Phenotypic and functional characterization of aqueous humor derived extracellular vesicles.

Extracellular vesicles (EVs) are double membrane vesicles, abundant in all biological fluids. However, the characterization of EVs in aqueous humor (AH) is still limited. The aim of the present work was to characterize EVs isolated from AH (AH-EVs) in terms of surface markers of cellular origin and functional properties. We obtained AHs from patients with cataract undergoing surgical phacoemulsification and insertion of intraocular lenses (n = 10). Nanoparticle tracking analysis, electron microscopy, super resolution microscopy and bead-based cytofluorimetry were used to characterize EVs from AH. Subsequently, we investigated the effects of AH-EVs on viability, proliferation and wound healing of human immortalized keratinocyte (HaCaT) cells in vitro in comparison with the effect of mesenchymal stromal cell-EVs (MSC-EVs). AH-EVs had a mean size of around 100 nm and expressed the classical tetraspanins (CD9, CD63 and CD81). Super resolution microscopy revealed co-expression of CD9, CD63 and CD81. Moreover, cytofluorimetric analysis highlighted the expression of mesenchymal, stem, epithelial and endothelial markers. In the in vitro wound healing assay on HaCaT cells, AH-EVs induced a significantly faster wound repair, comparable to the effects of MSC-EVs, and promoted HaCaT cell viability and proliferation. We provide evidence, herein, of the possible AH-EV origin from stromal cells, limbal epithelial/stem cells, ciliary epithelium and corneal endothelium. In addition, we showed their in vitro proliferative and regenerative capacities.

HLSC-Derived Extracellular Vesicles Attenuate Liver Fibrosis and Inflammation in a Murine Model of Non-alcoholic Steatohepatitis.

Extracellular vesicles (EVs) are membrane vesicles released virtually by all cell types. Several studies have shown that stem cell-derived EVs may mimic both in vitro and in vivo the biological effects of the cells. We recently demonstrated that non-alcoholic steatohepatitis (NASH) is inhibited by treatment with human liver stem cells (HLSCs). The aim of the present study was to evaluate whether EVs released by HLSCs influence the progression of NASH, induced by a diet deprived of methionine and choline, in immunocompromised mice. EV treatment was initiated after 2 weeks of diet with a biweekly administration of three different doses. Bio-distribution evaluated by optical imaging showed a preferential accumulation in normal and, in particular, in fibrotic liver. EV treatment significantly improved liver function and reduced signs of liver fibrosis and inflammation at both morphological and molecular levels. In particular, we observed that, out of 29 fibrosis-associated genes upregulated in NASH liver, 28 were significantly downregulated by EV treatment. In conclusion, HLSC-derived EVs display anti-fibrotic and anti-inflammatory effects in a model of chronic liver disease, leading to an improvement of liver function.

Human Liver Stem Cell Derived Extracellular Vesicles Alleviate Kidney Fibrosis by Interfering with the ß-Catenin Pathway through miR29b.

Human liver stem-cell-derived extracellular vesicles (HLSC-EVs) exhibit therapeutic properties in various pre-clinical models of kidney injury. We previously reported an overall improvement in kidney function following treatment with HLSC-EVs in a model of aristolochic acid nephropathy (AAN). Here, we provide evidence that HLSC-EVs exert anti-fibrotic effects by interfering with ß-catenin signalling. A mouse model of AAN and an in vitro pro-fibrotic model were used. The ß-catenin mRNA and protein expression, together with the pro-fibrotic markers α-SMA and collagen 1, were evaluated in vivo and in vitro following treatment with HLSC-EVs. Expression and functional analysis of miR29b was performed in vitro following HLSC-EV treatments through loss-of-function experiments. Results showed that expression of ß-catenin was amplified both in vivo and in vitro, and ß-catenin gene silencing in fibroblasts prevented AA-induced up-regulation of pro-fibrotic genes, revealing that ß-catenin is an important factor in fibroblast activation. Treatment with HLSC-EVs caused increased expression of miR29b, which was significantly inhibited in the presence of α-amanitin. The suppression of the miR29b function with a selective inhibitor abolished the anti-fibrotic effects of HLSC-EVs, resulting in the up-regulation of ß-catenin and pro-fibrotic α-Sma and collagen type 1 genes. Together, these data suggest a novel HLSC-EV-dependent regulatory mechanism in which ß-catenin is down regulated by HLSC-EVs-induced miR29b expression.

