MAGEA4 Coated Extracellular Vesicles Are Stable and Can Be Assembled In Vitro

Extracellular vesicles (EVs) are valued candidates for the development of new tools for medical applications. Vesicles carrying melanoma-associated antigen A (MAGEA) proteins, a subfamily of cancer-testis antigens, are particularly promising tools in the fight against cancer. Here, we have studied the biophysical and chemical properties of MAGEA4-EVs and show that they are stable under common storage conditions such as keeping at +4 °C and −80 °C for at least 3 weeks after purification. The MAGEA4-EVs can be freeze-thawed two times without losing MAGEA4 in detectable quantities. The attachment of MAGEA4 to the surface of EVs cannot be disrupted by high salt concentrations or chelators, but the vesicles are sensitive to high pH. The MAGEA4 protein can bind to the surface of EVs in vitro, using robust passive incubation. In addition, EVs can be loaded with recombinant proteins fused to the MAGEA4 open reading frame within the cells and also in vitro. The high stability of MAGEA4-EVs ensures their potential for the development of EV-based anti-cancer applications.     

Extracellular Vesicles: Interplay with the Extracellular Matrix and Modulated Cell Responses

The discovery that cells secrete extracellular vesicles (EVs), which carry a variety of regulatory proteins, nucleic acids, and lipids, has shed light on the sophisticated manner by which cells can communicate and accordingly function. The bioactivity of EVs is not only defined by their internal content, but also through their surface associated molecules, and the linked downstream signaling effects they elicit in target cells. The extracellular matrix (ECM) contains signaling and structural molecules that are central to tissue maintenance and repair. Recently, a subset of EVs residing within the extracellular matrix has been identified. Although some roles have been proposed for matrix-bound vesicles, their role as signaling molecules within the ECM is yet to be explored. Given the close association of EVs and the ECM, it is not surprising that EVs partly mediate repair and regeneration by modulating matrix deposition and degradation through their cellular targets. This review addresses unique EV features that allow them to interact with and navigate through the ECM, describes how their release and content is influenced by the ECM, and emphasizes the emerging role of stem-cell derived EVs in tissue repair and regeneration through their matrix-modulating properties.     

Extracellular Vesicles in Blood: Sources,Effects, and Applications

Extracellular vesicles (EVs) are important players for intercellular communication. EVs are secreted by almost all cell types; they can transfer information between nearby or distant cells, and they are highly abundant in body fluids. In this review, we describe the general characteristics of EVs, as well as isolation and characterization approaches. Then, we focus on one of the most relevant sources of EVs: the blood. Indeed, apart from EVs secreted by blood cells, EVs of diverse origins travel in the bloodstream. We present the numerous types of EVs that have been found in circulation. Besides, the implications of blood-derived EVs in both physiological and pathological processes are summarized, highlighting their potential as biomarkers for the diagnosis, treatment monitoring, and prognosis of several diseases, and also as indicators of physiological modifications. Finally, the applications of EVs introduced in the circulatory system are discussed. We describe the use of EVs from distinct origins, naturally produced or engineered, autologous, allogeneic, or even from different species and the effects they have when introduced in circulation. Therefore, the present work provides a comprehensive overview of the components, effects, and applications of EVs in blood.     

Educating EVs to Improve Bone Regeneration: Getting Closer to the Clinic

The incidence of bone-related disorders is continuously growing as the aging of the population in developing countries continues to increase. Although therapeutic interventions for bone regeneration exist, their effectiveness is questioned, especially under certain circumstances, such as critical size defects. This gap of curative options has led to the search for new and more effective therapeutic approaches for bone regeneration; among them, the possibility of using extracellular vesicles (EVs) is gaining ground. EVs are secreted, biocompatible, nano-sized vesicles that play a pivotal role as messengers between donor and target cells, mediated by their specific cargo. Evidence shows that bone-relevant cells secrete osteoanabolic EVs, whose functionality can be further improved by several strategies. This, together with the low immunogenicity of EVs and their storage advantages, make them attractive candidates for clinical prospects in bone regeneration. However, before EVs reach clinical translation, a number of concerns should be addressed. Unraveling the EVs’ mode of action in bone regeneration is one of them; the molecular mediators driving their osteoanabolic effects in acceptor cells are now beginning to be uncovered. Increasing the functional and bone targeting abilities of EVs are also matters of intense research. Here, we summarize the cell sources offering osteoanabolic EVs, and the current knowledge about the molecular cargos that mediate bone regeneration. Moreover, we discuss strategies under development to improve the osteoanabolic and bone-targeting potential of EVs.     

