Mammals do not possess the ability to spontaneously repair or regenerate damaged retinal tissue. In contrast to teleost fish which are capable of retina regeneration through the action of Müller glia, mammals undergo a process of reactive gliosis and scarring that inhibits replacement of lost neurons. Thus, it is important to discover novel methods for stimulating mammalian Müller glia to dedifferentiate and produce progenitor cells that can replace lost retinal neurons. Inducing an endogenous regenerative pathway mediated by Müller glia would provide an attractive alternative to stem cell injections or gene therapy approaches. Extracellular vesicles (EVs) are now recognized to serve as a novel form of cell-cell communication through the transfer of cargo from donor to recipient cells or by the activation of signaling cascades in recipient cells. EVs have been shown to promote proliferation and regeneration raising the possibility that delivery of EVs could be a viable treatment for visual disorders. Here, we provide protocols to isolate EVs for use in retina regeneration experiments.

The current study analyzed the intersecting biophysical, biochemical, and functional properties of extracellular particles (EPs) with the human immunodeficiency virus type-1 (HIV-1) beyond the currently accepted size range for HIV-1. We isolated five fractions (Frac-A through Frac-E) from HIV-infected cells by sequential differential ultracentrifugation (DUC). All fractions showed a heterogeneous size distribution with median particle sizes greater than 100 nm for Frac-A through Frac-D but not for Frac-E, which contained small EPs with an average size well below 50 nm. Synchronized and released cultures contained large infectious EPs in Frac-A, with markers of amphisomes and viral components. Additionally, Frac-E uniquely contained EPs positive for CD63, HSP70, and HIV-1 proteins. Despite its small average size, Frac-E contained membrane-protected viral integrase, detectable only after SDS treatment, indicating that it is enclosed in vesicles. Single particle analysis with dSTORM further supported these findings as CD63, HIV-1 integrase, and the viral surface envelope (Env) glycoprotein (gp) colocalized on the same Frac-E particles. Surprisingly, Frac-E EPs were infectious, and infectivity was significantly reduced by immunodepleting Frac-E with anti-CD63, indicating the presence of this protein on the surface of infectious small EPs in Frac-E. To our knowledge, this is the first time that extracellular vesicle (EV) isolation methods have identified infectious small HIV-1 particles (smHIV-1) that are under 50 nm. Collectively, our data indicate that the crossroads between EPs and HIV-1 potentially extend beyond the currently accepted biophysical properties of HIV-1, which may have further implications for viral pathogenesis.

Since its original discovery over a decade ago, extracellular RNA (exRNA) has been found in all biological fluids. Furthermore, extracellular microRNA has been shown to be involved in communication between various cell types. Importantly, the exRNA is protected from RNases degradation by certain carriers including membrane vesicles and non-vesicular protein nanoparticles. Each type of carrier has its unique exRNA profile, which may vary depending on cell type and physiological conditions. To clarify putative mechanisms of intercellular communication mediated by exRNA, the RNA profile of each carrier has to be characterized. While current methods of biofluids fractionation are continuously improving, they fail to completely separate exRNA carriers. Likewise, most popular library preparation approaches for RNA sequencing do not allow obtaining exhaustive and unbiased data on exRNA transcriptome. In this mini review we discuss ongoing progress in the field of exRNA, with the focus on exRNA carriers, analyze the key methodological challenges and provide recommendations on how the latter could be overcome.

Although extracellular vesicles (EVs) were discovered over 40 years ago, there has been a resurgence of interest in secreted vesicles and their attendant cargo as novel modes of intracellular communication. In addition to vesicles, two amembranous nanoparticles, exomeres and supermeres, have been isolated and characterized recently. In this rapidly expanding field, it has been challenging to assign cargo and specific functions to a particular carrier. Refinement of isolation methods, well-controlled studies, and guidelines detailed by Minimal Information for Studies of Extracellular Vesicles (MISEV) are being employed to \"bring order to chaos.\" In this review, we will briefly summarize three types of extracellular carriers - small EVs (sEVs), exomeres, and supermeres - in the context of colorectal cancer (CRC). We found that a number of GPI-anchored proteins (GPI-APs) are overexpressed in CRC, are enriched in exosomes (a distinct subset of sEVs), and can be detected in exomeres and supermeres. This affords the opportunity to elaborate on GPI-AP biogenesis, modifications, and trafficking using DPEP1, a GPI-AP upregulated in CRC, as a prime example. We have cataloged the GPI-anchored proteins secreted in CRC and will highlight features of select CRC-associated GPI-anchored proteins we have detected. Finally, we will discuss the remaining challenges and future opportunities in studying these secreted GPI-APs in CRC.

