Mesenchymal Stem Cell-Derived Extracellular Vesicles: Opportunities and Challenges for Clinical Translation.
ABSTRACT: Extracellular vesicles (EVs), including exosomes and microvesicles, derived from mesenchymal stem/stromal cells (MSCs) exert similar effects as their parental cells, and are of interest for various therapeutic applications. EVs can act through uptake by the target cells followed by release of their cargo inside the cytoplasm, or through interaction of membrane-bound ligands with receptors expressed on target cells to stimulate downstream intracellular pathways. EV-based therapeutics may be directly used as substitutes of intact cells or after modification for targeted drug delivery. However, for the development of EV-based therapeutics, several production, isolation, and characterization requirements have to be met and the quality of the final product has to be tested before its clinical implementation. In this review, we discuss the challenges associated with the development of EV-based therapeutics and the regulatory specifications for their successful clinical translation.
Project description:Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.
Project description:Extracellular vesicles (EVs) such as exosomes and microvesicles released from cells are potential biomarkers for blood-based diagnostic applications. To exploit EVs as diagnostic biomarkers, an effective pre-analytical process is necessary. However, recent studies performed with blood-borne EVs have been hindered by the lack of effective purification strategies. In this study, an efficient EV isolation method was developed by using polyethylene glycol/dextran aqueous two phase system (ATPS). This method provides high EV recovery efficiency (~70%) in a short time (~15 min). Consequently, it can significantly increase the diagnostic applicability of EVs.
Project description:Cancer cells actively release extracellular vesicles (EVs), including exosomes and microvesicles, into surrounding tissues. These EVs play pleiotropic roles in cancer progression and metastasis, including invasion, angiogenesis, and immune modulation. However, the proteomic differences between primary and metastatic cancer cell-derived EVs remain unclear. Here, we conducted comparative proteomic analysis between EVs derived from human primary colorectal cancer cells (SW480) and their metastatic derivatives (SW620). Using label-free quantitation, we identified 803 and 787 proteins in SW480 EVs and SW620 EVs, respectively. Based on comparison between the estimated abundance of EV proteins, we identified 368 SW480 EV-enriched and 359 SW620 EV-enriched proteins. SW480 EV-enriched proteins played a role in cell adhesion, but SW620 EV-enriched proteins were associated with cancer progression and functioned as diagnostic indicators of metastatic cancer; they were overexpressed in metastatic colorectal cancer and played roles in multidrug resistance. As the first proteomic analysis comparing primary and metastatic cancer-derived EVs, this study increases our understanding of the pathological function of EVs in the metastatic process and provides useful biomarkers for cancer metastasis.
Project description:During the past decade, extracellular vesicles (EVs), which include apoptotic bodies, microvesicles, and exosomes, have emerged as important players in cell-to-cell communication in normal physiology and pathological conditions. EVs encapsulate and convey various bioactive molecules that are further transmitted to neighboring or more distant cells, where they induce various signaling cascades. The message delivered to the target cells is dependent on EV composition, which, in turn, is determined by the cell of origin and the surrounding microenvironment during EV biogenesis. Among their multifaceted role in the modulation of biological responses, the involvement of EVs in vascular development, growth, and maturation has been widely documented and their potential therapeutic application in regenerative medicine or angiogenesis-related diseases is drawing increasing interest. EVs derived from various cell types have the potential to deliver complex information to endothelial cells and to induce either pro- or antiangiogenic signaling. As dynamic systems, in response to changes in the microenvironment, EVs adapt their cargo composition to fine-tune the process of blood vessel formation. This article reviews the current knowledge on the role of microvesicles and exosomes from various cellular origins in angiogenesis, with a particular emphasis on the underlying mechanisms, and discusses the main challenges and prerequisites for their therapeutic applications.
