Inter-laboratory multiplex bead-based surface protein profiling of MSC-derived EV preparations identifies MSC-EV surface marker signatures.

Mesenchymal stromal cells (MSCs) are promising regenerative therapeutics that primarily exert their effects through secreted extracellular vesicles (EVs). These EVs - being small and non-living - are easier to handle and possess advantages over cellular products. Consequently, the therapeutic potential of MSC-EVs is increasingly investigated. However, due to variations in MSC-EV manufacturing strategies, MSC-EV products should be considered as highly diverse. Moreover, the diverse array of EV characterisation technologies used for MSC-EV characterisation further complicates reliable interlaboratory comparisons of published data. Consequently, this study aimed to establish a common method that can easily be used by various MSC-EV researchers to characterise MSC-EV preparations to facilitate interlaboratory comparisons. To this end, we conducted a comprehensive inter-laboratory assessment using a novel multiplex bead-based EV flow cytometry assay panel. This assessment involved 11 different MSC-EV products from five laboratories with varying MSC sources, culture conditions, and EV preparation methods. Through this assay panel covering a range of mostly MSC-related markers, we identified a set of cell surface markers consistently positive (CD44, CD73 and CD105) or negative (CD11b, CD45 and CD197) on EVs of all explored MSC-EV preparations. Hierarchical clustering analysis revealed distinct surface marker profiles associated with specific preparation processes and laboratory conditions. We propose CD73, CD105 and CD44 as robust positive markers for minimally identifying MSC-derived EVs and CD11b, CD14, CD19, CD45 and CD79 as reliable negative markers. Additionally, we highlight the influence of culture medium components, particularly human platelet lysate, on EV surface marker profiles, underscoring the influence of culture conditions on resulting EV products. This standardisable approach for MSC-EV surface marker profiling offers a tool for routine characterisation of manufactured EV products in pre-clinical and clinical research, enhances the quality control of MSC-EV preparations, and hopefully paves the way for higher consistency and reproducibility in the emerging therapeutic MSC-EV field.

Process development for the production of mesenchymal stromal cell-derived extracellular vesicles in conventional 2D systems.

BACKGROUND: In recent years, the importance of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) has increased significantly. For their widespread use, a standardized EV manufacturing is needed which often includes conventional, static 2D systems. For these system critical process parameters need to be determined. METHODS: We studied the impact of process parameters on MSC proliferation, MSC-derived particle production including EVs, EV- and MSC-specific marker expression, and particle functionality in a HaCaT cell migration assay. RESULTS: We found that cell culture growth surface and media affected MSCs and their secretory behavior. Interestingly, the materials that promoted MSC proliferation did not necessarily result in the most functional MSC-derived particles. In addition, we found that MSCs seeded at 4 × 103 cells cm-2 produced particles with improved functional properties compared to higher seeding densities. MSCs in a highly proliferative state did not produce the most particles, although these particles were significantly more effective in promoting HaCaT cell migration. The same correlation was found when investigating the cultivation temperature. A physiological temperature of 37°C was not optimal for particle yield, although it resulted in the most functional particles. We observed a proliferation-associated particle production and found potential correlations between particle production and glucose consumption, enabling the estimation of final particle yields. CONCLUSIONS: Our findings suggest that parameters, which must be defined prior to each individual cultivation and do not require complex and expensive equipment, can significantly increase MSC-derived particle production including EVs. Integrating these parameters into a standardized EV process development paves the way for robust and efficient EV manufacturing for early clinical phases.

Liquid Biopsy in Organ Damage: small extracellular vesicle chip-based assessment of polytrauma.

