Recovery of extracellular vesicles from human breast milk is influenced by sample collection and vesicle isolation procedures (original) (raw)
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The Journal of nutrition, 2017
Extracellular vesicles (EVs) in milk harbor a variety of compounds, including lipids, proteins, noncoding RNAs, and mRNAs. Among the various classes of EVs, exosomes are of particular interest, because cargo sorting in exosomes is a regulated, nonrandom process and exosomes play essential roles in cell-to-cell communication. Encapsulation in exosomes confers protection against enzymatic and nonenzymatic degradation of cargos and provides a pathway for cellular uptake of cargos by endocytosis of exosomes. Compelling evidence suggests that exosomes in bovine milk are transported by intestinal cells, vascular endothelial cells, and macrophages in human and rodent cell cultures, and bovine-milk exosomes are delivered to peripheral tissues in mice. Evidence also suggests that cargos in bovine-milk exosomes, in particular RNAs, are delivered to circulating immune cells in humans. Some microRNAs and mRNAs in bovine-milk exosomes may regulate the expression of human genes and be translated ...
Journal of extracellular vesicles, 2017
Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk ...
Molecular & Cellular Proteomics, 2016
Breast milk contains several macromolecular components with distinctive functions, whereby milk fat globules and casein micelles mainly provide nutrition to the newborn, and whey contains molecules that can stimulate the newborn's developing immune system and gastrointestinal tract. Although extracellular vesicles (EV) have been identified in breast milk, their physiological function and composition has not been addressed in detail. EV are submicron sized vehicles released by cells for intercellular communication via selectively incorporated lipids, nucleic acids, and proteins. Because of the difficulty in separating EV from other milk components, an in-depth analysis of the proteome of human milk-derived EV is lacking. In this study, an extensive LC-MS/MS proteomic analysis was performed of EV that had been purified from breast milk of seven individual donors using a recently established, optimized density-gradient-based EV isolation protocol. A total of 1963 proteins were identified in milk-derived EV, including EV-associated proteins like CD9, Annexin A5, and Flotillin-1, with a remarkable overlap between the different donors. Interestingly, 198 of the identified proteins are not present in the human EV database Vesiclepedia, indicating that milk-derived EV harbor proteins not yet identified in EV of different origin. Similarly, the proteome of milk-derived EV was compared with that of other milk components. For this, data from 38 published milk proteomic studies were combined in order to construct the total milk proteome, which consists of 2698 unique proteins. Remarkably, 633 proteins identified in milk-derived EV have not yet been identified in human milk to date. Interestingly, these novel proteins include proteins involved in regulation of cell growth and controlling inflammatory signaling pathways, suggesting that milk-derived EVs could support the newborn's developing gastrointestinal tract and immune system. Overall, this study provides an expansion of the whole milk proteome and illustrates that milk-derived EV are macromolecular components with a unique functional proteome. Molecular & Cellular Proteomics 15:
Exosomes with Immune Modulatory Features Are Present in Human Breast Milk
The Journal of Immunology, 2007
Breast milk is a complex liquid with immune-competent cells and soluble proteins that provide immunity to the infant and affect the maturation of the infant's immune system. Exosomes are nanovesicles (30 -100 nm) with an endosome-derived limiting membrane secreted by a diverse range of cell types. Because exosomes carry immunorelevant structures, they are suggested to participate in directing the immune response. We hypothesized that human breast milk contain exosomes, which may be important for the development of the infant's immune system. We isolated vesicles from the human colostrum and mature breast milk by ultracentrifugations and/or immuno-isolation on paramagnetic beads. We found that the vesicles displayed a typical exosome-like size and morphology as analyzed by electron microscopy. Furthermore, they floated at a density between 1.10 and 1.18 g/ml in a sucrose gradient, corresponding to the known density of exosomes. In addition, MHC classes I and II, CD63, CD81, and CD86 were detected on the vesicles by flow cytometry. Western blot and mass spectrometry further confirmed the presence of several exosome-associated molecules. Functional analysis revealed that the vesicle preparation inhibited anti-CD3-induced IL-2 and IFN-␥ production from allogeneic and autologous PBMC. In addition, an increased number of Foxp3 ؉ CD4 ؉ CD25 ؉ T regulatory cells were observed in PBMC incubated with milk vesicle preparations. We conclude that human breast milk contains exosomes with the capacity to influence immune responses.
ABSTRACTThe promise of extracellular vesicles (EVs)-based liquid biopsy resides in the identification of specific signatures of EVs of interest. Knowing the EV profile of a body fluid can facilitate the identification of EV-based biomarkers of diseases. To this end, we characterised purified EVs from paired human milk and serum by surface protein profiling of cellular markers in association with gold standard EV markers (tetraspanins CD9, CD63 and CD81). By using the MACSPlex bead-based flow-cytometry assay with pan-tetraspanin detection (i.e. simultaneous CD9, CD63 and CD81 detection), besides specific breast epithelial cell signatures in milk EVs and platelet signatures in serum EVs, we also identified body fluid-specific markers of immune cells and stem cells. Interestingly, comparison of pan-tetraspanin and single tetraspanin detection unveiled both body fluid-specific tetraspanin distributions and specific tetraspanin distributions associated with certain cellular markers, whic...
PloS one, 2015
Extracellular vesicles, including exosomes, have been identified in all biological fluids and rediscovered as an important part of the intercellular communication. Breast milk also contains extracellular vesicles and the proposed biological function is to enhance the antimicrobial defense in newborns. It is, however, unknown whether extracellular vesicles are still present in commercial milk and, more importantly, whether they retained their bioactivity. Here, we characterize the extracellular vesicles present in semi-skimmed cow milk available for consumers and study their effect on T cells. Extracellular vesicles from commercial milk were isolated and characterized. Milk-derived extracellular vesicles contained several immunomodulating miRNAs and membrane protein CD63, characteristics of exosomes. In contrast to RAW 267.4 derived extracellular vesicles the milk-derived extracellular vesicles were extremely stable under degrading conditions, including low pH, boiling and freezing. ...
