells.

BACKGROUND: Endothelial cells (ECs) are primed to respond to various signaling cues. For example, TGF (transforming growth factor)-ß has major effects on EC function and phenotype by driving ECs towards a more mesenchymal state (ie, triggering endothelial to mesenchymal activation), a dynamic process associated with cardiovascular diseases. Although transcriptional regulation triggered by TGF-ß in ECs is well characterized, post-transcriptional regulatory mechanisms induced by TGF-ß remain largely unknown. METHODS: Using RNA interactome capture, we identified global TGF-ß driven changes in RNA-binding proteins in ECs. We investigated specific changes in the RNA-binding patterns of hnRNP H1 (heterogeneous nuclear ribonucleoprotein H1) and Csde1 (cold shock domain containing E1) using RNA immunoprecipitation and overlapped this with RNA-sequencing data after knockdown of either protein for functional insight. Using a modified proximity ligation assay, we visualized the specific interactions between hnRNP H1 and Csde1 and target RNAs in situ both in vitro and in mouse heart sections. RESULTS: Characterization of TGF-ß-regulated RBPs (RNA-binding proteins) revealed hnRNP H1 and Csde1 as key regulators of the cellular response to TGF-ß at the post-transcriptional level, with loss of either protein-promoting mesenchymal activation in ECs. We found that TGF-ß drives an increase in binding of hnRNP H1 to its target RNAs, offsetting mesenchymal activation, but a decrease in Csde1 RNA-binding, facilitating this process. Both, hnRNP H1 and Csde1, dynamically bind and regulate specific subsets of mRNAs related to mesenchymal activation and endothelial function. CONCLUSIONS: Together, we show that RBPs play a key role in the endothelial response to TGF-ß stimulation at the post-transcriptional level and that the RBPs hnRNP H1 and Csde1 serve to maintain EC function and counteract mesenchymal activation. We propose that TGF-ß profoundly modifies RNA-protein interaction entailing feedback and feed-forward control at the post-transcriptional level, to fine-tune mesenchymal activation in ECs.

Molecular profiling of sponge deflation reveals an ancient relaxant-inflammatory response.

A hallmark of animals is the coordination of whole-body movement. Neurons and muscles are central to this, yet coordinated movements also exist in sponges that lack these cell types. Sponges are sessile animals with a complex canal system for filter-feeding. They undergo whole-body movements resembling "contractions" that lead to canal closure and water expulsion. Here, we combine 3D optical coherence microscopy, pharmacology, and functional proteomics to elucidate anatomy, molecular physiology, and control of these movements. We find them driven by the relaxation of actomyosin stress fibers in epithelial canal cells, which leads to whole-body deflation via collapse of the incurrent and expansion of the excurrent system, controlled by an Akt/NO/PKG/A pathway. A concomitant increase in reactive oxygen species and secretion of proteinases and cytokines indicate an inflammation-like state reminiscent of vascular endothelial cells experiencing oscillatory shear stress. This suggests an ancient relaxant-inflammatory response of perturbed fluid-carrying systems in animals.

Redox Proteomic Profile of Tirapazamine-Resistant Murine Hepatoma Cells.

3-Amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine, TPZ) and other heteroaromatic N-oxides (ArN→O) exhibit tumoricidal, antibacterial, and antiprotozoal activities. Their action is attributed to the enzymatic single-electron reduction to free radicals that initiate the prooxidant processes. In order to clarify the mechanisms of aerobic mammalian cytotoxicity of ArN→O, we derived a TPZ-resistant subline of murine hepatoma MH22a cells (resistance index, 5.64). The quantitative proteomic of wild-type and TPZ-resistant cells revealed 5818 proteins, of which 237 were up- and 184 down-regulated. The expression of the antioxidant enzymes aldehyde- and alcohol dehydrogenases, carbonyl reductases, catalase, and glutathione reductase was increased 1.6-5.2 times, whereas the changes in the expression of glutathione peroxidase, superoxide dismutase, thioredoxin reductase, and peroxiredoxins were less pronounced. The expression of xenobiotics conjugating glutathione-S-transferases was increased by 1.6-2.6 times. On the other hand, the expression of NADPH:cytochrome P450 reductase was responsible for the single-electron reduction in TPZ and for the 2.1-fold decrease. These data support the fact that the main mechanism of action of TPZ under aerobic conditions is oxidative stress. The unchanged expression of intranuclear antioxidant proteins peroxiredoxin, glutaredoxin, and glutathione peroxidase, and a modest increase in the expression of DNA damage repair proteins, tend to support non-site-specific but not intranuclear oxidative stress as a main factor of TPZ aerobic cytotoxicity.

The Mechanism of Dendrite Formation in a Solid-State Transformation of High Aluminum Fe-Al Alloys.