A First Phenotypic and Functional Characterization of Placental Extracellular Vesicles from Women with Multiple Sclerosis.

Pregnancy is a unique situation of physiological immunomodulation, as well as a strong Multiple Sclerosis (MS) disease modulator whose mechanisms are still unclear. Both maternal (decidua) and fetal (trophoblast) placental cells secrete extracellular vesicles (EVs), which are known to mediate cellular communication and modulate the maternal immune response. Their contribution to the MS disease course during pregnancy, however, is unexplored. Here, we provide a first phenotypic and functional characterization of EVs isolated from cultures of term placenta samples of women with MS, differentiating between decidua and trophoblast. In particular, we analyzed the expression profile of 37 surface proteins and tested the functional role of placental EVs on mono-cultures of CD14+ monocytes and co-cultures of CD4+ T and regulatory T (Treg) cells. Results indicated that placental EVs are enriched for surface markers typical of stem/progenitor cells, and that conditioning with EVs from samples of women with MS is associated to a moderate decrease in the expression of proinflammatory cytokines by activated monocytes and in the proliferation rate of activated T cells co-cultured with Tregs. Overall, our findings suggest an immunomodulatory potential of placental EVs from women with MS and set the stage for a promising research field aiming at elucidating their role in MS remission.

Role of extracellular vesicles in stem cell biology.

The extracellular vesicles (EVs) are membrane vesicles carrying proteins, nucleic acids, and bioactive lipids of the cell of origin. These vesicles released within the extracellular space and entering into the circulation may transfer their cargo to neighboring or distant cells and induce phenotypical and functional changes that may be relevant in several physiopathological conditions. In an attempt to define the biological properties of EVs, several investigations have focused on their cargo and on the effects elicited in recipient cells. EVs have been involved in modulation of tumor microenvironment and behavior, as well as in the immune and inflammatory response. In the present review, we address the paracrine action of EVs released by stem cells and their potential involvement in the activation of regenerative programs in injured cells.

The Role of Extracellular Vesicles as Paracrine Effectors in Stem Cell-Based Therapies.

Stem cells act in a paracrine manner through the secretion of biologically active cargo that acts on cells locally and systemically. These active molecules include not only soluble factors but also extracellular vesicles (EVs) that have recently emerged as a mechanism of cell-to-cell communication. EVs act as vehicles that transfer molecules between originator and recipient cells, thereby modifying the phenotype and function of the latter. As EVs released from stem cells may successfully activate regenerative processes in injured cells, their application as a form of therapy can be envisaged. EVs exert these proregenerative effects through the modulation of relevant cellular processes including proliferation, angiogenesis, oxidative stress, inflammation, and immunotolerance, among others. In this chapter, we review the preclinical studies that report the effect of stem cell-derived EVs in various pathological models of human disease.

Renal Regenerative Potential of Extracellular Vesicles Derived from miRNA-Engineered Mesenchymal Stromal Cells.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) possess pro-regenerative potential in different animal models with renal injury. EVs contain different molecules, including proteins, lipids and nucleic acids. Among the shuttled molecules, miRNAs have a relevant role in the pro-regenerative effects of EVs and are a promising target for therapeutic interventions. The aim of this study was to increase the content of specific miRNAs in EVs that are known to be involved in the pro-regenerative effect of EVs, and to assess the capacity of modified EVs to contribute to renal regeneration in in vivo models with acute kidney injuries. To this purpose, MSCs were transiently transfected with specific miRNA mimics by electroporation. Molecular analyses showed that, after transfection, MSCs and derived EVs were efficiently enriched in the selected miRNAs. In vitro and in vivo experiments indicated that EVs engineered with miRNAs maintained their pro-regenerative effects. Of relevance, engineered EVs were more effective than EVs derived from naïve MSCs when used at suboptimal doses. This suggests the potential use of a low amount of EVs (82.5 × 106) to obtain the renal regenerative effect.

Detection of cellular prion protein in exosomes derived from ovine plasma.

Prion protein (PrP) is present at extremely low levels in the blood of animals and its detection is complicated by the poor sensitivity of current standard methodologies. Interesting results have been obtained with recent advanced technologies that are able to detect minute amounts of the pathological PrP (PrPSc), but their efficiency is reduced by various factors present in blood. In this study, we were able to extract cellular PrP (PrPC) from plasma-derived exosomes by a simple, fast method without the use of differential ultracentrifugation and to visualize it by Western blotting, reducing the presence of most plasma proteins. This result confirms that blood is capable of releasing PrP in association with exosomes and could be useful to better study its role in the pathogenesis of transmissible spongiform encephalopathies.