The Role of Extracellular Vesicles from Human Macrophages on Host-Pathogen Interaction

The nano-sized membrane enclosed extracellular vesicles (EVs) released by virtually all cell types play an essential role in intercellular communication via delivering bio-molecules, such as nucleic acids, proteins, lipids, and other molecules to recipient cells. By mediating an active and steady-state cell-to-cell communication, EVs contribute to regulating and preserving cellular homeostasis. On the other hand, EVs can also spread pathogen-derived molecules during infections, subverting the host immune responses during infections and thus worsening pathophysiological processes. In recent years, the biological functioning of EVs has become a widespread research field in basic and clinical branches of medical sciences due to their potential role in therapeutic applications for several diseases. This review aims to summarize the main recent findings regarding the implication of EVs shed by human macrophages (MΦ-EVs) and how they can modulate the host immune response to control or increase the damage caused by infectious agents. We will also present the methods used to describe MΦ-EVs, as well as the potential of these EVs as disease diagnostic tools for some human pathogens. We believe that an in-depth understanding of the host–pathogen interactions mediated by MΦ-EVs may trigger the development of innovative therapeutic strategies against infectious diseases.     

Focus on Extracellular Vesicles: Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles

The intense research focus on stem and progenitor cells could be attributed to their differentiation potential to generate new cells to replace diseased or lost cells in many highly intractable degenerative diseases, such as Alzheimer disease, multiple sclerosis, and heart diseases. However, experimental and clinical studies have increasingly attributed the therapeutic efficacy of these cells to their secretion. While stem and progenitor cells secreted many therapeutic molecules, none of these molecules singly or in combination could recapitulate the functional effects of stem cell transplantations. Recently, it was reported that extracellular vesicles (EVs) could recapitulate the therapeutic effects of stem cell transplantation. Based on the observations reported thus far, the prevailing hypothesis is that stem cell EVs exert their therapeutic effects by transferring biologically active molecules such as proteins, lipids, mRNA, and microRNA from the stem cells to injured or diseased cells. In this respect, stem cell EVs are similar to EVs from other cell types. They are both primarily vehicles for intercellular communication. Therefore, the differentiating factor is likely due to the composition of their cargo. The cargo of EVs from different cell types are known to include a common set of proteins and also proteins that reflect the cell source of the EVs and the physiological or pathological state of the cell source. Hence, elucidation of the stem cell EV cargo would provide an insight into the multiple physiological or biochemical changes necessary to affect the many reported stem cell-based therapeutic outcomes in a variety of experimental models and clinical trials.     

Small Extracellular Vesicles Derived from Human Chorionic MSCs as Modern Perspective towards Cell-Free Therapy

Mesenchymal stem cells (MSCs) are of great interest to scientists due to their application in cell therapy of many diseases, as well as regenerative medicine and tissue engineering. Recently, there has been growing evidence surrounding the research based on extracellular vesicles (EVs), especially small EVs (sEVs)/exosomes derived from MSCs. EVs/exosomes can be secreted by almost all cell types and various types of EVs show multiple functions. In addition, MSCs-derived exosomes have similar characteristics and biological activities to MSCs and their therapeutic applications are considered as a safe strategy in cell-free therapy. The aim of this study was the characterization of MSCs isolated from the chorion (CHo-MSCs) of human full-term placenta, as well as the isolation and analysis of small EVs obtained from these cells. Accordingly, in this study, the ability of small EVs’ uptake is indicated by synovial fibroblasts, osteoblasts and periosteum-derived MSCs. Improvement in the understanding of the structure, characteristics, mechanism of action and potential application of MSCs-derived small EVs can provide new insight into improved therapeutic strategies.     

