Comparison of morphology, protein concentration, and size distribution of bone marrow and Wharton's jelly-derived mesenchymal stem cells exosomes isolated by ultracentrifugation and polymer-based precipitation techniques.

Exosomes which are tiny extracellular vesicles (30-150 nm), transport vital proteins and gene materials such as miRNA, mRNA, or DNA, whose role in cell communication and epithelia regulation is critical. Many techniques have been developed as a result of studying exosomes' biochemical and physicochemical properties, although there is still no standard method to isolate exosomes simply with high yield. Commercial kits have gained popularity for exosome extraction despite concerns about their effectiveness in scientific research. On the other hand, ultracentrifugation remains the gold standard isolation method. This study compares these two common exosome isolation methods to determine their impact on the quality and quantity of exosomes isolated from bone marrow (BM) and Wharton's jelly (WJ)-derived mesenchymal stem cells. Isolated exosomes from the two sources of the cell's conditioned medium by two methods (polymer kit and ultracentrifuge) were characterized using western blotting, scanning electron microscopy (SEM), dynamic light scattering (DLS), and the Bradford assay. Western blot analysis confirmed separation efficiency based on CD81 and CD63 markers, with the absence of calnexin serving as a negative control. The Morphology of exosomes studied by SEM image analysis revealed a similar round shape appearance and their sizes (30-150 nm) were the same in both isolation techniques. The DLS analysis of the sample results was consistent with the SEM ones, showing a similar size range and very low disparity. The exosome protein content concentration analysis revealed that exosomes isolated by the polymer-based kits contained higher protein concentration density and purity (p <0.001). In general, though the protein yield was higher when the polymer-based kits were used, there were no significant differences in morphology, or size between WJ-derived and BM-derived exosomes, regardless of the isolation method employed.

Autophagy Promotes Enrichment of Raft Components within Extracellular Vesicles Secreted by Human 2FTGH Cells.

Autophagy plays a key role in removing protein aggregates and damaged organelles. In addition to its conventional degradative functions, autophagy machinery contributes to the release of cytosolic proteins through an unconventional secretion pathway. In this research, we analyzed autophagy-induced extracellular vesicles (EVs) in HT1080-derived human fibrosarcoma 2FTGH cells using transmission electron microscopy and atomic force microscopy (AFM). We preliminary observed that autophagy induces the formation of a subset of large heterogeneous intracellular vesicular structures. Moreover, AFM showed that autophagy triggering led to a more visible smooth cell surface with a reduced amount of plasma membrane protrusions. Next, we characterized EVs secreted by cells following autophagy induction, demonstrating that cells release both plasma membrane-derived microvesicles and exosomes. A self-forming iodixanol gradient was performed for cell subfractionation. Western blot analysis showed that endogenous LC3-II co-fractionated with CD63 and CD81. Then, we analyzed whether raft components are enriched within EV cargoes following autophagy triggering. We observed that the raft marker GD3 and ER marker ERLIN1 co-fractionated with LC3-II; dual staining by immunogold electron microscopy and coimmunoprecipitation revealed GD3-LC3-II association, indicating that autophagy promotes enrichment of raft components within EVs. Introducing a new brick in the crosstalk between autophagy and the endolysosomal system may have important implications for the knowledge of pathogenic mechanisms, suggesting alternative raft target therapies in diseases in which the generation of EV is active.

Comparison of extracellular vesicle isolation methods for the study of exosome cargo within Toxocara canis and Toxocara cati excretory secretory (TES) products.

Toxocara is a genus of nematodes, which infects a variety of hosts, principally dogs and cats, with potential zoonotic risks to humans. Toxocara spp. larvae are capable of migrating throughout the host tissues, eliciting eosinophilic and granulomatous reactions, while surviving for extended periods of time, unchanged, in the host. It is postulated that larvae are capable of altering the host's immune response through the release of excretory-secretory products, containing both proteins and extracellular vesicles (EVs). The study of EVs has increased exponentially in recent years, largely due to their potential use as a diagnostic tool, and in molecular therapy. To this end, there have been multiple isolation methods described for the study of EVs. Here, we use nanoparticle tracking to compare the yield, size distribution, and % labelling of EV samples acquired through various reported methods, from larval cultures of Toxocara canis and T. cati containing Toxocara excretory-secretory products (TES). The methods tested include ultracentrifugation, polymer precipitation, magnetic immunoprecipitation, size exclusion chromatography, and ultrafiltration. Based on these findings, ultrafiltration produces the best results in terms of yield, expected particle size, and % labelling of sample. Transmission electron microscopy confirmed the presence of EVs with characteristic cup-shaped morphology. These findings can serve as a guide for those investigating EVs, particularly those released from multicellular organisms, such as helminths, for which few comparative analyses have been performed.