Background: Despite major advances in medicine, blood-borne biomarkers are urgently needed to support decision-making, including polytrauma. Here, we assessed serum-derived extracellular vesicles (EVs) as potential markers of decision-making in polytrauma. Objective: Our Liquid Biopsy in Organ Damage (LiBOD) study aimed to differentiate polytrauma with organ injury from polytrauma without organ injury. We analysed of blood-borne small EVs at the individual level using a combination of immunocapture and high-resolution imaging. Methods: To this end, we isolated, purified, and characterized small EVs according to the latest Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines from human blood collected within 24 h post-trauma and validated our results using a porcine polytrauma model. Results: We found that small EVs derived from monocytes CD14+ and CD14+CD61+ were significantly elevated in polytrauma with organ damage. To be precise, our findings revealed that CD9+CD14+ and CD14+CD61+ small EVs exhibited superior performance compared to CD9+CD61+ small EVs in accurately indicating polytrauma with organ damage, reaching a sensitivity and a specificity of 0.81% and 0.97%, respectively. The results in humans were confirmed in an independent porcine model of polytrauma. Conclusion: These findings suggest that these specific types of small EVs may serve as valuable, non-invasive, and objective biomarkers for assessing and monitoring the severity of polytrauma and associated organ damage.

A Bioreactor-Based Yellow Fever Virus-like Particle Production Process with Integrated Process Analytical Technology Based on Transient Transfection.

Yellow Fever (YF) is a severe disease that, while preventable through vaccination, lacks rapid intervention options for those already infected. There is an urgent need for passive immunization techniques using YF-virus-like particles (YF-VLPs). To address this, we successfully established a bioreactor-based production process for YF-VLPs, leveraging transient transfection and integrating Process Analytical Technology. A cornerstone of this approach was the optimization of plasmid DNA (pDNA) production to a yield of 11 mg/L using design of experiments. Glucose, NaCl, yeast extract, and a phosphate buffer showed significant influence on specific pDNA yield. The preliminary work for VLP-production in bioreactor showed adjustments to the HEK cell density, the polyplex formation duration, and medium exchanges effectively elevated transfection efficiencies. The additive Pluronic F-68 was neutral in its effects, and anti-clumping agents (ACA) adversely affected the transfection process. Finally, we established the stirred-tank bioreactor process with integrated dielectric spectroscopy, which gave real-time insight in relevant process steps, e.g., cell growth, polyplex uptake, and harvest time. We confirmed the presence and integrity of YF-VLP via Western blot, imaging flow cytometry measurement, and transmission electron microscopy. The YF-VLP production process can serve as a platform to produce VLPs as passive immunizing agents against other neglected tropical diseases.

Immunomodulatory potential of mesenchymal stromal cell-derived extracellular vesicles in chondrocyte inflammation.

Introduction: Osteoarthritis (OA) affects a large percentage of the population worldwide. Current surgical and nonsurgical concepts for treating OA only result in symptom-modifying effects. However, there is no disease-modifying therapy available. Extracellular vesicles released by mesenchymal stem/stromal cells (MSC-EV) are promising agents to positively influence joint homeostasis in the osteoarthritic surroundings. This pilot study aimed to investigate the effect of characterized MSC-EVs on chondrogenesis in a 3D chondrocyte inflammation model with the pro-inflammatory cytokine TNFα. Methods: Bovine articular chondrocytes were expanded and transferred into pellet culture at passage 3. TNFα, human MSC-EV preparations (MSC-EV batches 41.5-EVi1 and 84-EVi), EVs from human platelet lysate (hPL4-EV), or the combination of TNFα and EVs were supplemented. To assess the effect of MSC-EVs in the chondrocyte inflammation model after 14 days, DNA, glycosaminoglycan (GAG), total collagen, IL-6, and NO release were quantified, and gene expression of anabolic (COL-II, aggrecan, COMP, and PRG-4), catabolic (MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5), dedifferentiation (COL-I), hypertrophy (COL-X, VEGF), and inflammatory (IL-8) markers were analyzed; histological evaluation was performed using safranin O/Fast Green staining and immunohistochemistry of COL I and II. For statistical evaluation, nonparametric tests were chosen with a significance level of p < 0.05. Results: TNFα supplementation resulted in catabolic stimulation with increased levels of NO and IL-6, upregulation of catabolic gene expression, and downregulation of anabolic markers. These findings were supported by a decrease in matrix differentiation (COL-II). Supplementation of EVs resulted in an upregulation of the chondrogenic marker PRG-4. All MSC-EV preparations significantly increased GAG retention per pellet. In contrast, catabolic markers and IL-8 expression were upregulated by 41.5-EVi1. Regarding protein levels, IL-6 and NO release were increased by 41.5-EVi1. Histologic and immunohistochemical evaluations indicated a higher differentiation potential of chondrocytes treated with 84-EVi. Discussion: MSC-EVs can positively influence chondrocyte matrix production in pro-inflammatory surroundings, but can also stimulate inflammation. In this study MSC-EV 41.5-EVi1 supplementation increased chondrocyte inflammation, whereas MSC-84-EVi supplementation resulted a higher chondrogenic potential of chondrocytes in 3D pellet culture. In summary, the selected MSC-EVs exhibited promising chondrogenic effects indicating their significant potential for the treatment of OA; however, the functional heterogeneity in MSC-EV preparations has to be solved.