暂无摘要。

The study of extracellular vesicles (EVs) and nanoparticles (NPs) is rapidly expanding because recent discoveries have revealed a much greater complexity and diversity than was appreciated only a few years ago. New types of EVs and NPs have recently been described. Proteins and nucleic acids previously thought to be packaged in exosomes appear to be more enriched in different types of EVs and in two recently identified amembranous NPs, exomeres and supermeres. Thus, our understanding of the cell biology and intercellular communication facilitated by the release of EVs and NPs is in a state of flux. In this review, we describe the different types of EVs and NPs, highlight recent advances, and present major outstanding questions.

Exosomes and exomeres are the smallest microparticles ranging from 20 to 130 nm in diameter. They are found in almost all biological fluids. Exosomes and exomeres are of considerable interest since they can be involved in intercellular signaling and are biological markers of the state of cells, which can be used for diagnostics. The nomenclature of exosomes remains poorly developed. Most researchers try to classify them based on the mode of formation, physicochemical characteristics, and the presence of tetrasporin markers CD9, CD63, and CD81. The data presented in this work show that although exomeres carry tetrasporin biomarkers, they differ from exosomes strongly in lipid composition, especially in cholesterol content. The production of exomeres by cells is associated with the synthesis of cholesterol in cells and is expressed or suppressed by regulators of the synthesis of mevalonate, an intermediate product of cholesterol metabolism. In addition, the work shows that the concentration of extracellular particles in the body correlates with the concentration of cholesterol in the plasma, but weakly correlates with the concentration of cholesterol in lipoproteins. This suggests that not all plasma cholesterol is associated with lipoproteins, as previously thought.

Raman spectroscopy together with comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GCxGC-TOFMS) was employed to characterize exomere- (<50 nm) and exosome-sized (50-80 nm) EVs isolated from human plasma by the novel on-line immunoaffinity chromatography - asymmetric flow field-flow fractionation method. CD9+, CD63+, and CD81+ EVs were selected to represent general EV subpopulations secreted into plasma, while CD61+ EVs represented the specific EV subset derived from platelets. Raman spectroscopy could distinguish EVs from non-EV particles, including apolipoprotein B-100-containing lipoproteins, signifying its potential in EV purity assessment. Moreover, platelet-derived (CD61+) EVs of both exomere and exosome sizes were discriminated from other EV subpopulations due to different biochemical compositions. Further investigations demonstrated composition differences between exomere- and exosome-sized EVs, confirming the applicability of Raman spectroscopy in distinguishing EVs, not only from different origins but also sizes. In addition, fatty acids that act as building blocks for lipids and membranes in EVs were studied by GCxGC-TOF-MS. The results achieved highlighted differences in EV fatty acid compositions in both esterified (membrane lipids) and non-esterified (free fatty acids) fractions, indicating possible differences in membrane structures, biological functions, and roles in cell-to-cell communications of EV subpopulations.

We developed a modified protocol, based on differential ultracentrifugation (dUC), to isolate extracellular vesicles and particles (specifically exomeres) (EVPs) from various human and murine sources, including cell lines, surgically resected tumors and adjacent tissues, and bodily fluids, such as blood, lymphatic fluid, and bile. The diversity of these samples requires robust and highly reproducible protocols and refined isolation technology, such as asymmetric-flow field-flow fractionation (AF4). Our isolation protocol allows for preparation of EVPs for various downstream applications, including proteomic profiling. For complete details on the use and execution of this protocol, please refer to Hoshino et al. (2020).

Extracellular vesicles (EVs) are described as membranous vesicles that are secreted by various cell types. EVs can be categorised as exosomes, ectosomes, apoptotic bodies, large oncosomes and migrasomes. EVs are heterogeneous in nature according to their origin, mode of release, size, and biochemical contents. Herein, we discuss a recently discovered subpopulation of EVs called \'exomeres\'. Unlike the other subtypes of EVs, exomeres are defined as non-membranous nanovesicles with a size ≤50 nm. They can be isolated using asymmetric-flow field-flow fractionation as well as ultracentrifugation. The cargo of exomeres are beginning to be unravelled and are highlighted to be enriched with proteins implicated in regulating metabolic pathways. Consistent with other types of EVs, exomeres also contain nucleic acids and lipids which can be delivered to recipient cells. These discoveries highlight the complex heterogeneity of EVs and thereby necessitates further attention to understand the nature of each subpopulation more exclusively. Overall, this chapter describes the current knowledge on exomeres.