Cow's milk is economically important to the agricultural industry with the nutritive value of milk being routinely measured. This does not give full insight into normal mammary tissue turnover during the course of lactation, which could be important for both an understanding of milk production and animal welfare. We have previously demonstrated that submicron particles, including extracellular vesicles (EVs), can be measured in unprocessed cow's milk by flow cytometry and that they correlate with stage of lactation. A number of different techniques are available to measure EVs and other milk-derived particles. The purpose of this study was to compare two different methodologies and the value of fluorescent staining for the phospholipid phosphatidylserine (PS), which is exposed on the surface of EVs (but not other milk-derived particles). We used two different flow cytometers and nanotracker analysis to detect milk-derived particles in whole and skimmed milk samples. Our findings indicate significant correlation, after staining for PS, suggesting potential for larger multicenter studies in the future.
Identification of protein markers for extracellular vesicle (EV) subsets in cow's milk
Journal of Proteomics
Extracellular vesicles (EVs), like exosomes, are small membrane vesicles involved in cell-to-cell communications that modulate numerous biological processes. We previously discovered a new EV subset in milk (sedimenting at 35,000 g; 35K) that protected its cargo (RNAs and proteins) during simulated digestion and was more enriched in microRNAs than exosomes (sedimenting at 100K). Here, we used LC-MS/MS to push further the comparison between these two pellets. Commonly used EV markers were not differentially enriched between the pellets, questioning their use with cow's milk EVs. Similarly, the majority of the quantified proteins were equally enriched between the two pellets. Nevertheless, 20 proteins were specific to 35K, while 41 were specifically enriched in 100K (p<0.05), suggesting their potential use as specific markers. Loaded with these proteins, the EVs in these pellets might regulate translation, proliferation and cell survival for 35K, and metabolism, extracellular matrix turnover and immunity for 100K. This approach also brought new insights into milk EV-associated integrins and their possible role in specifically targeting recipient cell types. These findings may help better discriminate between milk EVs, improve our understanding of milk EV-associated protein function and their possible use as therapeutic tools for the management of immunity-and metabolism-associated disorders.
Scientific Reports, 2023
Cell-derived extracellular vesicles (EVs) are currently in the limelight as potential disease biomarkers. The promise of EV-based liquid biopsy resides in the identification of specific disease-associated EV signatures. Knowing the reference EV profile of a body fluid can facilitate the identification of such disease-associated EV-biomarkers. With this aim, we purified EVs from paired human milk and serum samples and used the MACSPlex bead-based flow-cytometry assay to capture EVs on bead-bound antibodies specific for a certain surface protein, followed by EV detection by the tetraspanins CD9, CD63, and CD81. Using this approach we identified body fluid-specific EV signatures, e.g. breast epithelial cell signatures in milk EVs and platelet signatures in serum EVs, as well as body fluid-specific markers associated to immune cells and stem cells. Interestingly, comparison of pan-tetraspanin detection (simultaneous CD9, CD63 and CD81 detection) and single tetraspanin detection (detection by CD9, CD63 or CD81) also unveiled body fluid-specific tetraspanin distributions on EVs. Moreover, certain EV surface proteins were associated with a specific tetraspanin distribution, which could be indicative of the biogenesis route of this EV subset. Altogether, the identified body fluid-specific EV profiles can contribute to study EV profile deviations in these fluids during disease processes. Extracellular vesicles (EVs) are submicron lipid bilayer-delimited particles naturally released by cells that act as mediators of inter-cellular communication by targeting biologically active molecules to adjacent and distant cells 1. Cells in body tissues communicate by releasing EVs into proximal body fluids, such as breast milk and blood 2. Circulating EVs can originate from cells present in the body fluids, cells lining the cavities of extruded fluids or from tissue-resident cells 2 , and for this reason they can carry body fluid-specific and tissue-specific signatures. Additionally, the molecular make-up of EVs can be affected by the status of their originating cells and, as such, EVs can be enriched or depleted for specific surface proteins, resulting in specific protein biomarker profiles associated with (patho)physiological conditions 3,4. Nowadays, EV-based biomarker discovery attracts a lot of attention for monitoring disease and health status. The promise of EV-based biomarkers resides in the unique combination of different EV molecules, resulting in a "combined" biomarker that outperforms single component-based biomarkers. To identify EV-based biomarkers of disease or disturbed homeostasis, knowing the "normal" molecular reference profile of EVs in different body fluids is of utmost importance. However, the discovery of such body fluid-specific EV profiles is complicated due to the colloidal properties of body fluids, containing non-EV particles with overlapping characteristics of EVs 5,6. For example, lipoproteins in blood and casein micelles in milk, which co-isolate to various degrees with EVs 7,8 , can act as confounders in (semi)-quantitative EV analyses. Hence for comparative analyses of EVs present in different body fluids, a tailored protocol for EV isolation might overcome these problems. In immunoassays developed for EV phenotyping, the tetraspanins CD9, CD63 and CD81 are commonly used as bona fide EV-associated markers for "total" EV detection. These tetraspanins have primary functions in EV formation, cargo selection/sorting and EV release and uptake 9. Via their extracellular domains, tetraspanins associate with other tetraspanins and surface proteins thereby forming "tetraspanin webs" resulting in membrane domains with a variety of surface protein profiles 10. Importantly, in recent years, it has been reported that