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.

Cell-to-cell spread inhibiting antibodies constitute a correlate of protection against herpes simplex virus type 1 reactivations: A retrospective study.

Background: Herpes simplex viruses (HSV) cause ubiquitous human infections. For vaccine development, knowledge concerning correlates of protection is essential. Therefore, we investigated (I) if humans are in principle capable producing cell-to-cell spread inhibiting antibodies against HSV and (II) whether this capacity is associated with a reduced HSV-1 reactivation risk. Methods: We established a high-throughput HSV-1-ΔgE-GFP reporter virus-based assay and evaluated 2,496 human plasma samples for HSV-1 glycoprotein E (gE) independent cell-to-cell spread inhibiting antibodies. Subsequently, we conducted a retrospective survey among the blood donors to analyze the correlation between the presence of cell-to-cell spread inhibiting antibodies in plasma and the frequency of HSV reactivations. Results: In total, 128 of the 2,496 blood donors (5.1%) exhibited high levels of HSV-1 gE independent cell-to-cell spread inhibiting antibodies in the plasma. None of the 147 HSV-1 seronegative plasmas exhibited partial or complete cell-to-cell spread inhibition, demonstrating the specificity of our assay. Individuals with cell-to-cell spread inhibiting antibodies showed a significantly lower frequency of HSV reactivations compared to subjects without sufficient levels of such antibodies. Conclusion: This study contains two important findings: (I) upon natural HSV infection, some humans produce cell-to-cell spread inhibiting antibodies and (II) such antibodies correlate with protection against recurrent HSV-1. Moreover, these elite neutralizers may provide promising material for immunoglobulin therapy and information for the design of a protective vaccine against HSV-1.

CD73 activity of mesenchymal stromal cell-derived extracellular vesicle preparations is detergent-resistant and does not correlate with immunomodulatory capabilities.

BACKGROUND AIMS: Extracellular vesicles (EVs) derived from human mesenchymal stromal cells (MSCs) show immunomodulatory activity in different assays both in vitro and in vivo. In previous work, the authors compared the immunomodulatory potential of independent MSC-EV preparations in a multi-donor mixed lymphocyte reaction (mdMLR) assay and an optimized steroid-refractory acute graft-versus-host disease (aGVHD) mouse model. The authors observed that only a proportion of the MSC-EV preparations showed immunomodulatory capabilities and demonstrated that only MSC-EV preparations with mdMLR immunomodulating activities were able to suppress aGVHD symptoms in vivo and vice versa. Since the mdMLR assay is complex and depends on primary human cells of different donors, the authors sought to establish an assay that is much easier to standardize and fulfills the requirements for becoming qualified as a potency assay. METHODS: The bona fide MSC antigen CD73 possesses ecto-5'-nucleotidase activity that cleaves pro-inflammatory extracellular adenosine monophosphate into anti-inflammatory adenosine and free phosphate. To test whether the ecto-5'-nucleotidase activity of the MSC-EV preparations reflected their immunomodulatory potential, the authors adopted an enzymatic assay that monitors the ecto-5'-nucleotidase activity of CD73 in a quantitative manner and compared the activity of well-characterized MSC-EV preparations containing or lacking mdMLR immunomodulatory activity. RESULTS: The authors showed that the ecto-5'-nucleotidase activity of the MSC-EV preparations did not correlate with their ability to modulate T-cell responses in the mdMLR assay and thus with their potency in improving disease symptomatology in the optimized mouse aGVHD model. Furthermore, the ecto-5'-nucleotidase activity was resistant to EV-destroying detergent treatment. CONCLUSIONS: Ecto-5'-nucleotidase activity neither reflects the potency of the authors' MSC-EV preparations nor provides any information about the integrity of the respective EVs. Thus, ecto-5'-nucleotidase enzyme activity is not indicative for the immunomodulatory potency of the authors' MSC-EV products. The development of appropriate potency assays for MSC-EV products remains challenging.