Extraction and identification of exosomes from three different sources of human ovarian granulosa cells and analysis of their differential miRNA expression profiles.

OBJECTIVE: As important functional cells in the ovary, ovarian granulosa cells are involved in the regulation of oocyte growth and development and play an important role in the study of female fertility preservation. Based on the importance of granulosa cell functionalism, in this study, we analyzed the exosome secretion capacity of human ovarian granulosa cells (SVOG/KGN-cell line, PGC-primary cells) and the differences in their miRNA expression. METHODS: Cells were identified by hematoxylin-eosin staining (HE) and FSHR immunofluorescence staining; CCK8 and colony-forming assay were performed to compare cell proliferation capacity; exosomes were extracted and identified by ultra-high speed centrifugation, transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot analysis (WB), and the expression profile of each cellular exosomal miRNA was analyzed by miRNA high-throughput sequencing. RESULTS: The proliferative abilities of the three granulosa cells differed, but all had the ability to secrete exosomes. In the exosomes of SVOG, KGN, and PGC cells, 218, 327, and 471 miRNAs were detected, respectively. When compared to the exosomal miRNAs of PGC cells, 111 miRNAs were significantly different in SVOG, and 70 miRNAs were washed two significantly different in KGN cells. These differential miRNA functions were mainly enriched in the cell cycle, cell division/differentiation, multicellular biogenesis, and protein binding. CONCLUSION: Human ovarian granulosa cells of different origins are capable of secreting exosomes, but there are still some differences in their exosomes and exosomal miRNAs, and experimental subjects should be selected rationally according to the actual situation.

A method for extraction of exosomes from breast tumour cells and characterisation by transmission electron microscopy.

Exosomes can not only be used as markers of tumour metastasis but also be used for noninvasive diagnosis of clinical diseases, which holds significant medical research value. Observing the morphology and distribution of exosomes using transmission electron microscopy (TEM) is highly significant. In this study, we use breast tumour cell (MDA-MB-231) exosomes as a representative sample and focus on the extraction and purification of exosomes, as well as the investigation of optimal conditions for the observation of exosomes using TEM. Through comparative tests, we obtained the optimal dilution concentration and staining method for TEM of exosomes, the best dilution concentration is 100 times, the best negative staining time is 1.5 min. Western blotting and scanning electron microscopy (SEM) confirmed the presence of exosome. The particle size experiment shows that the size of exosomes is mainly distributed between 60 nm and 150 nm. This method provides a reference for TEM sample preparation of subcellular structures and small molecular biomaterials other than exosomes.

Biogenesis and release of endothelial extracellular vesicles: Morphological aspects.

BACKGROUND: Cell-cell communication through extracellular vesicles (EVs) including exosomes, microvesicles and apoptotic bodies has been shown to be important in physiological homoeostasis as well as pathological processes such as atherosclerosis. However, while the cellular machinery controlling EV formation and composition has been studied during the past decade, less is known about the morphological process of their formation and release. METHODS: Using different electron microscopic approaches including transmission-, scanning-, immun-, and serial block face electron microscopy we studied the morphogenetic events of EV formation and release. We analysed the different steps of EV formation and release in cultured myocardial endothelial (MyEnd) and aortic endothelial (AoEnd) cell lines under serum starvation and under inflammatory conditions. RESULTS: We show that in a narrow time frame, the number of active cells and microvesicle (MV) producing cells increased in dependence of time spent in cultivation and additional stimulation by TNF-α. However, MV secretion was a highly heterogeneous process which couldn´t be seen in all cells cultivated under the same conditions. Release of MVs could be observed all over the cells' surface with no preferred release site. While no single specific microscopic approach applied was sufficient to provide a comprehensive analysis of EV biogenesis, we show that the limitations of one technique could be compensated by the qualities of the respective other applied techniques, thus enabling us to provide a detailed ultrastructural analysis of MV and exosome biogenesis. Surprisingly, exosome release in endothelial cells occurred via a yet undescribed process indicating that MVBs were incorporated into a novel distinct cellular compartment covered by fenestrated endothelium before exosome release. Lastly, we could show that TNF-α stimulation of AoEnd cells leads not only to the upregulation of CD44 in parental cells, but also to incorporation of CD44 into the membranes of generated MVs and exosomes. CONCLUSIONS: Taken together, our data contribute to a better understanding of biogenesis and release of EVs. We conclude that under inflammatory conditions, EVs can mediate the transfer of CD44 from endothelial cells to target cells at distant sites including vessel wall cells and this could be a mechanism by which MVs may change the and thus contribute to the development and progression of atherosclerotic lesions.