Extracellular Vesicle Measurements with Nanoparticle Tracking Analysis: A Different Appreciation of Up and Down Secretion

As is the case with most eucaryotic cells, cancer cells are able to secrete extracellular vesicles (EVs) as a communication means towards their environment and surrounding cells. EVs are represented by microvesicles and smaller vesicles called exosomes, which are known for their involvement in cancer aggressiveness. The release of such EVs requires the intervention of trafficking-associated proteins, mostly represented by the RAB-GTPases family. In particular, RAB27A is known for its role in addressing EVs-to-be secreted towards the the plasma membrane. In this study, shRNAs targeting RAB27A were used in colorectal (CRC) and glioblastoma (GB) cell lines in order to alter EVs secretion. To study and monitor EVs secretion in cell lines’ supernatants, nanoparticle tracking analysis (NTA) was used through the NanoSight NS300 device. Since it appeared that NanoSight failed to detect the decrease in the EVs secretion, we performed another approach to drop EVs secretion (RAB27A-siRNA, indomethacin, Nexihnib20). Similar results were obtained i.e., no variation in EVs concentration. Conversely, NTA allowed us to monitor EVs up-secretion following rotenone treatment or hypoxia conditions. Therefore, our data seemed to point out the insufficiency of using only this technique for the assessment of EVs secretion decrease.     

Helicobacter pylori Outer Membrane Vesicles and Extracellular Vesicles from Helicobacter pylori-Infected Cells in Gastric Disease Development

Extracellular vesicles (EVs) are cell-derived vesicles important in intercellular communication that play an essential role in host-pathogen interactions, spreading pathogen-derived as well as host-derived molecules during infection. Pathogens can induce changes in the composition of EVs derived from the infected cells and use them to manipulate their microenvironment and, for instance, modulate innate and adaptive inflammatory immune responses, both in a stimulatory or suppressive manner. Gastric cancer is one of the leading causes of cancer-related deaths worldwide and infection with Helicobacter pylori (H. pylori) is considered the main risk factor for developing this disease, which is characterized by a strong inflammatory component. EVs released by host cells infected with H. pylori contribute significantly to inflammation, and in doing so promote the development of disease. Additionally, H. pylori liberates vesicles, called outer membrane vesicles (H. pylori-OMVs), which contribute to atrophia and cell transformation in the gastric epithelium. In this review, the participation of both EVs from cells infected with H. pylori and H. pylori-OMVs associated with the development of gastric cancer will be discussed. By deciphering which functions of these external vesicles during H. pylori infection benefit the host or the pathogen, novel treatment strategies may become available to prevent disease.     

Plant Extracellular Vesicles and Nanovesicles: Focus on Secondary Metabolites,Proteins and Lipids with Perspectives on Their Potential and Sources

While human extracellular vesicles (EVs) have attracted a big deal of interest and have been extensively characterized over the last years, plant-derived EVs and nanovesicles have earned less attention and have remained poorly investigated. Although a series of investigations already revealed promising beneficial health effects and drug delivery properties, adequate (pre)clinical studies are rare. This fact might be caused by a lack of sources with appropriate qualities. Our study introduces plant cell suspension culture as a new and well controllable source for plant EVs. Plant cells, cultured in vitro, release EVs into the growth medium which could be harvested for pharmaceutical applications. In this investigation we characterized EVs and nanovesicles from distinct sources. Our findings regarding secondary metabolites indicate that these might not be packaged into EVs in an active manner but enriched in the membrane when lipophilic enough, since apparently lipophilic compounds were associated with nanovesicles while more hydrophilic structures were not consistently found. In addition, protein identification revealed a possible explanation for the mechanism of EV cell wall passage in plants, since cell wall hydrolases like 1,3-β-glucosidases, pectinesterases, polygalacturonases, β-galactosidases and β-xylosidase/α-L-arabinofuranosidase 2-like are present in plant EVs and nanovesicles which might facilitate cell wall transition. Further on, the identified proteins indicate that plant cells secrete EVs using similar mechanisms as animal cells to release exosomes and microvesicles.     

