Innovative Visualization and Quantification of Extracellular Vesicles Interaction with and Incorporation in Target Cells in 3D Microenvironments

Extracellular vesicles (EVs) showed therapeutic properties in several applications, many in regenerative medicine. A clear example is in the treatment of osteoarthritis (OA), where adipose-derived mesenchymal stem cells (ASCs)-EVs were able to promote regeneration and reduce inflammation in both synovia and cartilage. A still obscure issue is the effective ability of EVs to be internalized by target cells, rather than simply bound to the extracellular matrix (ECM) or plasma membrane, since the current detection or imaging technologies cannot fully decipher it due to technical limitations. In the present study, human articular chondrocytes (ACHs) and fibroblast-like synoviocytes (FLSs) isolated from the same OA patients were cocultured in 2D as well as in 3D conditions with fluorescently labeled ASC-EVs, and analyzed by flow cytometry or confocal microscopy, respectively. In contrast with conventional 2D, in 3D cultures, confocal microscopy allowed a clear detection of the tridimensional morphology of the cells and thus an accurate discrimination of EV interaction with the external and/or internal cell environment. In both 2D and 3D conditions, FLSs were more efficient in interacting with ASC-EVs and 3D imaging demonstrated a faster uptake process. The removal of the hyaluronic acid component from the ECM of both cell types reduced their interaction with ASC-EVs only in the 2D system, showing that 2D and 3D conditions can yield different outcomes when investigating events where ECM plays a key role. These results indicate that studying EVs binding and uptake both in 2D and 3D guarantees a more precise and complementary characterization of the molecular mechanisms involved in the process. The implementation of this strategy can become a valuable tool not only for basic research, but also for release assays and potency prediction for clinical EV batches.

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Extracellular Vesicles: An Emerging Mechanism Governing the Secretion and Biological Roles of Tenascin-C

Frontiers in Immunology ◽

10.3389/fimmu.2021.671485 ◽

2021 ◽

Vol 12 ◽

Author(s):

Lucas Albacete-Albacete ◽

Miguel Sánchez-Álvarez ◽

Miguel Angel del Pozo

Keyword(s):

Extracellular Vesicles ◽

Molecular Mechanisms ◽

Cell Communication ◽

Cell Types ◽

Target Cells ◽

Tenascin C ◽

Signalling Molecules ◽

Inflammatory Processes ◽

Bona Fide ◽

Cellular Components

ECM composition and architecture are tightly regulated for tissue homeostasis. Different disorders have been associated to alterations in the levels of proteins such as collagens, fibronectin (FN) or tenascin-C (TnC). TnC emerges as a key regulator of multiple inflammatory processes, both during physiological tissue repair as well as pathological conditions ranging from tumor progression to cardiovascular disease. Importantly, our current understanding as to how TnC and other non-collagen ECM components are secreted has remained elusive. Extracellular vesicles (EVs) are small membrane-bound particles released to the extracellular space by most cell types, playing a key role in cell-cell communication. A broad range of cellular components can be transported by EVs (e.g. nucleic acids, lipids, signalling molecules and proteins). These cargoes can be transferred to target cells, potentially modulating their function. Recently, several extracellular matrix (ECM) proteins have been characterized as bona fide EV cargoes, exosomal secretion being particularly critical for TnC. EV-dependent ECM secretion might underpin diseases where ECM integrity is altered, establishing novel concepts in the field such as ECM nucleation over long distances, and highlighting novel opportunities for diagnostics and therapeutic intervention. Here, we review recent findings and standing questions on the molecular mechanisms governing EV–dependent ECM secretion and its potential relevance for disease, with a focus on TnC.

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Current methods for the isolation of extracellular vesicles

Biological Chemistry ◽

10.1515/hsz-2013-0141 ◽

2013 ◽

Vol 394 (10) ◽

pp. 1253-1262 ◽

Cited By ~ 279

Author(s):

Fatemeh Momen-Heravi ◽

Leonora Balaj ◽

Sara Alian ◽

Pierre-Yves Mantel ◽

Allison E. Halleck ◽

...