Impact of mesenchymal stromal cell-derived vesicular cargo on B-cell acute lymphoblastic leukemia progression.

Leukemia cells reciprocally interact with their surrounding bone marrow microenvironment (BMM), rendering it hospitable to leukemia cell survival, for instance through the release of small extracellular vesicles (sEVs). In contrast, we show here that BMM deficiency of pleckstrin homology domain family M member 1 (PLEKHM1), which serves as a hub between fusion and secretion of intracellular vesicles and is important for vesicular secretion in osteoclasts, accelerates murine BCR-ABL1+ B-cell acute lymphoblastic leukemia (B-ALL) via regulation of the cargo of sEVs released by BMM-derived mesenchymal stromal cells (MSCs). PLEKHM1-deficient MSCs and their sEVs carry increased amounts of syntenin and syndecan-1, resulting in a more immature B-cell phenotype and an increased number/function of leukemia-initiating cells (LICs) via focal adhesion kinase and AKT signaling in B-ALL cells. Ex vivo pretreatment of LICs with sEVs derived from PLEKHM1-deficient MSCs led to a strong trend toward acceleration of murine and human BCR-ABL1+ B-ALL. In turn, inflammatory mediators such as recombinant or B-ALL cell-derived tumor necrosis factor α or interleukin-1ß condition murine and human MSCs in vitro, decreasing PLEKHM1, while increasing syntenin and syndecan-1 in MSCs, thereby perpetuating the sEV-associated circuit. Consistently, human trephine biopsies of patients with B-ALL showed a reduced percentage of PLEKHM1+ MSCs. In summary, our data reveal an important role of BMM-derived sEVs for driving specifically BCR-ABL1+ B-ALL, possibly contributing to its worse prognosis compared with BCR-ABL1- B-ALL, and suggest that secretion of inflammatory cytokines by cancer cells in general may similarly modulate the tumor microenvironment.

Enhancement of Immunosuppressive Activity of Mesenchymal Stromal Cells by Platelet-Derived Factors is Accompanied by Apoptotic Priming.

The pro-inflammatory phase of bone healing, initiated by platelet activation and eventually hematoma formation, impacts bone marrow mesenchymal stromal cells (MSCs) in unknown ways. Here, we created platelet-rich plasma (PRP) hydrogels to study how platelet-derived factors modulate functional properties of encapsulated MSCs in comparison to a non-inflammatory fibrin (FBR) hydrogel environment. MSCs were isolated from human bone marrow, while PRP was collected from pooled apheresis thrombocyte concentrates and used for hydrogel preparation. After their encapsulation in hydrogels for 72 h, retrieved MSCs were analyzed for immunomodulatory activities, apoptosis, stem cell properties, senescence, CD9+, CD63+ and CD81+ extracellular vesicle (EV) release, and metabolism-related changes. PRP-hydrogels stimulated immunosuppressive functions of MSCs, along with their upregulated susceptibility to cell death in communication with PBMCs and augmented caspase 3/7 activity. We found impaired clonal growth and cell cycle progression, and more pronounced ß-galactosidase activity as well as accumulation of LC3-II-positive vacuoles in PRP-MSCs. Stimuli derived from PRP-hydrogels upregulated AKT and reduced mTOR phosphorylation in MSCs, which suggests an initiation of survival-related processes. Our results showed that PRP-hydrogels might represent a metabolically stressful environment, inducing acidification of MSCs, reducing polarization of the mitochondrial membrane and increasing lipid accumulation. These features were not detected in FBR-MSCs, which showed reduced CD63+ and CD81+ EV production and maintained clonogenicity. Our data revealed that PRP-derived hematoma components cause metabolic adaptation of MSCs followed by increased immune regulatory functions. For the first time, we showed that PRP stimuli represent a survival challenge and "apoptotic priming" that are detrimental for stem cell-like growth of MSCs and important for their therapeutic consideration.

