Exosomes are vesicles of endocytic origin released by many cells. These vesicles can mediate communication between cells, facilitating processes such as antigen presentation. Here, we show that exosomes from a mouse and a human mast cell line (MC/9 and HMC-1, respectively), as well as primary bone marrow-derived mouse mast cells, contain RNA. Microarray assessments revealed the presence of mRNA from approximately 1300 genes, many of which are not present in the cytoplasm of the donor cell. In vitro translation proved that the exosome mRNAs were functional. Quality control RNA analysis of total RNA derived from exosomes also revealed presence of small RNAs, including microRNAs. The RNA from mast cell exosomes is transferable to other mouse and human mast cells. After transfer of mouse exosomal RNA to human mast cells, new mouse proteins were found in the recipient cells, indicating that transferred exosomal mRNA can be translated after entering another cell. In summary, we show that exosomes contain both mRNA and microRNA, which can be delivered to another cell, and can be functional in this new location. We propose that this RNA is called "exosomal shuttle RNA" (esRNA).

/pdf/exosome-mediated-transfer-of-mrnas-and-micrornas-is-a-novel-9o52kfcb0e.pdf

Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells

Cells release into the extracellular environment diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles, respectively. These extracellular vesicles (EVs) represent an important mode of intercellular communication by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, and RNA. Deficiencies in our knowledge of the molecular mechanisms for EV formation and lack of methods to interfere with the packaging of cargo or with vesicle release, however, still hamper identification of their physiological relevance in vivo. In this review, we focus on the characterization of EVs and on currently proposed mechanisms for their formation, targeting, and function.

https://rupress.org/jcb/article-pdf/200/4/373/1262520/jcb_201211138.pdf

Extracellular vesicles: exosomes, microvesicles, and friends.

Exosomes are small membrane vesicles found in cell culture supernatants and in different biological fluids. Exosomes form in a particular population of endosomes, called multivesicular bodies (MVBs), by inward budding into the lumen of the compartment. Upon fusion of MVBs with the plasma membrane, these internal vesicles are secreted. Exosomes possess a defined set of membrane and cytosolic proteins. The physiological function of exosomes is still a matter of debate, but increasing results in various experimental systems suggest their involvement in multiple biological processes. Because both cell-culture supernatants and biological fluids contain different types of lipid membranes, it is critical to perform high-quality exosome purification. This unit describes different approaches for exosome purification from various sources, and discusses methods to evaluate the purity and homogeneity of the purified exosome preparations.

/pdf/isolation-and-characterization-of-exosomes-from-cell-culture-4k118gm22m.pdf

Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids

Extracellular vesicles are a heterogeneous group of cell-derived membranous structures comprising exosomes and microvesicles, which originate from the endosomal system or which are shed from the plasma membrane, respectively They are present in biological fluids and are involved in multiple physiological and pathological processes Extracellular vesicles are now considered as an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids and genetic material Knowledge of the cellular processes that govern extracellular vesicle biology is essential to shed light on the physiological and pathological functions of these vesicles as well as on clinical applications involving their use and/or analysis However, in this expanding field, much remains unknown regarding the origin, biogenesis, secretion, targeting and fate of these vesicles

/pdf/shedding-light-on-the-cell-biology-of-extracellular-vesicles-52ri6w7hp6.pdf

Shedding light on the cell biology of extracellular vesicles.

In the 1980s, exosomes were described as vesicles of endosomal origin secreted from reticulocytes. Interest increased around these extracellular vesicles, as they appeared to participate in several cellular processes. Exosomes bear proteins, lipids, and RNAs, mediating intercellular communication between different cell types in the body, and thus affecting normal and pathological conditions. Only recently, scientists acknowledged the difficulty of separating exosomes from other types of extracellular vesicles, which precludes a clear attribution of a particular function to the different types of secreted vesicles. To shed light into this complex but expanding field of science, this review focuses on the definition of exosomes and other secreted extracellular vesicles. Their biogenesis, their secretion, and their subsequent fate are discussed, as their functions rely on these important processes.

/pdf/biogenesis-secretion-and-intercellular-interactions-of-4pskmvujl3.pdf

Biogenesis, Secretion, and Intercellular Interactions of Exosomes and Other Extracellular Vesicles

Exosomes are secreted spherical structures that are limited by a lipid bilayer and contain cytosolic components from the producing cell. Exosomes form in intracellular multivesicular compartments of the endocytic pathway, and are secreted upon fusion of these compartments with the plasma membrane. Secretion of exosomes had been described initially in reticulocytes differentiating into red blood cells, and proposed to allow elimination of useless proteins. But exosomes became the object of increasing interest from immunologists in the late 90s, when antigen presenting cells where shown to secrete exosomes bearing functional Major Histocompatibility Complex molecules, able to induce activation of cognate T cells. Since then, a large amount of work has strengthen the idea that exosomes secreted by antigen presenting cells could represent « minimal antigen presenting units ». In this review, we describe the biophysical and biochemical specificities of exosomes, give an overview of their antigen presenting functions, and discuss their use for immunotherapies.

Exosomes: Naturally Occurring Minimal Antigen-Presenting Units

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. This article provides protocols with top ticks and pitfalls for preparation and successful generation of mouse and human DC from different cellular sources, such as murine BM and HoxB8 cells, as well as human CD34+ cells from cord blood, BM, and peripheral blood or peripheral blood monocytes. We describe murine cDC1, cDC2, and pDC generation with Flt3L and the generation of BM-derived DC with GM-CSF. Protocols for human DC generation focus on CD34+ cell culture on OP9 cell layers for cDC1, cDC2, cDC3, and pDC subset generation and DC generation from peripheral blood monocytes (MoDC). Additional protocols include enrichment of murine DC subsets, CRISPR/Cas9 editing, and clinical grade human DC generation. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

/pdf/guidelines-for-mouse-and-human-dc-generation-28cdannx.pdf

Guidelines for mouse and human DC generation.

A new model has been proposed by Li et al. for the role of DC subsets in the semidirect pathway of allorecognition.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/imcb.12361

Cross-dressed cDC1s instruct T cells in allorecognition.

Exosomes from bronchoalveolar fluid of tolerized mice prevent allergen-specific allergic reaction

The protocol described here has been developed to detect RNA at the single cell level. Fluorescent probes hybridize to target RNAs and are detected by flow cytometry after multiple amplification steps. Different types of RNA can be detected such as mRNA, long noncoding RNA, viral RNA or telomere RNA and up to 4 different target probes can be used simultaneously. We used this protocol to specifically measure the expression of two transcription factor mRNAs, MAFB and IRF4, in human monocytes.

/pdf/visualization-of-rna-at-the-single-cell-level-by-fluorescent-21blf65tco.pdf

Elodie Segura

Papers

Exosomes: Naturally Occurring Minimal Antigen-Presenting Units

Guidelines for mouse and human DC generation.

Cross-dressed cDC1s instruct T cells in allorecognition.

Exosomes from bronchoalveolar fluid of tolerized mice prevent allergen-specific allergic reaction

Visualization of RNA at the Single Cell Level by Fluorescent in situ Hybridization Coupled to Flow Cytometry