Milk-derived Exosomes Research Articles

Lipidomic Profiling of Human Milk Derived Exosomes and Their Emerging Roles in the Prevention of Necrotizing Enterocolitis.

Human milk can prevent the development of necrotizing enterocolitis (NEC). Human milk is rich in cargo-carrying exosomes that participate in intercellular communication. This study investigated the effects of term and preterm human milk-derived exosomes, and elucidated their lipid expression profiles. Milk from healthy mothers is collected who have delivered full-term or preterm infants, and exosomes are isolated and quantified. Administration of term and preterm milk exosomes significantly enhances epithelial proliferation and migration in vitro, and ameliorates the severity of NEC in vivo. A total of 395 lipids are identified in term and preterm human milk-derived exosomes. Bioinformatics analysis and western blotting reveal that top 50 lipids regulate intestinal epithelial cell function via the Extracellular-Signal-Regulated Kinase/Mitogen Activated Protein Kinase (ERK/MAPK) pathway. This study reveals for the first time the lipidomic complexities in exosomes derived from preterm and term milk. The results provide novel mechanistic insight on how human milk prevents the development of NEC.

Exosomal circRNAs contribute to intestinal development via the VEGF signalling pathway in human term and preterm colostrum.

Human breast milk (HBM) provides essential nutrients for newborn growth and development, and contains a variety of biologically active ingredients that can affect gastrointestinal tract and immune system development in breastfed infants. HBM also contains mRNAs, microRNAs and lncRNAs, most of which are encapsulated in milk-derived exosomes and exhibit various important infant development related biological functions. While previous studies have shown that exosomal circRNAs are involved in the intestinal epithelial cells’ proliferation and repair. However, the effect of HBM exosomal circRNAs on intestinal development is not clear. In this study, we identified 6756 circRNAs both in preterm colostrum (PC) and term colostrum (TC), of which 66 were upregulated, and 42 were downregulated (|fold change>2|, p < 0.05) in PC. Pathway analysis showed that the VEGF signalling pathway was involved, and network analysis revealed that the differentially expressed circRNAs bound various miRNAs. Further analyses showed that has_circRNA_405708 and has_circRNA_104707 were involved in the VEGF signalling pathway, and that they all bound various mirRNAs. Exosomes found in preterm colostrum (PC) and term colostrum (TC) promoted VEGF protein expression and induced the proliferation and migration of small intestinal epithelial cells (FHCs). Exosomal circRNAs found in human colostrum (HC) binding to related miRNAs may regulate VEGF signalling, and intestinal development.

Human breast milk-derived exosomes may help maintain intestinal epithelial barrier integrity.

This study explores the functions of exosomes derived from human breast milk (HBM) in vivo and in vitro. HBM-derived exosomes were collected from healthy lactating mothers. In vitro analysis were divided into five groups: (1) a control with no added agents, (2) exosomes added, (3) stimulated with lipopolysaccharide (LPS), (4) pretreated with exosomes and stimulated with LPS, and (5) pretreated with exosome-free HBM and stimulated with LPS. For in vivo analysis, mouse pups were randomly assigned to four groups: (1) a control group of breastfed pups, (2) necrotizing enterocolitis-induced (NEC) pups, (3) pups pretreated with HBM-derived exosomes 6 h before being induced by NEC, and (4) pups pretreated with exosome-free HBM 6 h before NEC induction. Expression of zonula occludens-1 (ZO-1), claudin-1, and occludin were decreased in groups 3 and 5. In the animal model, mice pups in group 3 showed milder intestinal tissue injury than those in group 2 or 4 and had lower levels of the proinflammatory cytokines and higher levels of epithelial tight-junction proteins than groups 2 and 4. HBM-derived exosomes exert beneficial effects in preventing NEC by reducing inflammation and injury to the intestinal epithelium as well as by restoring intestinal tight-junction proteins. HBM-derived exosomes can help protect the epithelial tight-junction proteins ZO-1, claudin, and occludin from inflammatory attack. This study sought (1) to analyze whether there were differences in exosome levels between the human breast milk (HBM) of mothers who had delivered preterm or at term and (2) to investigate whether these exosomes could help sustain the intestinal epithelial tight-junction proteins ZO-1, claudin-1, and occludin in the presence of NEC in vitro and in vivo.