Keyword(s):

Extracellular Vesicles ◽

Molecular Mechanisms ◽

Magnetic Beads ◽

Cell Types ◽

Target Cells ◽

Cell Of Origin ◽

Novel Technologies ◽

Advantages And Disadvantages ◽

Isolation Methods ◽

Technical Advances

Abstract Extracellular vesicles (EVs), including microvesicles and exosomes, are nano- to micron-sized vesicles, which may deliver bioactive cargos that include lipids, growth factors and their receptors, proteases, signaling molecules, as well as mRNA and non-coding RNA, released from the cell of origin, to target cells. EVs are released by all cell types and likely induced by mechanisms involved in oncogenic transformation, environmental stimulation, cellular activation, oxidative stress, or death. Ongoing studies investigate the molecular mechanisms and mediators of EVs-based intercellular communication at physiological and oncogenic conditions with the hope of using this information as a possible source for explaining physiological processes in addition to using them as therapeutic targets and disease biomarkers in a variety of diseases. A major limitation in this evolving discipline is the hardship and the lack of standardization for already challenging techniques to isolate EVs. Technical advances have been accomplished in the field of isolation with improving knowledge and emerging novel technologies, including ultracentrifugation, microfluidics, magnetic beads and filtration-based isolation methods. In this review, we will discuss the latest advances in methods of isolation methods and production of clinical grade EVs as well as their advantages and disadvantages, and the justification for their support and the challenges that they encounter.

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Pluripotency and Epigenetic Factors in Mouse Embryonic Stem Cell Fate Regulation

Molecular and Cellular Biology ◽

10.1128/mcb.00266-15 ◽

2015 ◽

Vol 35 (16) ◽

pp. 2716-2728 ◽

Cited By ~ 57

Author(s):

Lluis Morey ◽

Alexandra Santanach ◽

Luciano Di Croce

Keyword(s):

Cell Fate ◽

Cancer Progression ◽

Molecular Mechanisms ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cell ◽

Basic Research ◽

Cell Types ◽

Pluripotency Factors ◽

Perfect System

Embryonic stem cells (ESCs) are characterized by their ability to self-renew and to differentiate into all cell types of a given organism. Understanding the molecular mechanisms that govern the ESC state is of great interest not only for basic research—for instance, ESCs represent a perfect system to study cellular differentiationin vitro—but also for their potential implications in human health, as these mechanisms are likewise involved in cancer progression and could be exploited in regenerative medicine. In this minireview, we focus on the latest insights into the molecular mechanisms mediated by the pluripotency factors as well as their roles during differentiation. We also discuss recent advances in understanding the function of the epigenetic regulators, Polycomb and MLL complexes, in ESC biology.

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Biology, Pathophysiological Role, and Clinical Implications of Exosomes: A Critical Appraisal

Cells ◽

10.3390/cells8020099 ◽

2019 ◽

Vol 8 (2) ◽

pp. 99 ◽

Cited By ~ 22

Author(s):

Arif Jan ◽

Safikur Rahman ◽

Shahanavaj Khan ◽

Sheikh Tasduq ◽

Inho Choi

Keyword(s):

Plasma Membranes ◽

Molecular Mechanisms ◽

Cell Types ◽

Target Cells ◽

Host Immune System ◽

Messenger Rnas ◽

Extracellular Milieu ◽

Tumor Microenvironments ◽

Physiological Processes ◽

Antigen Presenting

Exosomes are membrane-enclosed entities of endocytic origin, which are generated during the fusion of multivesicular bodies (MVBs) and plasma membranes. Exosomes are released into the extracellular milieu or body fluids; this process was reported for mesenchymal, epithelial, endothelial, and different immune cells (B-cells and dendritic cells), and was reported to be correlated with normal physiological processes. The compositions and abundances of exosomes depend on their tissue origins and cell types. Exosomes range in size between 30 and 100 nm, and shuttle nucleic acids (DNA, messenger RNAs (mRNAs), microRNAs), proteins, and lipids between donor and target cells. Pathogenic microorganisms also secrete exosomes that modulate the host immune system and influence the fate of infections. Such immune-modulatory effect of exosomes can serve as a diagnostic biomarker of disease. On the other hand, the antigen-presenting and immune-stimulatory properties of exosomes enable them to trigger anti-tumor responses, and exosome release from cancerous cells suggests they contribute to the recruitment and reconstitution of components of tumor microenvironments. Furthermore, their modulation of physiological and pathological processes suggests they contribute to the developmental program, infections, and human diseases. Despite significant advances, our understanding of exosomes is far from complete, particularly regarding our understanding of the molecular mechanisms that subserve exosome formation, cargo packaging, and exosome release in different cellular backgrounds. The present study presents diverse biological aspects of exosomes, and highlights their diagnostic and therapeutic potentials.