Characterisation of Extracellular Vesicles from Equine Mesenchymal Stem Cells

Extracellular vesicles (EVs) are nanosized lipid bilayer-encapsulated particles secreted by virtually all cell types. EVs play an essential role in cellular crosstalk in health and disease. The cellular origin of EVs determines their composition and potential therapeutic effect. Mesenchymal stem/stromal cell (MSC)-derived EVs have shown a comparable therapeutic potential to their donor cells, making them a promising tool for regenerative medicine. The therapeutic application of EVs circumvents some safety concerns associated with the transplantation of viable, replicating cells and facilitates the quality-controlled production as a ready-to-go, off-the-shelf biological therapy. Recently, the International Society for Extracellular Vesicles (ISEV) suggested a set of minimal biochemical, biophysical and functional standards to define extracellular vesicles and their functions to improve standardisation in EV research. However, nonstandardised EV isolation methods and the limited availability of cross-reacting markers for most animal species restrict the application of these standards in the veterinary field and, therefore, the species comparability and standardisation of animal experiments. In this study, EVs were isolated from equine bone-marrow-derived MSCs using two different isolation methods, stepwise ultracentrifugation and size exclusion chromatography, and minimal experimental requirements for equine EVs were established and validated. Equine EVs were characterised using a nanotracking analysis, fluorescence-triggered flow cytometry, Western blot and transelectron microscopy. Based on the ISEV standards, minimal criteria for defining equine EVs are suggested as a baseline to allow the comparison of EV preparations obtained by different laboratories.     

Extracellular Vesicles in Myeloid Neoplasms

Myeloid neoplasms arise from malignant primitive cells, which exhibit growth advantage within the bone marrow microenvironment (BMM). The interaction between these malignant cells and BMM cells is critical for the progression of these diseases. Extracellular vesicles (EVs) are lipid bound vesicles secreted into the extracellular space and involved in intercellular communication. Recent studies have described RNA and protein alterations in EVs isolated from myeloid neoplasm patients compared to healthy controls. The altered expression of various micro-RNAs is the best-described feature of EVs of these patients. Some of these micro-RNAs induce growth-related pathways such as AKT/mTOR and promote the acquisition of stem cell-like features by malignant cells. Another well-described characteristic of EVs in myeloid neoplasms is their ability to suppress healthy hematopoiesis either via direct effect on healthy CD34+ cells or via alteration of the differentiation of BMM cells. These results support a role of EVs in the pathogenesis of myeloid neoplasms. mainly through mediating the interaction between malignant and BMM cells, and warrant further study to better understand their biology. In this review, we describe the reported alterations of EV composition in myeloid neoplasms and the recent discoveries supporting their involvement in the development and progression of these diseases.     

Pregnancy-Related Extracellular Vesicles Revisited

Extracellular vesicles (EVs) are small vesicles ranging from 20–200 nm to 10 μm in diameter that are discharged and taken in by many different types of cells. Depending on the nature and quantity of their content—which generally includes proteins, lipids as well as microRNAs (miRNAs), messenger-RNA (mRNA), and DNA—these particles can bring about functional modifications in the receiving cells. During pregnancy, placenta and/or fetal-derived EVs have recently been isolated, eliciting interest in discovering their clinical significance. To date, various studies have associated variations in the circulating levels of maternal and fetal EVs and their contents, with complications including gestational diabetes and preeclampsia, ultimately leading to adverse pregnancy outcomes. Furthermore, EVs have also been identified as messengers and important players in viral infections during pregnancy, as well as in various congenital malformations. Their presence can be detected in the maternal blood from the first trimester and their level increases towards term, thus acting as liquid biopsies that give invaluable insight into the status of the feto-placental unit. However, their exact roles in the metabolic and vascular adaptations associated with physiological and pathological pregnancy is still under investigation. Analyzing peer-reviewed journal articles available in online databases, the purpose of this review is to synthesize current knowledge regarding the utility of quantification of pregnancy related EVs in general and placental EVs in particular as non-invasive evidence of placental dysfunction and adverse pregnancy outcomes, and to develop the current understanding of these particles and their applicability in clinical practice.     