CD9- and CD81-positive extracellular vesicles provide a marker to monitor glioblastoma cell response to photon-based and proton-based radiotherapy.

Glioblastoma multiforme (GBM) is the most aggressive tumor of the central nervous system with a poor prognosis. In the treatment of GBM tumors, radiotherapy plays a major role. Typically, GBM tumors cannot be cured by irradiation because of intrinsic resistance machanisms. An escalation of the irradiation dose in the GBM tumor is difficult due to the high risk of severe side effects in the brain. In the last decade, the development of new irradiation techniques, including proton-based irradiation, promised new chances in the treatment of brain tumors. In contrast to conventional radiotherapy, irradiation with protons allows a dosimetrically more confined dose deposition in the tumor while better sparing the normal tissue surrounding the tumor. A systematic comparison of both irradiation techniques on glioblastoma cells has not been performed so far. Despite the improvements in radiotherapy, it remains challenging to predict the therapeutical response of GBM tumors. Recent publications suggest extracellular vesicles (EVs) as promising markers predicting tumor response. Being part of an ancient intercellular communication system, virtually all cells release specifically composed EVs. The assembly of EVs varies between cell types and depends on environmental parameters. Here, we compared the impact of photon-based with proton-based radiotherapy on cell viability and phenotype of four different glioblastoma cell lines. Furthermore, we characterized EVs released by different glioblastoma cells and correlated released EVs with the cellular response to radiotherapy. Our results demonstrated that glioblastoma cells reacted more sensitive to irradiation with protons than photons, while radiation-induced cell death 72 h after single dose irradiation was independent of the irradiation modality. Moreover, we detected CD9 and CD81-positive EVs in the supernatant of all glioblastoma cells, although at different concentrations. The amount of released CD9 and CD81-positive EVs increased after irradiation when cells became apoptotic. Although secreted EVs of non-irradiated cells were not predictive for radiosensitivity, their increased EV release after irradiation correlated with the cytotoxic response to radiotherapy 72 h after irradiation. Thus, our data suggest a novel application of EVs in the surveillance of anti-cancer therapies.

Characterizing the secretome of licensed hiPSC-derived MSCs.

Although mesenchymal stromal cells (MSCs) from primary tissues have been successfully applied in the clinic, their expansion capabilities are limited and results are variable. MSCs derived from human-induced pluripotent stem cells (hiMSCs) are expected to overcome these limitations and serve as a reproducible and sustainable cell source. We have explored characteristics and therapeutic potential of hiMSCs in comparison to hBMSCs. RNA sequencing confirmed high resemblance, with average Pearson correlation of 0.88 and Jaccard similarity index of 0.99, and similar to hBMSCs the hiMSCs released extracellular vesicles with in vitro immunomodulatory properties. Potency assay with TNFα and IFNγ demonstrated an increase in well-known immunomodulatory genes such as IDO1, CXCL8/IL8, and HLA-DRA which was also highlighted by enhanced secretion in the media. Notably, expression of 125 genes increased more than 1000-fold. These genes were predicted to be regulated by NFΚB signaling, known to play a central role in immune response. Altogether, our data qualify hiMSCs as a promising source for cell therapy and/or cell-based therapeutic products. Additionally, the herewith generated database will add to our understanding of the mode of action of regenerative cell-based therapies and could be used to identify relevant potency markers.

Detailed Characterization of Small Extracellular Vesicles from Different Cell Types Based on Tetraspanin Composition by ExoView R100 Platform.