Bovine Milk-Derived Exosomes as a Drug Delivery Vehicle for miRNA-Based Therapy

MicroRNAs (miRNAs) are small non-coding RNAs with a known role as mediators of gene expression in crucial biological processes, which converts them into high potential contenders in the ongoing search for effective therapeutic strategies. However, extracellular RNAs are unstable and rapidly degraded, reducing the possibility of successfully exerting a biological function in distant target cells. Strategies aimed at enhancing the therapeutic potential of miRNAs include the development of efficient, tissue-specific and nonimmunogenic delivery methods. Since miRNAs were discovered to be naturally transported within exosomes, a type of extracellular vesicle that confers protection against RNase degradation and increases miRNA stability have been proposed as ideal delivery vehicles for miRNA-based therapy. Although research in this field has grown rapidly in the last few years, a standard, reproducible and cost-effective protocol for exosome isolation and extracellular RNA delivery is lacking. We aimed to evaluate the use of milk-derived extracellular vesicles as vehicles for extracellular RNA drug delivery. With this purpose, exosomes were isolated from raw bovine milk, combining ultracentrifugation and size exclusion chromatography (SEC) methodology. Isolated exosomes were then loaded with exogenous hsa-miR148a-3p, a highly expressed miRNA in milk exosomes. The suitability of exosomes as delivery vehicles for extracellular RNAs was tested by evaluating the absorption of miR-148a-3p in hepatic (HepG2) and intestinal (Caco-2) cell lines. The potential exertion of a biological effect by miR-148a-3p was assessed by gene expression analysis, using microarrays. Results support that bovine milk is a cost-effective source of exosomes which can be used as nanocarriers of functional miRNAs with a potential use in RNA-based therapy. In addition, we show here that a combination of ultracentrifugation and SEC technics improve exosome enrichment, purity, and integrity for subsequent use.

Synergistic Effects of Milk-Derived Exosomes and Galactose on α-Synuclein Pathology in Parkinson's Disease and Type 2 Diabetes Mellitus.

Epidemiological studies associate milk consumption with an increased risk of Parkinson’s disease (PD) and type 2 diabetes mellitus (T2D). PD is an α-synucleinopathy associated with mitochondrial dysfunction, oxidative stress, deficient lysosomal clearance of α-synuclein (α-syn) and aggregation of misfolded α-syn. In T2D, α-syn promotes co-aggregation with islet amyloid polypeptide in pancreatic β-cells. Prion-like vagal nerve-mediated propagation of exosomal α-syn from the gut to the brain and pancreatic islets apparently link both pathologies. Exosomes are critical transmitters of α-syn from cell to cell especially under conditions of compromised autophagy. This review provides translational evidence that milk exosomes (MEX) disturb α-syn homeostasis. MEX are taken up by intestinal epithelial cells and accumulate in the brain after oral administration to mice. The potential uptake of MEX miRNA-148a and miRNA-21 by enteroendocrine cells in the gut, dopaminergic neurons in substantia nigra and pancreatic β-cells may enhance miRNA-148a/DNMT1-dependent overexpression of α-syn and impair miRNA-148a/PPARGC1A- and miRNA-21/LAMP2A-dependent autophagy driving both diseases. MiRNA-148a- and galactose-induced mitochondrial oxidative stress activate c-Abl-mediated aggregation of α-syn which is exported by exosome release. Via the vagal nerve and/or systemic exosomes, toxic α-syn may spread to dopaminergic neurons and pancreatic β-cells linking the pathogenesis of PD and T2D.

Milk exosomes with enhanced mucus penetrability for oral delivery of siRNA.