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Stem cells and extracellular vesicles: biological regulators of physiology and disease

AJP Cell Physiology ◽

10.1152/ajpcell.00017.2019 ◽

2019 ◽

Vol 317 (2) ◽

pp. C155-C166 ◽

Cited By ~ 7

Author(s):

Theodor Borgovan ◽

Lorin Crawford ◽

Chibuikem Nwizu ◽

Peter Quesenberry

Keyword(s):

Stem Cells ◽

Extracellular Vesicles ◽

Cell Types ◽

Target Cells ◽

Malignant Phenotype ◽

Marrow Mesenchymal Stem Cells ◽

Physiological Homeostasis ◽

Clinical Advances ◽

Machine Learning Models

Many different subpopulations of subcellular extracellular vesicles (EVs) have been described. EVs are released from all cell types and have been shown to regulate normal physiological homeostasis, as well as pathological states by influencing cell proliferation, differentiation, organ homing, injury and recovery, as well as disease progression. In this review, we focus on the bidirectional actions of vesicles from normal and diseased cells on normal or leukemic target cells; and on the leukemic microenvironment as a whole. EVs from human bone marrow mesenchymal stem cells (MSC) can have a healing effect, reversing the malignant phenotype in prostate and colorectal cancer, as well as mitigating radiation damage to marrow. The role of EVs in leukemia and their bimodal cross talk with the encompassing microenvironment remains to be fully characterized. This may provide insight for clinical advances via the application of EVs as potential therapy and the employment of statistical and machine learning models to capture the pleiotropic effects EVs endow to a dynamic microenvironment, possibly allowing for precise therapeutic intervention.

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Extracellular vesicles : novel mediators of conceptus-maternal interactions in sheep

10.32469/10355/66309 ◽

2018 ◽

Author(s):

◽

Gregory W. Burns

Keyword(s):

Extracellular Vesicles ◽

Pregnancy Loss ◽

Cell Types ◽

Target Cells ◽

Uterine Receptivity ◽

Mediated Communication ◽

University Of Missouri ◽

Dynamic Component ◽

Uterine Endometrium ◽

Endometrial Epithelium

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Infertility and pregnancy loss are common problems affecting reproductive efficiency, health and development in livestock. Pregnancy loss occurs most commonly during the first weeks of gestation and may arise due to asynchrony between the conceptus and uterus or endometrial dysfunction, resulting in defective conceptus elongation, implantation and/or placentation. Extracellular vesicles (EVs), a term including exosomes and microvesicles, are membrane-bound nanoparticles released from diverse cell types that deliver nucleic acids and proteins to target cells. Available studies support the central hypothesis that EVs are a component of uterine histotroph and mediate crosstalk between the developing conceptus and uterine endometrium prior to implantation. Studies were conducted here to: (1) identify and characterize EVs from uterine flush of cyclic and pregnant ewes; (2) examine potential of vesicle mediated communication during early pregnancy from endometrium and elongating conceptus derived EVs; and (3) determine progesterone regulation of EV cargo, endometrial gene expression, and total EV number in the uterine lumen of cyclic sheep. Results from these studies established that: (1) EVs are a component of the uterine histotroph in sheep with pregnancy associated differences; (2) EVs emanate from the uterine endometrium and elongating conceptus; (3) the endometrial epithelium and conceptus trophectoderm uptake labeled EVs indicating a role in intercellular communication during the establishment of pregnancy; (4) uterine EV content increases more than five-fold from day 10 to 14 of the estrous cycle; (5) the endometrial epithelia produce EVs with multivesicular endosomes, the progenitors of EVs, localized to the luminal and glandular epithelium; and (6) progesterone treatment increases the number of uterine EVs and alters their miRNA cargo. Collectively, these studies have established that EVs are a dynamic component of the uterine histotroph, produced by both the uterine epithelium and conceptus trophectoderm, with the ability to traffic between maternal and embryonic tissues and support the idea that EVs mediate communication that underpins conceptus development required for the successful establishment of pregnancy. These studies provide evidence of EVs as novel mediators of communication between the developing conceptus and endometrium. Uterine EVs may provide useful biomarkers for uterine receptivity or indicate endometrial dysfunction given their dynamic cargo and robust stability in biofluids.