Small extracellular vesicles (sEV) hold enormous potential as biomarkers, drug carriers, and therapeutic agents. However, due to previous limitations in the phenotypic characterization of sEV at the single vesicle level, knowledge of cell type-specific sEV signatures remains sparse. With the introduction of next-generation sEV analysis devices, such as the single-particle interferometric reflectance imaging sensor (SP-IRIS)-based ExoView R100 platform, single sEV analyses are now possible. While the tetraspanins CD9, CD63, and CD81 were generally considered pan-sEV markers, it became clear that sEV of different cell types contain several combinations and amounts of these proteins on their surfaces. To gain better insight into the complexity and heterogeneity of sEV, we used the ExoView R100 platform to analyze the CD9/CD63/CD81 phenotype of sEV released by different cell types at a single sEV level. We demonstrated that these surface markers are sufficient to distinguish cell-type-specific sEV phenotypes. Furthermore, we recognized that tetraspanin composition in some sEV populations does not follow a random pattern. Notably, the tetraspanin distribution of sEV derived from mesenchymal stem cells (MSCs) alters depending on cell culture conditions. Overall, our data provide an overview of the cell-specific characteristics of sEV populations, which will increase the understanding of sEV physiology and improve the development of new sEV-based therapeutic approaches.

Imaging flow cytometry challenges the usefulness of classically used extracellular vesicle labeling dyes and qualifies the novel dye Exoria for the labeling of mesenchymal stromal cell-extracellular vesicle preparations.

BACKGROUND AIMS: Extracellular vesicles (EVs) are involved in mediating intercellular communication processes. An important goal within the EV field is the study of the biodistribution of EVs and the identification of their target cells. Considering that EV uptake is assumed to be important for EVs in mediating intercellular communication processes, labeling with fluorescent dyes has emerged as a broadly distributed strategy for the identification of EV target cells and tissues. However, the accuracy and specificity of commonly utilized labeling dyes have not been sufficiently analyzed. METHODS: By combining recent advances in imaging flow cytometry for the phenotypic analysis of single EVs and aiming to identify target cells for EVs within therapeutically relevant mesenchymal stromal cell (MSC)-EV preparations, the authors explored the EV labeling efficacy of various fluorescent dyes, specifically carboxyfluorescein diacetate succinimidyl ester, calcein AM, PKH67, BODIPY TR ceramide (Thermo Fisher Scientific, Darmstadt, Germany) and a novel lipid dye called Exoria (Exopharm Limited, Melbourne, Australia). RESULTS: The authors' analyses qualified Exoria as the only dye that specifically labeled EVs within the MSC-EV preparations. Furthermore, the authors demonstrated that Exoria labeling did not interfere with the immunomodulatory properties of the MSC-EV preparations as tested in a multi-donor mixed lymphocyte reaction assay. Within this assay, labeled EVs were differentially taken up by different immune cell types. CONCLUSIONS: Overall, the results qualify Exoria as an appropriate dye for the labeling of EVs derived from the authors' MSC-EV preparations. This study also demonstrates the need for the development of next-generation EV characterization tools that are able to localize and confirm the specificity of EV labeling.

Postischemic Neuroprotection Associated With Anti-Inflammatory Effects by Mesenchymal Stromal Cell-Derived Small Extracellular Vesicles in Aged Mice.

BACKGROUND AND PURPOSE: Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) were shown to induce ischemic neuroprotection in mice by modulating the brain infiltration of leukocytes and, specifically polymorphonuclear neutrophils. So far, effects of MSC-sEVs were only studied in young ischemic rodents. We herein examined the effects of MSC-sEVs in aged mice. METHODS: Male and female C57Bl6/j mice (8-10 weeks or 15-24 months) were exposed to transient intraluminal middle cerebral artery occlusion. Vehicle or sEVs (equivalent of 2×106 MSCs) were intravenously administered. Neurological deficits, ischemic injury, blood-brain barrier integrity, brain leukocyte infiltration, and blood leukocyte responses were evaluated over up to 7 days. RESULTS: MSC-sEV delivery reduced neurological deficits, infarct volume, brain edema, and neuronal injury in young and aged mice of both sexes, when delivered immediately postreperfusion or with 6 hours delay. MSC-sEVs decreased leukocyte and specifically polymorphonuclear neutrophil, monocyte, and macrophage infiltrates in ischemic brains of aged mice. In peripheral blood, the number of monocytes and activated T cells was significantly reduced by MSC-sEVs. CONCLUSIONS: MSC-sEVs induce postischemic neuroprotection and anti-inflammation in aged mice.

Mesenchymal stromal cell-derived small extracellular vesicles promote neurological recovery and brain remodeling after distal middle cerebral artery occlusion in aged rats.

Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) promote neurological recovery after middle cerebral artery occlusion (MCAO) in young rodents. Ischemic stroke mainly affects aged humans. MSC-sEV effects on stroke recovery in aged rodents had not been assessed. In a head-to-head comparison, we exposed young (4-5 months) and aged (19-20 months) male Sprague-Dawley rats to permanent distal MCAO. At 24 h, 3 and 7 days post-stroke, vehicle or MSC-sEVs (2 × 106 or 2 × 107 MSC equivalents/kg) were intravenously administered. Neurological deficits, ischemic injury, brain inflammatory responses, post-ischemic angiogenesis, and endogenous neurogenesis were evaluated over 28 days. Post-MCAO, aged vehicle-treated rats exhibited more severe motor-coordination deficits evaluated by rotating pole and cylinder tests and larger brain infarcts than young vehicle-treated rats. Although infarct volume was not influenced by MSC-sEVs, sEVs at both doses effectively reduced motor-coordination deficits in young and aged rats. Brain macrophage infiltrates in periinfarct tissue, which were evaluated as marker of a recovery-aversive inflammatory environment, were significantly stronger in aged than young vehicle-treated rats. sEVs reduced brain macrophage infiltrates in aged, but not young rats. The tolerogenic shift in immune balance paved the way for structural brain tissue remodeling. Hence, sEVs at both doses increased periinfarct angiogenesis evaluated by CD31/BrdU immunohistochemistry in young and aged rats, and low-dose sEVs increased neurogenesis in the subventricular zone examined by DCX/BrdU immunohistochemistry. Our study provides robust evidence that MSC-sEVs promote functional neurological recovery and brain tissue remodeling in aged rats post-stroke. This study encourages further proof-of-concept studies in clinic-relevant stroke settings.

Isolation of native EVs from primary biofluids-Free-flow electrophoresis as a novel approach to purify ascites-derived EVs.

Although extracellular vesicles (EVs) have been extensively characterized, efficient purification methods, especially from primary biofluids, remain challenging. Here we introduce free-flow electrophoresis (FFE) as a novel approach for purifying EVs from primary biofluids, in particular from the peritoneal fluid (ascites) of ovarian cancer patients. FFE represents a versatile, fast, matrix-free approach for separating different analytes with inherent differences in charge density and/or isoelectric point (pI). Using a series of buffered media with different pH values allowed us to collect 96 fractions of ascites samples. To characterize the composition of the individual fractions, we used state-of-the-art methods such as nanoflow and imaging flow cytometry (nFCM and iFCM) in addition to classical approaches. Of note, tetraspanin-positive events measured using nFCM were enriched in a small number of distinct fractions. This observation was corroborated by Western blot analysis and electron microscopy, demonstrating only minor contamination with soluble proteins and lipid particles. In addition, these gently purified EVs remain functional. Thus, FFE represents a new, efficient and fast method for separating native and highly purified EVs from complicated primary samples.

Single Extracellular Vesicle Analysis Performed by Imaging Flow Cytometry and Nanoparticle Tracking Analysis Evaluate the Accuracy of Urinary Extracellular Vesicle Preparation Techniques Differently.

Small extracellular vesicles isolated from urine (uEVs) are increasingly recognized as potential biomarkers. Meanwhile, different uEV preparation strategies exist. Conventionally, the performance of EV preparation methods is evaluated by single particle quantification, Western blot, and electron microscopy. Recently, we introduced imaging flow cytometry (IFCM) as a next-generation single EV analysis technology. Here, we analyzed uEV samples obtained with different preparation procedures using nanoparticle tracking analysis (NTA), semiquantitative Western blot, and IFCM. IFCM analyses demonstrated that urine contains a predominant CD9+ sEV population, which exceeds CD63+ and CD81+ sEV populations. Furthermore, we demonstrated that the storage temperature of urine samples negatively affects the recovery of CD9+ sEVs. Although overall reduced, the highest CD9+ sEV recovery was obtained from urine samples stored at -80 °C and the lowest from those stored at -20 °C. Upon comparing the yield of the different uEV preparations, incongruencies between NTA and IFCM data became apparent. Results obtained by both NTA and IFCM were consistent with Western blot analyses for EV marker proteins; however, NTA results correlated with the amount of the impurity marker uromodulin. Despite demonstrating that the combination of ultrafiltration and size exclusion chromatography appears as a reliable uEV preparation technique, our data challenge the soundness of traditional NTA for the evaluation of different EV preparation methods.