The mechanism of solid-state dendrite formation in high-aluminum Fe-Al alloys is not clear. Applying an in-situ observation technique, the real-time formation and growth of FeAl solid-state dendrites during the eutectoid decomposition of the high-temperature phase Fe5Al8 is visualized. In-situ experiments by HT-CSLM reveal that proeutectoid FeAl usually does not preferentially nucleate at grain boundaries regardless of rapid or slow cooling conditions. The critical radii for generating morphological instability are 1.2 µm and 0.9 µm for slow and rapid cooling, respectively. The morphology after both slow and rapid cooling exhibits dendrites, while there are differences in the size and critical instability radius Rc, which are attributed to the different supersaturation S and the number of protrusions l. The combination of crystallographic and thermodynamic analysis indicates that solid-state dendrites only exist on the hypoeutectoid side in high-aluminum Fe-Al alloys. A large number of lattice defects in the parent phase provides an additional driving force for nucleation, leading to coherent nucleation from the interior of the parent phase grains based on the orientation relationship {3¯30}Fe5Al8//{1¯10}FeAl, <111¯>Fe5Al8//<111¯>FeAl. The maximum release of misfit strain energy leads to the preferential growth of the primary arm of the nucleus along <111¯> {1¯10}. During the rapid cooling process, a large supersaturation is induced in the matrix, driving the Al atoms to undergo unstable uphill diffusion and causing variations in the concentration gradient as well as generating constitutional undercooling, ultimately leading to morphological instability and the growth of secondary arms.

The RNA-binding protein landscapes differ between mammalian organs and cultured cells.

System-wide approaches have unveiled an unexpected breadth of the RNA-bound proteomes of cultured cells. Corresponding information regarding RNA-binding proteins (RBPs) of mammalian organs is still missing, largely due to technical challenges. Here, we describe ex vivo enhanced RNA interactome capture (eRIC) to characterize the RNA-bound proteomes of three different mouse organs. The resulting organ atlases encompass more than 1300 RBPs active in brain, kidney or liver. Nearly a quarter (291) of these had formerly not been identified in cultured cells, with more than 100 being metabolic enzymes. Remarkably, RBP activity differs between organs independent of RBP abundance, suggesting organ-specific levels of control. Similarly, we identify systematic differences in RNA binding between animal organs and cultured cells. The pervasive RNA binding of enzymes of intermediary metabolism in organs points to tightly knit connections between gene expression and metabolism, and displays a particular enrichment for enzymes that use nucleotide cofactors. We describe a generically applicable refinement of the eRIC technology and provide an instructive resource of RBPs active in intact mammalian organs, including the brain.

Wnt/ß-catenin signaling induces axial elasticity patterns of Hydra extracellular matrix.

The extracellular matrix (ECM) plays crucial roles in animal development and diseases. Here, we report that Wnt/ß-catenin signaling induces the ECM remodeling during Hydra axis formation. We determined the micro- and nanoscopic arrangement of fibrillar type I collagen along Hydra's body axis using high-resolution microscopy and X-ray scattering. Elasticity mapping of the ECM ex vivo revealed distinctive elasticity patterns along the body axis. A proteomic analysis of the ECM showed that these elasticity patterns correlate with a gradient-like distribution of metalloproteases along the body axis. Activation of the Wnt/ß-catenin pathway in wild-type and transgenic animals alters these patterns toward low ECM elasticity patterns. This suggests a mechanism whereby high protease activity under control of Wnt/ß-catenin signaling causes remodeling and softening of the ECM. This Wnt-dependent spatiotemporal coordination of biochemical and biomechanical cues in ECM formation was likely a central evolutionary innovation for animal tissue morphogenesis.

Independent human mesenchymal stromal cell-derived extracellular vesicle preparations differentially attenuate symptoms in an advanced murine graft-versus-host disease model.

BACKGROUND AIMS: Extracellular vesicles (EVs) harvested from conditioned media of human mesenchymal stromal cells (MSCs) suppress acute inflammation in various disease models and promote regeneration of damaged tissues. After successful treatment of a patient with acute steroid-refractory graft-versus-host disease (GVHD) using EVs prepared from conditioned media of human bone marrow-derived MSCs, this study focused on improving the MSC-EV production for clinical application. METHODS: Independent MSC-EV preparations all produced according to a standardized procedure revealed broad immunomodulatory differences. Only a proportion of the MSC-EV products applied effectively modulated immune responses in a multi-donor mixed lymphocyte reaction (mdMLR) assay. To explore the relevance of such differences in vivo, at first a mouse GVHD model was optimized. RESULTS: The functional testing of selected MSC-EV preparations demonstrated that MSC-EV preparations revealing immunomodulatory capabilities in the mdMLR assay also effectively suppress GVHD symptoms in this model. In contrast, MSC-EV preparations, lacking such in vitro activities, also failed to modulate GVHD symptoms in vivo. Searching for differences of the active and inactive MSC-EV preparations, no concrete proteins or miRNAs were identified that could serve as surrogate markers. CONCLUSIONS: Standardized MSC-EV production strategies may not be sufficient to warrant manufacturing of MSC-EV products with reproducible qualities. Consequently, given this functional heterogeneity, every individual MSC-EV preparation considered for the clinical application should be evaluated for its therapeutic potency before administration to patients. Here, upon comparing immunomodulating capabilities of independent MSC-EV preparations in vivo and in vitro, we found that the mdMLR assay was qualified for such analyses.