Extracellular Vesicles Taken up by Astrocytes

Extracellular vesicles (EVs) are composed of lipid bilayer membranes and contain various molecules, such as mRNA and microRNA (miRNA), that regulate the functions of the recipient cell. Recent studies have reported the importance of EV-mediated intercellular communication in the brain. The brain contains several types of cells, including neurons and glial cells. Among them, astrocytes are the most abundant glial cells in the mammalian brain and play a wide range of roles, from structural maintenance of the brain to regulation of neurotransmission. Furthermore, since astrocytes can take up EVs, it is possible that EVs originating from inside and outside the brain affect astrocyte function, which in turn affects brain function. However, it has not been fully clarified whether the specific targeting mechanism of EVs to astrocytes as recipient cells exists. In recent years, EVs have attracted attention as a cell-targeted therapeutic approach in various organs, and elucidation of the targeting mechanism of EVs to astrocytes may pave the way for new therapies for brain diseases. In this review, we focus on EVs in the brain that affect astrocyte function and discuss the targeting mechanism of EVs to astrocytes.     

Characterization and Therapeutic Use of Extracellular Vesicles Derived from Platelets

Autologous blood products, such as platelet-rich plasma (PRP), are gaining increasing interest in different fields of regenerative medicine. Although growth factors, the main components of PRP, are thought to stimulate reparation processes, the exact mechanism of action and main effectors of PRP are not fully understood. Plasma contains a high amount of extracellular vesicles (EVs) produced by different cells, including anucleated platelets. Platelet-derived EVs (PL-EVs) are the most abundant type of EVs in circulation. Numerous advantages of PL-EVs, including their ability to be released locally, their ease of travel through the body, their low immunogenicity and tumourigenicity, the modulation of signal transduction as well as the ease with which they can be obtained, has attracted increased attention n. This review focuses briefly on the biological characteristics and isolation methods of PL-EVs, including exosomes derived from platelets (PL-EXOs), and their involvement in the pathology of diseases. Evidence that shows how PL-EVs can be used as a novel tool in medicine, particularly in therapeutic and regenerative medicine, is also discussed in this review.     

Extracellular Vesicles as Emerging Players in Intercellular Communication: Relevance in Mast Cell-Mediated Pathophysiology

Mast cells are major effector cells in eliciting allergic responses. They also play a significant role in establishing innate and adaptive immune responses, as well as in modulating tumor growth. Mast cells can be activated upon engagement of the high-affinity receptor FcεRI with specific IgE to multivalent antigens or in response to several FcεRI-independent mechanisms. Upon stimulation, mast cells secrete various preformed and newly synthesized mediators. Emerging evidence indicates their ability to be a rich source of secreted extracellular vesicles (EVs), including exosomes and microvesicles, which convey biological functions. Mast cell-derived EVs can interact with and affect other cells located nearby or at distant sites and modulate inflammation, allergic response, and tumor progression. Mast cells are also affected by EVs derived from other cells in the immune system or in the tumor microenvironment, which may activate mast cells to release different mediators. In this review, we summarize the latest data regarding the ability of mast cells to release or respond to EVs and their role in allergic responses, inflammation, and tumor progression. Understanding the release, composition, and uptake of EVs by cells located near to or at sites distant from mast cells in a variety of clinical conditions, such as allergic inflammation, mastocytosis, and lung cancer will contribute to developing novel therapeutic approaches.     

Extracellular Vesicles as Signal Carriers in Malignant Thyroid Tumors?

Extracellular vesicles (EVs) are small, membranous structures involved in intercellular communication. Here, we analyzed the effects of thyroid cancer-derived EVs on the properties of normal thyroid cells and cells contributing to the tumor microenvironment. EVs isolated from thyroid cancer cell lines (CGTH, FTC-133, 8505c, TPC-1 and BcPAP) were used for treatment of normal thyroid cells (NTHY), as well as monocytes and endothelial cells (HUVEC). EVs’ size/number were analyzed by flow cytometry and confocal microscopy. Gene expression, protein level and localization were investigated by qRT-PCR, WB and ICC/IF, respectively. Proliferation, migration and tube formation were analyzed. When compared with NTHY, CGTH and BcPAP secreted significantly more EVs. Treatment of NTHY with cancer-derived EVs changed the expression of tetraspanin genes, but did not affect proliferation and migration. Cancer-derived EVs suppressed tube formation by endothelial cells and did not affect the phagocytic index of monocytes. The number of 6 μm size fraction of cancer-derived EVs correlated negatively with the CD63 and CD81 expression in NTHY cells, as well as positively with angiogenesis in vitro. Thyroid cancer-derived EVs can affect the expression of tetraspanins in normal thyroid cells. It is possible that 6 μm EVs contribute to the regulation of NTHY gene expression and angiogenesis.     