Bovine milk-derived exosomes have recently emerged as a promising nano-vehicle for the encapsulation and delivery of macromolecular biotherapeutics. Here we engineer high purity bovine milk exosomes (mExo) with modular surface tunability for oral delivery of small interfering RNA (siRNA). We utilize a low-cost enrichment method combining casein chelation with differential ultracentrifugation followed by size exclusion chromatography, yielding mExo of high concentration and purity. Using in vitro models, we demonstrate that negatively charged hydrophobic mExos can penetrate multiple biological barriers to oral drug delivery. A hydrophilic polyethylene glycol (PEG) coating was introduced on the mExo surface via passive, stable hydrophobic insertion of a conjugated lipid tail, which significantly reduced mExo degradation in acidic gastric environment and enhanced their permeability through mucin by over 3× compared to unmodified mExo. Both mExo and PEG-mExo exhibited high uptake by intestinal epithelial cells and mediated functional intracellular delivery of siRNA, thereby suppressing the expression of the target green fluorescence protein (GFP) gene by up to 70%. We also show that cationic chemical transfection is significantly more efficient in loading siRNA into mExo than electroporation. The simplicity of isolating high purity mExo in high concentrations and equipping them with tunable surface properties, demonstrated here, paves way for the development of mExo as an effective, scalable platform technology for oral drug delivery of siRNA.

Isolation and Characterisation of Bactrian Camel Milk-Derived Exosomes

Isolation and Characterisation of Bactrian Camel Milk-Derived Exosomes

Yak milk–derived exosomal microRNAs regulate intestinal epithelial cells on proliferation in hypoxic environment

Intestinal epithelial cells (IEC) act as an important intestinal barrier whose function can be impaired upon induction by hypoxia. Although intestinal barrier injuries are preventable by milk-derived exosomal microRNAs (miRNAs), the underlying mechanism remains poorly understood. This study aimed to characterize the effect of yak and cow milk-derived exosomal miRNA on the barrier function of IEC-6 under hypoxic conditions, and explore the mechanism of yak milk exosomal miRNA to relieve the hypoxia stress. First, by Illumina HiSeq 2500 (Illumina Inc., San Diego, CA) sequencing, the miRNA expression was systematically screened, and differential expression of 130 miRNAs was identified with 51 being upregulated and 79 downregulated in yak and cow milk-derived exosomes. Furthermore, the top 20 miRNAs that had a relatively consistent high expression in yak milk exosome were identified, and bta-miR-34a was found to be an effective regulator for alleviating hypoxic injury of IEC-6. In vitro assay of the role of bta-miR-34a on survival of IEC-6 in hypoxia by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) confirmed its effectiveness to significantly increase IEC-6 survival up to 13% for 12 h, and up to 9.5% for 24 h. Investigation on the regulatory relationship between bta-miRNA-34a and the hypoxia-inducible factor/apoptosis signaling pathway provided insights into the possible mechanisms by which bta-miR-34a activated the hypoxia-inducible factor and apoptosis signaling pathway, thus promoting IEC-6 survival. The results of this study suggest an important relationship between miRNA expression and intestine barrier integrity, which facilitated further understanding of the physiological function of yak and cow milk exosomal miRNAs, as well as mechanisms of hypoxia-driven epithelial homeostasis.

Comprehensive Analysis of the Glycome and Glycoproteome of Bovine Milk-Derived Exosomes.

Bovine milk-derived exosomes (BMDEs) have potential applications in the pharmaceutical industry as drug delivery carriers. A comprehensive analysis of protein glycosylation in exosomes is necessary to elucidate the process of targeted delivery. In this work, free oligosaccharides (FOSs), O-glycans, and N-glycans in BMDEs and whey were first analyzed through multiple derivation strategies. In summary, 13 FOSs, 44 O-glycans, and 94 N-glycans were identified in bovine milk. To analyze site-specific glycosylation of glycoproteins, a one-step method was used to enrich and characterize intact glycopeptides. A total of 1359 proteins including 114 glycoproteins were identified and most of these were located in the exosomes. Approximately 95 glycopeptides were initially discovered and 5 predicted glycosites were confirmed in BMDEs. Collectively, these findings revealed the characterization and distribution of glycans and glycoproteins in BMDEs, providing insight into the potential applications of BMDEs in drug delivery and food science.

Exosomes exist in nipple discharge of breast cancer.

Exosomes exist in nipple discharge of breast cancer.

More than Nutrition: Therapeutic Potential of Breast Milk-Derived Exosomes in Cancer.