Role of HLA-G and extracellular vesicles in renal cancer stem cell-induced inhibition of dendritic cell differentiation.

BACKGROUND: Tumor immune-escape has been related to the ability of cancer cells to inhibit T cell activation and dendritic cell (DC) differentiation. We previously identified a tumor initiating population, expressing the mesenchymal marker CD105, which fulfills the criteria for definition as cancer stem cells (CD105(+) CSCs) able to release extracellular vesicles (EVs) that favor tumor progression and metastases. The aim of the present study was to compare the ability of renal CSCs and derived EVs to modulate the behavior of monocyte-derived DCs with a non-tumor initiating renal cancer cell population (CD105(-) TCs) and their EVs. METHODS: Maturation of monocyte-derived DCs was studied in presence of CD105(+) CSCs and CD105(-) TCs and their derived EVs. DC differentiation experiments were evaluated by cytofluorimetric analysis. T cell proliferation and ELISA assays were performed. Monocytes and T cells were purified from peripheral blood mononuclear cells obtained from healthy donors. RESULTS: The results obtained demonstrate that both CD105(+) CSCs and CD105(-) TCs impaired the differentiation process of DCs from monocytes. However, the immune-modulatory effect of CD105(+) CSCs was significantly greater than that of CD105(-) TCs. EVs derived from CD105(+) CSCs and in less extent, those derived from CD105(-) TCs retained the ability to impair monocyte maturation and T cell activation. The mechanism has been mainly related to the expression of HLA-G by tumor cells and to its release in a form associated to EVs. HLA-G blockade significantly reduced the inhibitory effect of EVs on DC differentiation. CONCLUSIONS: In conclusion, the results of the present study indicate that renal cancer cells and in particular CSCs and derived EVs impair maturation of DCs and T cell immune response by a mechanism involving HLA-G.

Edible Plant-Derived Extracellular Vesicles for Oral mRNA Vaccine Delivery.

Nucleic acid delivery through extracellular vesicles (EVs) is a well-preserved evolutionary mechanism in all life kingdoms including eukaryotes, prokaryotes, and plants. EVs naturally allow horizontal transfer of native as well as exogenous functional mRNAs, which once incorporated in EVs are protected from enzymatic degradation. This observation has prompted researchers to investigate whether EVs from different sources, including plants, could be used for vaccine delivery. Several studies using human or bacterial EVs expressing mRNA or recombinant SARS-CoV-2 proteins showed induction of a humoral and cell mediated immune response. Moreover, EV-based vaccines presenting the natural configuration of viral antigens have demonstrated advantages in conferring long-lasting immunization and lower toxicity than synthetic nanoparticles. Edible plant-derived EVs were shown to be an alternative to human EVs for vaccine delivery, especially via oral administration. EVs obtained from orange juice (oEVs) loaded with SARS-CoV-2 mRNAs protected their cargo from enzymatic degradation, were stable at room temperature for one year, and were able to trigger a SARS-CoV-2 immune response in mice. Lyophilized oEVs containing the S1 mRNA administered to rats via gavage induced a specific humoral immune response with generation of blocking antibodies, including IgA and Th1 lymphocyte activation. In conclusion, mRNA-containing oEVs could be used for developing new oral vaccines due to optimal mucosal absorption, resistance to stress conditions, and ability to stimulate a humoral and cellular immune response.

Role of stem-cell-derived microvesicles in the paracrine action of stem cells.

The paracrine theory has recently changed the view of the biological action of stem cells and of the subsequent potential application of stem cells in regenerative medicine. Indeed, most of the beneficial effects of stem-cell-based therapy have been attributed to soluble factors released from stem cells. In this context, MVs (microvesicles) released as exosomes from the endosomal compartment, or as shedding vesicles from the cell surface, may play a relevant role in the intercellular communication between stem and injured cells. By transferring proteins, bioactive lipids, mRNA and microRNA, MVs act as vehicles of information that may lead to alteration of the phenotype of recipient cells. The exchange of information between stem cells and tissue-injured cells is reciprocal. The MV-mediated transfer of tissue-specific information from the injured cells to stem cells may reprogramme the latter to gain phenotypic and functional characteristics of the cell of origin. On the other hand, MVs released from stem cells may confer a stem-cell-like phenotype to injured cells, with the consequent activation of self-regenerative programmes. In fact, MVs released from stem cells retain several biological activities that are able to reproduce the beneficial effects of stem cells in a variety of experimental models.