Project description:Extracellular vesicles (EVs) are small lipid bilayer-delimited nanoparticles released from all types of cells examined thus far. Several groups of EVs, including exosomes, microvesicles, and apoptotic bodies, have been identified according to their size and biogenesis. With extensive investigations on EVs over the last decade, it is now recognized that EVs play a pleiotropic role in various physiological processes as well as pathological conditions through mediating intercellular communication. Most notably, EVs have been shown to be involved in cancer initiation and progression and EV signaling in cancer are viewed as potential therapeutic targets. Furthermore, as membrane nanoparticles, EVs are natural products with some of them, such as tumor exosomes, possessing tumor homing propensity, thus leading to strategies utilizing EVs as drug carriers to effectively deliver cancer therapeutics. In this review, we summarize recent reports on exploring EVs signaling as potential therapeutic targets in cancer as well as on developing EVs as therapeutic delivery carriers for cancer therapy. Findings from preclinical studies are primarily discussed, with early phase clinical trials reviewed. We hope to provide readers updated information on the development of EVs as cancer therapeutic targets or therapeutic carriers.
Project description:Extracellular vesicles (EVs) are traditionally divided into two major groups: (i) large vesicles originating from plasma membrane and called microvesicles, and (ii) small vesicles originating from the endoplasmic membrane and called exosomes. However, it is increasingly clear that the actual composition of a particular EV preparation cannot be adequately described with these two simple terms and is much more complex. Since the cell membrane origin of EVs predetermines their biological functions, the understanding of EV biogenesis is important for accurate interpretation of observed results. In the present study, we propose to take advantage of selective expression of some proteins in plasma or endosomal membranes and to use these proteins as plasma membrane-specific or endosomal membrane-specific markers. We have demonstrated that a quantitative mass spectrometry analysis allows simultaneous measurement of plasma membrane-specific and endosomal membrane-specific proteins in microvesicles and exosomes obtained after differential ultracentrifugation. Before mass spectrometry analysis, we also used sonicated platelets as a model of mixed EVs and multidetector asymmetrical-flow field-flow fractionation as an analytical method to verify a possible cross contamination of obtained microvesicles and exosomes. Based on the quantitative appearance of membrane-specific protein markers in EV preparations from human plasma and from human ARPE-19 cell medium, we concluded that there is no actual size limitation and both microvesicles and exosomes can be represented by large and small vesicles.
Project description:Extracellular vesicles (EVs) are submicron vesicles released from many cell types, including adipocytes. EVs are implicated in the pathogenesis of obesity-driven cardiovascular disease, although the characteristics of adipocyte-derived EVs are not well described. We sought to define the characteristics of adipocyte-derived EVs before and after adipogenesis, hypothesising that adipogenesis would affect EV structure, molecular composition and function. Using 3T3-L1 cells, EVs were harvested at day 0 and day 15 of differentiation. EV and cell preparations were visualised by electron microscopy and EVs quantified by nanoparticle tracking analysis (NTA). EVs were then assessed for annexin V positivity using flow cytometry; lipid and phospholipid composition using 2D thin layer chromatography and gas chromatography; and vesicular protein content by an immuno-phenotyping assay. Pre-adipogenic cells are connected via a network of protrusions and EVs at both time points display classic EV morphology. EV concentration is elevated prior to adipogenesis, particularly in exosomes and small microvesicles. Parent cells contain higher proportions of phosphatidylserine (PS) and show higher annexin V binding. Both cells and EVs contain an increased proportion of arachidonic acid at day 0. PREF-1 was increased at day 0 whilst adiponectin was higher at day 15 indicating EV protein content reflects the stage of adipogenesis of the cell. Our data suggest that EV production is higher in cells before adipogenesis, particularly in vesicles <300 nm. Cells at this time point possess a greater proportion of PS (required for EV generation) whilst corresponding EVs are enriched in signalling fatty acids, such as arachidonic acid, and markers of adipogenesis, such as PREF-1 and PPAR?.