Focus on Extracellular Vesicles: Physiological Role and Signalling Properties of Extracellular Membrane Vesicles

Extracellular vesicles (EVs) are a heterogeneous population of secreted membrane vesicles, with distinct biogenesis routes, biophysical properties and different functions both in physiological conditions and in disease. The release of EVs is a widespread biological process, which is conserved across species. In recent years, numerous studies have demonstrated that several bioactive molecules are trafficked with(in) EVs, such as microRNAs, mRNAs, proteins and lipids. The understanding of their final impact on the biology of specific target cells remains matter of intense debate in the field. Also, EVs have attracted great interest as potential novel cell-free therapeutics. Here we describe the proposed physiological and pathological functions of EVs, with a particular focus on their molecular content. Also, we discuss the advances in the knowledge of the mechanisms regulating the secretion of EV-associated molecules and the specific pathways activated upon interaction with the target cell, highlighting the role of EVs in the context of the immune system and as mediators of the intercellular signalling in the brain.     

Plant-Derived Extracellular Vesicles as Therapeutic Nanocarriers

Mammalian exosomes have emerged as a promising class of functional materials, inspiring novel applications as therapeutic vehicles and nutraceutical compounds. Despite this, their immunogenicity has been an issue of controversy within the scientific community. Although, exosome-like vesicles, innately formed in plants and inherent to eukaryotic cell-derived vesicles, could soothe most of the concerns, they are notably underutilized as therapeutic modalities. This review highlights all efforts published so far, on the use of plant-derived extracellular vesicles (EVs) as therapeutic delivery systems. A summary of the physicochemical characteristics of plant-derived EVs is provided along with their main biological composition and in vitro/in vivo evidence of their therapeutic efficacy provided where available. Despite only a hand full of clinical trials being underway, concerning these vesicles, they arguably possess significant potential as nanodelivery systems of natural origin.     

Epithelial Cell-Derived Extracellular Vesicles Trigger the Differentiation of Two Epithelial Cell Lines

Extracellular vesicles (EVs), specifically exosomes, carry a cell-type dependent cargo that is transported to the recipient cell and translated in the presence of a required machinery. Differences in the cargo carried by the corneal and conjunctival-derived EVs could be the agent that triggers the transdifferentiation of these two cell populations. Therefore, this study investigates the role of EVs in triggering the plasticity of corneal and conjunctival epithelial cells and identifies prospective miRNA and genes responsible for maintaining ocular surface homeostasis. The EVs were extracted from the conditioned media (after starving) of corneal epithelial (hTCEpi) and conjunctival (HCjE-Gi) cell lines using ultracentrifugation. HCjE-Gi cells were cultured with hTCEpi-derived EVs and vice-versa. The EVs were characterized as exosomes using Nanosight and Flow cytometry. KRT3 and KRT12 were used as associated corneal markers, whereas KRT7 and KRT13 were used as associated conjunctival markers with ΔNp63 as a differentiation marker. Shift of these markers was an indication of transdifferentiation. The cargo of the extracted exosomes from both the cell types was explored using next-generation sequencing. The hTCEpi-derived EVs induced conjunctival epithelial cells to express the corneal-associated markers KRT3 and KRT12, losing their conjunctival phenotype at both the mRNA and protein level. Simultaneously, HCjE-Gi-derived EVs induced corneal epithelial cells to express the conjunctival associated markers KRT7 and KRT13, losing their corneal phenotype. This process of differentiation was accompanied by an intermediate step of cell de-differentiation showed by up-regulation in the expression of epithelial stem cell marker ΔNp63, also shown on the ex vivo human cadaveric donor corneas. miRNA molecules (total of 11 including precursor and mature) with significant differences in their relative abundance between the two populations (p < 0.05) were found and investigated. miR-9-5p expression was higher in HCjE-Gi cells and HCjE-Gi-derived EVs when compared to hTCEpi cells and hTCEPi-derived EVs (p < 0.001). The results suggest that EVs released by the two cell types have the ability to influence the transdifferentiation of human conjunctival and corneal epithelial cells. miR-9-5p could have a role in stem cell homeostasis and cell differentiation via HES-1 gene.