Small extracellular vesicles obtained from hypoxic mesenchymal stromal cells have unique characteristics that promote cerebral angiogenesis, brain remodeling and neurological recovery after focal cerebral ischemia in mice.

Obtained from the right cell-type, mesenchymal stromal cell (MSC)-derived small extracellular vesicles (sEVs) promote stroke recovery. Within this process, microvascular remodeling plays a central role. Herein, we evaluated the effects of MSC-sEVs on the proliferation, migration, and tube formation of human cerebral microvascular endothelial cells (hCMEC/D3) in vitro and on post-ischemic angiogenesis, brain remodeling and neurological recovery after middle cerebral artery occlusion (MCAO) in mice. In vitro, sEVs obtained from hypoxic (1% O2), but not 'normoxic' (21% O2) MSCs dose-dependently promoted endothelial proliferation, migration, and tube formation and increased post-ischemic endothelial survival. sEVs from hypoxic MSCs regulated a distinct set of miRNAs in hCMEC/D3 cells previously linked to angiogenesis, three being upregulated (miR-126-3p, miR-140-5p, let-7c-5p) and three downregulated (miR-186-5p, miR-370-3p, miR-409-3p). LC/MS-MS revealed 52 proteins differentially abundant in sEVs from hypoxic and 'normoxic' MSCs. 19 proteins were enriched (among them proteins involved in extracellular matrix-receptor interaction, focal adhesion, leukocyte transendothelial migration, protein digestion, and absorption), and 33 proteins reduced (among them proteins associated with metabolic pathways, extracellular matrix-receptor interaction, focal adhesion, and actin cytoskeleton) in hypoxic MSC-sEVs. Post-MCAO, sEVs from hypoxic MSCs increased microvascular length and branching point density in previously ischemic tissue assessed by 3D light sheet microscopy over up to 56 days, reduced delayed neuronal degeneration and brain atrophy, and enhanced neurological recovery. sEV-induced angiogenesis in vivo depended on the presence of polymorphonuclear neutrophils. In neutrophil-depleted mice, MSC-sEVs did not influence microvascular remodeling. sEVs from hypoxic MSCs have distinct angiogenic properties. Hypoxic preconditioning enhances the restorative effects of MSC-sEVs.

CRISPR/Cas9-mediated knockout of c-REL in HeLa cells results in profound defects of the cell cycle.

Cervical cancer is the fourth common cancer in women resulting worldwide in 266,000 deaths per year. Belonging to the carcinomas, new insights into cervical cancer biology may also have great implications for finding new treatment strategies for other kinds of epithelial cancers. Although the transcription factor NF-κB is known as a key player in tumor formation, the relevance of its particular subunits is still underestimated. Here, we applied CRISPR/Cas9n-mediated genome editing to successfully knockout the NF-κB subunit c-REL in HeLa Kyoto cells as a model system for cervical cancers. We successfully generated a homozygous deletion in the c-REL gene, which we validated using sequencing, qPCR, immunocytochemistry, western blot analysis, EMSA and analysis of off-target effects. On the functional level, we observed the deletion of c-REL to result in a significantly decreased cell proliferation in comparison to wildtype (wt) without affecting apoptosis. The impaired proliferative behavior of c-REL-/- cells was accompanied by a strongly decreased amount of the H2B protein as well as a significant delay in the prometaphase of mitosis compared to c-REL+/+ HeLa Kyoto cells. c-REL-/- cells further showed significantly decreased expression levels of c-REL target genes in comparison to wt. In accordance to our proliferation data, we observed the c-REL knockout to result in a significantly increased resistance against the chemotherapeutic agents 5-Fluoro-2'-deoxyuridine (5-FUDR) and cisplatin. In summary, our findings emphasize the importance of c-REL signaling in a cellular model of cervical cancer with direct clinical implications for the development of new treatment strategies.