Rationale of mesenchymal stem cell therapy in kidney injury.

Numerous preclinical and clinical studies suggest that mesenchymal stem cells, also known as multipotent mesenchymal stromal cells (MSCs), may improve pathologic conditions involving different organs. These beneficial effects initially were ascribed to the differentiation of MSCs into organ parenchymal cells. However, at least in the kidney, this is a very rare event and the kidney-protective effects of MSCs have been attributed mainly to paracrine mechanisms. MSCs release a number of trophic, anti-inflammatory, and immune-modulatory factors that may limit kidney injury and favor recovery. In this article, we provide an overview of the biologic activities of MSCs that may be relevant for the treatment of kidney injury in the context of a case vignette concerning a patient at high immunologic risk who underwent a second kidney transplantation followed by the development of ischemia-reperfusion injury and acute allograft rejection. We discuss the possible beneficial effect of MSC treatment in the light of preclinical and clinical data supporting the regenerative and immunomodulatory potential of MSCs.

Human liver stem cell-derived microvesicles inhibit hepatoma growth in SCID mice by delivering antitumor microRNAs.

Microvesicles (MVs) play a pivotal role in cell-to-cell communication. Recent studies demonstrated that MVs may transfer genetic information between cells. Here, we show that MVs derived from human adult liver stem cells (HLSC) may reprogram in vitro HepG2 hepatoma and primary hepatocellular carcinoma cells by inhibiting their growth and survival. In vivo intratumor administration of MVs induced regression of ectopic tumors developed in SCID mice. We suggest that the mechanism of action is related to the delivery of microRNAs (miRNAs) from HLSC-derived MVs (MV-HLSC) to tumor cells on the basis of the following evidence: (a) the rapid, CD29-mediated internalization of MV-HLSC in HepG2 and the inhibition of tumor cell growth after MV uptake; (b) the transfer by MV-HLSC of miRNAs with potential antitumor activity that was downregulated in HepG2 cells with respect to normal hepatocytes; (c) the abrogation of the MV-HLSC antitumor effect after MV pretreatment with RNase or generation of MVs depleted of miRNAs; (d) the relevance of selected miRNAs was proven by transfecting HepG2 with miRNA mimics. The antitumor effect of MV-HLSC was also observed in tumors other than liver such as lymphoblastoma and glioblastoma. These results suggest that the delivery of selected miRNAs by MVs derived from stem cells may inhibit tumor growth and stimulate apoptosis.

Oral Delivery of mRNA Vaccine by Plant-Derived Extracellular Vesicle Carriers.

mRNA-based vaccines were effective in contrasting SARS-CoV-2 infection. However, they presented several limitations of storage and supply chain, and their parenteral administration elicited a limited mucosal IgA immune response. Extracellular vesicles (EVs) have been recognized as a mechanism of cell-to-cell communication well-preserved in all life kingdoms, including plants. Their membrane confers protection from enzyme degradation to encapsulated nucleic acids favoring their transfer between cells. In the present study, EVs derived from the juice of an edible plant (Citrus sinensis) (oEVs) were investigated as carriers of an orally administered mRNA vaccine coding for the S1 protein subunit of SARS-CoV-2 with gastro-resistant oral capsule formulation. The mRNA loaded into oEVs was protected and was stable at room temperature for one year after lyophilization and encapsulation. Rats immunized via gavage administration developed a humoral immune response with the production of specific IgM, IgG, and IgA, which represent the first mucosal barrier in the adaptive immune response. The vaccination also triggered the generation of blocking antibodies and specific lymphocyte activation. In conclusion, the formulation of lyophilized mRNA-containing oEVs represents an efficient delivery strategy for oral vaccines due to their stability at room temperature, optimal mucosal absorption, and the ability to trigger an immune response.

Plant-Derived Extracellular Vesicles as a Delivery Platform for RNA-Based Vaccine: Feasibility Study of an Oral and Intranasal SARS-CoV-2 Vaccine.