Project description:Extracellular vesicles (EVs) mediate targeted cellular interactions in normal and pathophysiological conditions and are increasingly recognised as potential biomarkers, therapeutic agents and drug delivery vehicles. Based on their size and biogenesis, EVs are classified as exosomes, microvesicles and apoptotic bodies. Due to overlapping size ranges and the lack of specific markers, these classes cannot yet be distinguished experimentally. Currently, it is a major challenge in the field to define robust and sensitive technological platforms being suitable to resolve EV heterogeneity, especially for small EVs (sEVs) with diameters below 200 nm, i.e. smaller microvesicles and exosomes. Most conventional flow cytometers are not suitable for the detection of particles being smaller than 300 nm, and the poor availability of defined reference materials hampers the validation of sEV analysis protocols. Following initial reports that imaging flow cytometry (IFCM) can be used for the characterisation of larger EVs, we aimed to investigate its usability for the characterisation of sEVs. This study set out to identify optimal sample preparation and instrument settings that would demonstrate the utility of this technology for the detection of single sEVs. By using CD63eGFP-labelled sEVs as a biological reference material, we were able to define and optimise IFCM acquisition and analysis parameters on an Amnis ImageStreamX MkII instrument for the detection of single sEVs. In addition, using antibody-labelling approaches, we show that IFCM facilitates robust detection of different EV and sEV subpopulations in isolated EVs, as well as unprocessed EV-containing samples. Our results indicate that fluorescently labelled sEVs as biological reference material are highly useful for the optimisation of fluorescence-based methods for sEV analysis. Finally, we propose that IFCM will help to significantly increase our ability to assess EV heterogeneity in a rigorous and reproducible manner, and facilitate the identification of specific subsets of sEVs as useful biomarkers in various diseases.
Project description:Extracellular vesicles (EVs) such as exosomes and microvesicles serve as messengers of intercellular network, allowing exchange of cellular components between cells. EVs carry lipids, proteins, and RNAs derived from their producing cells, and have potential as biomarkers specific to cell types and even cellular states. However, conventional methods (such as ultracentrifugation or polymeric precipitation) for isolating EVs have disadvantages regarding purity and feasibility. Here, we have developed a novel method for EV purification by using Tim4 protein, which specifically binds the phosphatidylserine displayed on the surface of EVs. Because the binding is Ca<sup>2+</sup>-dependent, intact EVs can be easily released from Tim4 by adding Ca<sup>2+</sup> chelators. Tim4 purification, which we have applied to cell conditioned media and biofluids, is capable of yielding EVs of a higher purity than those obtained using conventional methods. The lower contamination found in Tim4-purified EV preparations allows more EV-specific proteins to be detected by mass spectrometry, enabling better characterization and quantification of different EV populations' proteomes. Tim4 protein can also be used as a powerful tool for quantification of EVs in both ELISA and flow cytometry formats. Thus, the affinity of Tim4 for EVs will find abundant applications in EV studies.
Project description:Extracellular vesicles (EVs), including exosomes and microvesicles, are 30-800?nm vesicles that are released by most cell types, as biological packages for intercellular communication. Their importance in cancer and inflammation makes EVs and their cargo promising biomarkers of disease and cell-free therapeutic agents. Emerging high-resolution cytometric methods have created a pressing need for efficient fluorescent labeling procedures to visualize and detect EVs. Suitable labels must be bright enough for one EV to be detected without the generation of label-associated artifacts. To identify a strategy that robustly labels individual EVs, we used nanoFACS, a high-resolution flow cytometric method that utilizes light scattering and fluorescence parameters along with sample enumeration, to evaluate various labels. Specifically, we compared lipid-, protein-, and RNA-based staining methods and developed a robust EV staining strategy, with the amine-reactive fluorescent label, 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester, and size exclusion chromatography to remove unconjugated label. By combining nanoFACS measurements of light scattering and fluorescence, we evaluated the sensitivity and specificity of EV labeling assays in a manner that has not been described for other EV detection methods. Efficient characterization of EVs by nanoFACS paves the way towards further study of EVs and their roles in health and disease.