Human breast milk (HBM) is an irreplaceable source of nutrition for early infant growth and development. Breast-fed children are known to have a low prevalence and reduced risk of various diseases, such as necrotizing enterocolitis, gastroenteritis, acute lymphocytic leukemia, and acute myeloid leukemia. In recent years, HBM has been found to contain a microbiome, extracellular vesicles or exosomes, and microRNAs, as well as nutritional components and non-nutritional proteins, including immunoregulatory proteins, hormones, and growth factors. Especially, the milk-derived exosomes exert various physiological and therapeutic function in cell proliferation, inflammation, immunomodulation, and cancer, which are mainly attributed to their cargo molecules such as proteins and microRNAs. The exosomal miRNAs are protected from enzymatic digestion and acidic conditions, and play a critical role in immune regulation and cancer. In addition, the milk-derived exosomes are developed as drug carriers for delivering small molecules and siRNA to tumor sites. In this review, we examined the various components of HBM and their therapeutic potential, in particular of exosomes and microRNAs, towards cancer.

Cow and Human Milk-Derived Exosomes Ameliorate Colitis in DSS Murine Model

The aim of this study was to investigate the therapeutic effect of cow and human milk derived exosomes (MDEs) on colitis. We used gavage administration of fluorescent labeled MDEs to track their localization patterns in vivo and studied their therapeutic effect on colitis in a dextran sulfate sodium (DSS)-induced colitis model. MDEs attenuated the severity of colitis induced by DSS and statistically reduced the histopathological scoring grade and shortening of the colon. Likewise, treatment with MDEs reduced the expression of interleukin 6 and tumor necrosis factor-α. Moreover, miRNAs highly expressed in milk, such as miRNA-320, 375, and Let-7, were found to be more abundant in the colon of MDE-treated mice compared with untreated mice; contrastingly, the expression of their target genes, mainly DNA methyltransferase 1 (DNMT1) and DNMT3 were downregulated. Furthermore, the level of TGF-β was upregulated in the colon of MDE-treated mice. We demonstrated that MDEs have a therapeutic and anti-inflammatory effect on colitis, involving several complementary pathways in its mechanism of action. The therapeutic effects of MDEs might have implications for the possible addition of MDEs as a nutrient in enteral nutrition formulas for patients with inflammatory bowel disease.

The Potential of Milk-Derived Exosomes for Drug Delivery.

Exosomes, one of the extracellular vesicles, are widely present in all biological fluids and play an important role in intercellular communication. Due to their hydrophobic lipid bilayer and aqueous hydrophilic core structure, they are considered a possible alternative to liposome drug delivery systems. Not only do they protect the cargo like liposomes during delivery, but they are also less toxic and better tolerated. However, due to the lack of sources and methods for obtaining enough exosomes, the therapeutic application of exosomes as drug carriers is limited. A literature search was performed using the ScienceDirect and PubMed electronic databases to obtain information from published literature on milk exosomes related to drug delivery. Here, we briefly reviewed the current knowledge of exosomes, expounded the advantages of milk-derived exosomes over other delivery vectors, including higher yield, the oral delivery characteristic and additional therapeutic benefits. The purification and drug loading methods of milk exosomes, and the current application of milk exosomes were also introduced. The emergence of milk-derived exosomes is expected to break through the limitations of exosomes as therapeutic carriers of drugs. We hope to raise awareness of the therapeutic potential of milk-derived exosomes as a new drug delivery system.

Effect of mastitis on the function of milk-derived exosomes: observations from mammary epithelial cells

Effect of mastitis on the function of milk-derived exosomes: observations from mammary epithelial cells

MicroRNA Milk Exosomes: From Cellular Regulator to Genomic Marker.