Plant-derived extracellular vesicles (EVs) may represent a platform for the delivery of RNA-based vaccines, exploiting their natural membrane envelope to protect and deliver nucleic acids. Here, EVs extracted from orange (Citrus sinensis) juice (oEVs) were investigated as carriers for oral and intranasal SARS-CoV-2 mRNA vaccine. oEVs were efficiently loaded with different mRNA molecules (coding N, subunit 1 and full S proteins) and the mRNA was protected from degrading stress (including RNase and simulated gastric fluid), delivered to target cells and translated into protein. APC cells stimulated with oEVs loaded with mRNAs induced T lymphocyte activation in vitro. The immunization of mice with oEVs loaded with S1 mRNA via different routes of administration including intramuscular, oral and intranasal stimulated a humoral immune response with production of specific IgM and IgG blocking antibodies and a T cell immune response, as suggested by IFN-γ production by spleen lymphocytes stimulated with S peptide. Oral and intranasal administration also triggered the production of specific IgA, the mucosal barrier in the adaptive immune response. In conclusion, plant-derived EVs represent a useful platform for mRNA-based vaccines administered not only parentally but also orally and intranasally.

Therapeutic potential of mesenchymal stem cell-derived microvesicles.

Several studies have demonstrated that mesenchymal stem cells have the capacity to reverse acute and chronic kidney injury in different experimental models by paracrine mechanisms. This paracrine action may be accounted for, at least in part, by microvesicles (MVs) released from mesenchymal stem cells, resulting in a horizontal transfer of mRNA, microRNA and proteins. MVs, released as exosomes from the endosomal compartment, or as shedding vesicles from the cell surface, are now recognized as being an integral component of the intercellular microenvironment. By acting as vehicles for information transfer, MVs play a pivotal role in cell-to-cell communication. This exchange of information between the injured cells and stem cells has the potential to be bi-directional. Thus, MVs may either transfer transcripts from injured cells to stem cells, resulting in reprogramming of their phenotype to acquire specific features of the tissue, or conversely, transcripts could be transferred from stem cells to injured cells, restraining tissue injury and inducing cell cycle re-entry of resident cells, leading to tissue self-repair. Upon administration with a therapeutic regimen, MVs mimic the effect of mesenchymal stem cells in various experimental models by inhibiting apoptosis and stimulating cell proliferation. In this review, we discuss whether MVs released from mesenchymal stem cells have the potential to be exploited in novel therapeutic approaches in regenerative medicine to repair damaged tissues, as an alternative to stem cell-based therapy.

Generation of Spike-Extracellular Vesicles (S-EVs) as a Tool to Mimic SARS-CoV-2 Interaction with Host Cells.

Extracellular vesicles (EVs) and viruses share common features: size, structure, biogenesis and uptake. In order to generate EVs expressing the SARS-CoV-2 spike protein on their surface (S-EVs), we collected EVs from SARS-CoV-2 spike expressing human embryonic kidney (HEK-293T) cells by stable transfection with a vector coding for the S1 and S2 subunits. S-EVs were characterized using nanoparticle tracking analysis, ExoView and super-resolution microscopy. We obtained a population of EVs of 50 to 200 nm in size. Spike expressing EVs represented around 40% of the total EV population and co-expressed spike protein with tetraspanins on the surfaces of EVs. We subsequently used ACE2-positive endothelial and bronchial epithelial cells for assessing the internalization of labeled S-EVs using a cytofluorimetric analysis. Internalization of S-EVs was higher than that of control EVs from non-transfected cells. Moreover, S-EV uptake was significantly decreased by anti-ACE2 antibody pre-treatment. Furthermore, colchicine, a drug currently used in clinical trials, significantly reduced S-EV entry into the cells. S-EVs represent a simple, safe, and scalable model to study host-virus interactions and the mechanisms of novel therapeutic drugs.

Dual Role of Extracellular Vesicles in Sepsis-Associated Kidney and Lung Injury.