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Microbial extracellular RNAs and their roles in human diseases

Experimental Biology and Medicine ◽

10.1177/1535370220923585 ◽

2020 ◽

Vol 245 (10) ◽

pp. 845-850 ◽

Cited By ~ 3

Author(s):

Heon-Jin Lee

Keyword(s):

Extracellular Vesicles ◽

Cell Types ◽

Human Cells ◽

The Body ◽

Human Diseases ◽

Host Responses ◽

Target Cells ◽

Extracellular Rna ◽

Communication Signals ◽

Novel Therapeutic Strategies

Extracellular RNAs (exRNAs) are released by extracellular vesicles, small membranous nanoparticles secreted by all cell types. When transported into cells, exRNAs can modulate gene expression or cellular responses in the target cells since many small RNAs have regulatory functions. Indeed, it is widely acknowledged that endogenous exRNAs in the human body are related to various diseases. However, microbial exRNAs have been less studied, and their connection to host diseases has just begun to be explored. In this review, I will discuss analytical methods for exRNAs and the potential use of exRNAs as disease biomarkers. I also consider current progress in understanding the regulation of host mechanisms by microbial exRNAs as inter-kingdom communication, efforts to utilize extracellular vesicles as therapeutic vehicles loaded with engineered RNA cargos, and a putative connection between microbial exRNA-based regulation of host responses and human diseases such as Alzheimer’s. This overview aims to present novel insights into pathogenesis with regard to the function of microbial exRNAs as “disease-relevant travelers.” Impact statement The number of commensal bacteria in the body surpasses the number of actual human cells. Thus, various interactions between microbes and human cells constitute an inevitable phenomenon. Recent evidence has led to bacterial extracellular RNAs (exRNAs) being proposed as good candidates for microbe–host inter-kingdom communication tools as they can modulate the expression of host genes. However, research findings on the relevance of interactions between extracellular RNA and human diseases are still in their infancy. Nevertheless, substantial data suggest that microbial exRNAs are implicated in various human diseases both at local and distant sites. By exploring various scenarios for the involvement of microbial exRNAs in human diseases, we may better understand the role of exRNAs as “communication signals” for diseases and thereby develop novel therapeutic strategies by using them and their carrier extracellular vesicles.

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The Past, the Present, and the Future of the Size Exclusion Chromatography in Extracellular Vesicles Separation

Viruses ◽

10.3390/v13112272 ◽

2021 ◽

Vol 13 (11) ◽

pp. 2272

Author(s):

Hussein Kaddour ◽

Malik Tranquille ◽

Chioma M. Okeoma

Keyword(s):

Size Exclusion Chromatography ◽

Extracellular Vesicles ◽

Cell Types ◽

Size Exclusion ◽

Target Cells ◽

Purification And Characterization ◽

Extracellular Milieu ◽

Exclusion Chromatography ◽

Single Vesicle

Extracellular vesicles (EVs) are cell-derived membranous particles secreted by all cell types (including virus infected and uninfected cells) into the extracellular milieu. EVs carry, protect, and transport a wide array of bioactive cargoes to recipient/target cells. EVs regulate physiological and pathophysiological processes in recipient cells and are important in therapeutics/drug delivery. Despite these great attributes of EVs, an efficient protocol for EV separation from biofluids is lacking. Numerous techniques have been adapted for the separation of EVs with size exclusion chromatography (SEC)-based methods being the most promising. Here, we review the SEC protocols used for EV separation, and discuss opportunities for significant improvements, such as the development of novel particle purification liquid chromatography (PPLC) system capable of tandem purification and characterization of biological and synthetic particles with near-single vesicle resolution. Finally, we identify future perspectives and current issues to make PPLC a tool capable of providing a unified, automated, adaptable, yet simple and affordable particle separation resource.