Simple SummaryBovine milk contains proteins, minerals (e.g., calcium), vitamins (A, B, D, E and K), bioactive compounds and signaling molecules, as short non-coding RNA, either floating or contained in exosomes. Milk compositions and the milk-derived exosomes’ (EXO) cargo varies in relation to the health status of the mammary gland and the lactation stage. Stress, immune-depression and mastitis generate a differential microRNA (miRNA) expression, and consequently a modulation of the local immune cell response. This review aims to discuss the possible application of EXO cargo as a biomarker of animal resilience, and as a phenotype for genomic studies.Recent advances in ruminants’ milk-derived exosomes (EXO) have indicated a role of microRNAs (miRNAs) in cell-to-cell communication in dairy ruminants. The miRNAs EXO retain peculiar mechanisms of uptake from recipient cells, which enables the selective delivery of cargos, with a specific regulation of target genes. Although many studies have been published on the miRNAs contained in milk, less information is available on the role of miRNAs EXO, which are considered stable over time and resistant to digestion and milk processing. Several miRNAs EXO have been implicated in the cellular signaling pathway, as in the regulation of immune response. Moreover, they exert epigenetic control, as extenuating the expression of DNA methyltransferase 1. However, the study of miRNAs EXO is still challenging due to the difficulty of isolating EXO. In fact, there are not agreed protocols, and different methods, often time-consuming, are used, making it difficult to routinely process a large number of samples. The regulation of cell functions in mammary glands by miRNAs EXO, and their applications as genomic markers in livestock, is presented.

Short communication: Dietary bovine milk–derived exosomes improve bone health in an osteoporosis-induced mouse model

Osteoporosis is a systemic skeletal disease characterized by low bone mass and micro-architectural deterioration of bone tissue, with a consequent increase in bone fragility and fracture susceptibility. In an aged society with increased life expectancy, the incidence rate of osteoporosis is also rapidly increasing. Inadequate nutrition may negatively influence bone metabolism. Recently, many studies have investigated the functionality of milk-derived exosomes, which play important roles in cell-to-cell communication. However, there are few reports of how milk-derived exosomes influence osteoblast proliferation and differentiation. Here, we determined whether bovine colostrum-derived exosomes promote anti-osteoporosis in vitro and in vivo. Tartrate-resistant acid phosphatase-stained cells were significantly inhibited in Raw264.7 cells treated with exosomes, indicating reduced osteoclast differentiation. We induced osteoporosis in mice using glucocorticoid pellets after orally administering exosomes for 2 mo. Interestingly, the bone mineral density of exosome-fed mouse groups was significantly improved compared with the glucocorticoid-induced osteoporosis group without exosome treatment. In addition, Lactobacillus were decreased in the gut microbiota community of osteoporosis-induced mice, but the gut microbiota community composition was effectively restored by exosome intake. Taken together, we propose that exosomes isolated from bovine colostrum could be a potential candidate for osteoporosis prevention, bone remodeling improvement, and inhibition of bone resorption. To our knowledge, this is the first time that a protective effect of milk exosomes against osteoporosis has been demonstrated in vivo. Our results strongly suggest that bovine colostrum exosomes might be used as a prophylaxis to prevent the onset of osteoporosis. Indeed, our results offer promising alternative strategies in the nutritional management of age-related bone complications.

Proteomic Analysis Reveals Proteins and Pathways Associated with Lactation in Bovine Mammary Epithelial Cell-Derived Exosomes.

Milk-derived exosomes have been reported, which are involved in many biological processes. The exosomes derived from mammary glands are not known yet, and their relationship with mammary gland lactation and the origin of milk-derived exosomes are largely unclear. The present study aimed to investigate the proteome of exosomes derived from bovine mammary epithelial cells (BMECs) and compare them with milk-derived exosomes in the database. BMEC-derived exosomes were successfully separated from the culture supernatant of BMECs by a combined ultracentrifugation approach, and the purity of exosomes was identified by western blot analysis. Liquid chromatography with tandem mass spectrometry identified 638 proteins in BMEC-derived exosomes. The MS data were deposited into the PUBLIC repository ProteomeXchange, dataset identifier(s): https://www.iprox.org/page/PSV023.html;?url=1590961453176tKpa. Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these proteins were associated with specific biological processes and molecular functions of metabolism. Cross comparison of these proteins with the protein database of milk exosomes showed that 77 common expressed proteins (CEPs) were in both BMEC- and milk-derived exosomes. The KEGG pathway analysis for these CEPs showed that they were mainly involved in signaling pathways associated with milk biosynthesis in BMECs. Among these CEPs, six proteins have been previously reported to be associated with the lactation function. The western blot analysis detected that expression of these six proteins in BMEC-derived exosomes was increased after the stimulation of methionine and β-estradiol on BMECs. In summary, the proteome of BMEC-derived exosomes reveals that they are associated with milk biosynthesis in BMECs and might be a source of milk-derived exosomes.