Extracellular vesicles form a complex intercellular communication network, shuttling a variety of proteins, lipids, and nucleic acids, including regulatory RNAs, such as microRNAs. Transfer of these molecules to target cells allows for the modulation of sets of genes and mediates multiple paracrine and endocrine actions. EVs exert broad pro-inflammatory, pro-oxidant, and pro-apoptotic effects in sepsis, mediating microvascular dysfunction and multiple organ damage. This deleterious role is well documented in sepsis-associated acute kidney injury and acute respiratory distress syndrome. On the other hand, protective effects of stem cell-derived extracellular vesicles have been reported in experimental models of sepsis. Stem cell-derived extracellular vesicles recapitulate beneficial cytoprotective, regenerative, and immunomodulatory properties of parental cells and have shown therapeutic effects in experimental models of sepsis with kidney and lung involvement. Extracellular vesicles are also likely to play a role in deranged kidney-lung crosstalk, a hallmark of sepsis, and may be key to a better understanding of shared mechanisms underlying multiple organ dysfunction. In this review, we analyze the state-of-the-art knowledge on the dual role of EVs in sepsis-associated kidney/lung injury and repair. PubMed library was searched from inception to July 2022, using a combination of medical subject headings (MeSH) and keywords related to EVs, sepsis, acute kidney injury (AKI), acute lung injury (ALI), and acute respiratory distress syndrome (ARDS). Key findings are summarized into two sections on detrimental and beneficial mechanisms of actions of EVs in kidney and lung injury, respectively. The role of EVs in kidney-lung crosstalk is then outlined. Efforts to expand knowledge on EVs may pave the way to employ them as prognostic biomarkers or therapeutic targets to prevent or reduce organ damage in sepsis.

Microvesicles derived from human adult mesenchymal stem cells protect against ischaemia-reperfusion-induced acute and chronic kidney injury.

BACKGROUND: Several studies demonstrated that mesenchymal stem cells (MSCs) reverse acute kidney injury (AKI) by a paracrine mechanism rather than by MSC transdifferentiation. We recently demonstrated that microvesicles (MVs) released from MSCs may account for this paracrine mechanism by a horizontal transfer of messenger RNA and microRNA. METHODS: MVs isolated from MSCs were injected intravenously in rats (30 µg/rat) immediately after monolateral nephrectomy and renal artery and vein occlusion for 45 min. To evaluate the MV effects on AKI induced by ischaemia-reperfusion injury (IRI), the animals were divided into different groups: normal rats (n = 4), sham-operated rats (n = 6), IRI rats (n = 6), IRI + MV rats (n = 6), and IRI + RNase-MV rats (n = 6), and all animals were sacrificed at Day 2 after the operation. To evaluate the chronic kidney damage consequent to IRI, the rats were divided into different groups: sham-operated rats (n = 6) and IRI rats (n = 6), IRI + MV rats (n = 6), and all animal were sacrificed 6 months after the operation. RESULTS: We found that a single administration of MVs, immediately after IRI, protects rats from AKI by inhibiting apoptosis and stimulating tubular epithelial cell proliferation. The MVs also significantly reduced the impairment of renal function. Pretreatment of MVs with RNase to inactivate their RNA cargo abrogated these protective effects. Moreover, MVs by reducing the acute injury also protected from later chronic kidney disease. CONCLUSION: MVs released from MSCs protect from AKI induced by ischaemia reperfusion injury and from subsequent chronic renal damage. This suggest that MVs could be exploited as a potential new therapeutic approach.

Surface Marker Expression in Small and Medium/Large Mesenchymal Stromal Cell-Derived Extracellular Vesicles in Naive or Apoptotic Condition Using Orthogonal Techniques.

Extracellular vesicles released by mesenchymal stromal cells (MSC-EVs) are a promising resource for regenerative medicine. Small MSC-EVs represent the active EV fraction. A bulk analysis was applied to characterise MSC-EVs' identity and purity, with the assessment of single EV morphology, size and integrity using electron microscopy. We applied different methods to quantitatively analyse the size and surface marker expression in medium/large and small fractions, namely 10k and 100k fractions, of MSC-EVs obtained using sequential ultracentrifugation. Bone marrow, adipose tissue and umbilical cord MSC-EVs were compared in naive and apoptotic conditions. As detected by electron microscopy, the 100k EV size < 100 nm was confirmed by super-resolution microscopy and ExoView. Single-vesicle imaging using super-resolution microscopy revealed heterogeneous patterns of tetraspanins. ExoView allowed a comparative screening of single MSC-EV tetraspanin and mesenchymal markers. A semiquantitative bead-based cytofluorimetric analysis showed the segregation of immunological and pro-coagulative markers on the 10k MSC-EVs. Apoptotic MSC-EVs were released in higher numbers, without significant differences in the naive fractions in surface marker expression. These results show a consistent profile of MSC-EV fractions among the different sources and a safer profile of the 100k MSC-EV population for clinical application. Our study identified suitable applications for EV analytical techniques.