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Extracellular Vesicles in Modifying the Effects of Ionizing Radiation

International Journal of Molecular Sciences ◽

10.3390/ijms20225527 ◽

2019 ◽

Vol 20 (22) ◽

pp. 5527 ◽

Cited By ~ 4

Author(s):

Tünde Szatmári ◽

Rita Hargitai ◽

Géza Sáfrány ◽

Katalin Lumniczky

Keyword(s):

Ionizing Radiation ◽

Extracellular Vesicles ◽

Radiation Effects ◽

Cell Types ◽

Tumor Formation ◽

The Body ◽

Target Cells ◽

Cell Of Origin ◽

Radiation Induced ◽

Almost All

Extracellular vesicles (EVs) are membrane-coated nanovesicles actively secreted by almost all cell types. EVs can travel long distances within the body, being finally taken up by the target cells, transferring information from one cell to another, thus influencing their behavior. The cargo of EVs comprises of nucleic acids, lipids, and proteins derived from the cell of origin, thereby it is cell-type specific; moreover, it differs between diseased and normal cells. Several studies have shown that EVs have a role in tumor formation and prognosis. It was also demonstrated that ionizing radiation can alter the cargo of EVs. EVs, in turn can modulate radiation responses and they play a role in radiation-induced bystander effects. Due to their biocompatibility and selective targeting, EVs are suitable nanocarrier candidates of drugs in various diseases, including cancer. Furthermore, the cargo of EVs can be engineered, and in this way they can be designed to carry certain genes or even drugs, similar to synthetic nanoparticles. In this review, we describe the biological characteristics of EVs, focusing on the recent efforts to use EVs as nanocarriers in oncology, the effects of EVs in radiation therapy, highlighting the possibilities to use EVs as nanocarriers to modulate radiation effects in clinical applications.

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Micro RNAs are minor constituents of extracellular vesicles and are hardly delivered to target cells

10.1101/2020.05.20.106393 ◽

2020 ◽

Cited By ~ 6

Author(s):

Manuel Albanese ◽

Yen-Fu Adam Chen ◽

Corinna Hüls ◽

Kathrin Gärtner ◽

Takanobu Tagawa ◽

...

Keyword(s):

Extracellular Vesicles ◽

Mammalian Cells ◽

Cell Communication ◽

Cell Types ◽

Convincing Evidence ◽

Target Cells ◽

Mrna Targets ◽

Negative Results ◽

Micro Rnas ◽

Different Types

ABSTRACTMammalian cells release different types of vesicles, collectively termed extracellular vesicles (EVs). EVs contain cellular microRNAs (miRNAs) with an apparent potential to deliver their miRNA cargo to recipient cells to affect the stability of individual mRNAs and the cells’ transcriptome. The extent to which miRNAs are exported via the EV route and whether they contribute to cell-cell communication are controversial. To address these issues, we analyzed the capacity of EVs to deliver packaged miRNAs into target cells and to exert biological functions. We applied well-defined approaches to produce and characterize purified EVs with or without specific viral miRNAs. We found that only a small fraction of EVs carried miRNAs. EVs readily bound to different target cell types, but there was no EV-cell membrane fusion or delivery of cargo. Importantly, the functionality of cells exposed to miRNA-carrying EVs was not affected. These results suggest EV-borne miRNAs do not act as effectors and question their relevancy in paracrine cell-to-cell communication.AUTHOR SUMMARYThe majority of metazoan cells release vesicles of different types and origins, such as exosomes and microvesicles, now collectively termed extracellular vesicles (EVs). EVs have gained much attention because they contain microRNAs (miRNAs) and thus could regulate their specific mRNA targets in recipient or acceptor cells that take up EVs. Using a novel fusion assay with superior sensitivity and specificity, we revisited this claim but found no convincing evidence for an efficient functional uptake of EVs in many different cell lines and primary human blood cells. Even EVs engineered to fuse and deliver their miRNA cargo to recipient cells had no measurable effect on target mRNAs in very carefully controlled, quantitative experiments. Our negative results clearly indicate that EVs do not act as vehicles for miRNA-based cell-to-cell communication.

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