Radioactive Labeling of Milk-Derived Exosomes with 99mTc and In Vivo Tracking by SPECT Imaging.

Over the last decade, exosomes from diverse biological sources have been proposed as new natural platforms in drug delivery. Translation of these nanometric tools to clinical practice requires deep knowledge of their pharmacokinetic properties and biodistribution. The pharmacokinetic properties of exosomes are sometimes evaluated using biochemical and histological techniques that are considerably invasive. As an alternative, we present radiochemical labeling of milk-derived exosomes based on reduced 99mTc (IV) without modifying biological and physicochemical properties. This approach enables longitudinal tracking of natural exosomes by non-invasive single photon emission computed tomography (SPECT) imaging and the evaluation of their pharmacokinetic properties according to the route of administration.

Expression profiles of lincRNA and mRNA related to milk yield and milk composition traits in the milk-derived exosomes of Holstein and Doğu Anadolu Kırmızısı cows

This study aimed to demonstrate the expression profiles of lincRNAs and mRNAs affecting milk yield and composition traits in the milk-derived exosomes of Holstein and Doğu Anadolu Kırmızısı (DAK) cows. For this purpose, the locations of these specific lincRNAs and mRNAs were confirmed in quantitative trait loci. Then RT-PCR analysis was performed to identify the expression profiles of the lincRNAs and mRNAs. Lastly, correlation analysis was carried out between milk yield data from Holstein and DAK cows and expression levels of the lincRNAs and mRNAs. The findings showed that while lincRNAs and mRNAs associated with milk yield traits were upregulated in the Holstein cows exhibiting high milk yield in comparison to the DAK cows exhibiting low milk yield, lincRNA and mRNA associated with milk composition traits were downregulated in the Holstein cows with high milk yield compared to the DAK cows with low milk yield. These results suggest primary evidence for expression profiles of lincRNA and mRNA related to milk production traits in the milk-derived exosomes of Holstein and DAK cows. These lincRNAs and mRNAs, which are carried in the milkderived exosomes, could be utilized in animal breeding programs to enhance milk yield and composition traits.

P044 Milk derived exosomes has a therapeutic effect on experimental colitis

Abstract Background Inflammatory bowel disease (IBD) is a complex disorder that results from a dysregulated immune response in the gut. Emerging therapy for IBD treatment is mainly focused on regulation of the immune response. Exosomes are nanovesicle packing different molecules such as miRNAs that transfer their cargo to recipient cells. We and others found that mammalian milk contain high concentration of exosomes (milk-derived exosomes, MDE) carrying beneficial miRNAs such as miRNA-148. Furthermore, MDE are taken up by different cell type such as intestinal epithelial cells, modify target gene expression, promote proliferation and differentiation of colon epithelial cells. The aim of this study is to explore the therapeutic effect of MDE on colitis. Methods We used gavage administration of MDE labelled with DiR dye to track their localisation patterns in vivo. The therapeutic effect of MDE on colitis was study in mice model of SDS induced colitis, colon length, histopathological scoring grade, cytokine expression, stool consistency and miRNA expression were analysed. Results Imaging of mouse that have receive labelled MDE revealed an accumulation of fluorescent signal in the intestine. Moreover, fluorescent signal in the intestine and liver is time dependent. MDE reduced the histopathological scoring grade from 5.83 ± 1.47 to 0.6 ± 0.6, p &amp;lt; 0.05. The length of the colon of MDE-treated animals was 7.9 ± 0.19 in comparison to 6.92 ± 0.3 p &amp;lt; 0.05 of the untreated. The weight loss as a result of the colitis was reverted in MDE-treated mice. Likewise, MDE treatment reduced IL-6, TNF-α and caveolin expression from 3.83 to 0.78, 1.59 to 0.86 and 3.52 to 1.1, respectively. Highly expressed miRNAs (miRNA-320, 375, Let-7a and 6073) were found to be more abundant in colon of MDE treatment mice compared with the untreated. Conclusion This study demonstrated that MDE have a therapeutic effect on colitis in vivo. Proving the effect of MDE on colitis will have implications for the potential of adding MDE as a therapeutic nutrient to be included in the formulas for IBD patients.