Detection of plasma exosomal miRNA-205 as a biomarker for early diagnosis and an adjuvant indicator of ovarian cancer staging.

BACKGROUND: Ovarian cancer (OC) is one of the serious threats to the health of women worldwide, and accurate biomarkers are urgently demanded for early diagnosis of OC. We have previously confirmed that miR-205 promotes the invasion and metastasis of OC cells by inhibiting the expression of the tumor suppressor gene TCF21. In this study, we used liquid biopsy technology to detect the expression levels of the four genes, miR-205, CA125, HE4 and TCF21, in the exosomes of plasma of OC patients. Combined with analysis of clinicopathological parameters of OC patients, we aimed to provide efficient and non-invasive laboratory biomarkers for early diagnosis of OC. METHODS: 36 OC patients who were diagnosed in local hospitals from September 2020 to July 2021 were selected as OC group, 31 cases of surgically diagnosed with ovarian benign lesions were selected as benign group, and 32 healthy people who underwent physical examination during the same period were selected as a control group. We employed transmission electron microscope (TEM), Western blotting (WB), and nanoparticle tracking analysis (NTA) to identify biomarkers in the exosomes extracted from plasma of the three groups. The RNA levels of miR-205, CA125, HE4 and TCF21 genes in plasma exosomes were detected by real-time quantitative PCR (qRT-PCR) method. We used clinical pathological parameters and the Receiver Operating Characteristic (ROC) curves to evaluate the diagnostic efficacy for the genes detected in plasma exosomes. RESULTS: We found that the expression level of miR-205 in plasma exosomes of the OC group was significantly higher than that of the benign and control groups (P <  0.05), and the level of miR-205 was elevated during the III-IV periods of OC and lymph node metastasis. CONCLUSION: The level of miR-205 in plasma exosomes is a valuable tumor biomarker to improve OC diagnosis.

Exosomes derived from human placental mesenchymal stem cells ameliorate myocardial infarction via anti-inflammation and restoring gut dysbiosis.

BACKGROUND: Myocardial infarction (MI) represents a severe cardiovascular disease with limited therapeutic agents. This study was aimed to elucidate the role of the exosomes derived from human placental mesenchymal stem cells (PMSCs-Exos) in MI. METHODS: PMSCs were isolated and cultured in vitro, with identification by both transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). To further investigate the effects of PMSC-Exos on MI, C57BL/6 mice were randomly divided into Sham group, MI group, and PMSC-Exos group. After 4 weeks of the intervention, cardiac function was assessed by cardiac echocardiography, electrocardiogram and masson trichrome staining; lipid indicators were determined by automatic biochemical instrument; inflammatory cytokines were measured by cytometric bead array (CBA); gut microbiota, microbial metabolites short chain fatty acids (SCFAs) as well as lipopolysaccharide (LPS) were separately investigated by 16S rRNA high throughput sequencing, gas chromatography mass spectrometry (GC-MS) and tachypleus amebocyte lysate kit; transcriptome analysis was used to test the transcriptional components (mRNA\miRNA\cirRNA\lncRNA) of PMSC-Exos. RESULTS: We found that human PMSC-Exos were obtained and identified with high purity and uniformity. MI model was successfully established. Compared to MI group, PMSC-Exos treatment ameliorated myocardial fibrosis and left ventricular (LV) remodeling (P < 0.05). Moreover, PMSC-Exos treatment obviously decreased MI molecular markers (AST/BNP/MYO/Tn-I/TC), pro-inflammatory indicators (IL-1ß, IL-6, TNF-α, MCP-1), as well as increased HDL in comparison with MI group (all P < 0.05). Intriguingly, PMSC-Exos intervention notably modulated gut microbial community via increasing the relative abundances of Bacteroidetes, Proteobacteria, Verrucomicrobia, Actinobacteria, Akkermansia, Bacteroides, Bifidobacterium, Thauera and Ruminiclostridium, as well as decreasing Firmicutes (all P < 0.05), compared with MI group. Furthermore, PMSC-Exos supplementation increased gut microbiota metabolites SCFAs (butyric acid, isobutyric acid and valeric acid) and decreased LPS in comparison with MI group (all P < 0.05). Correlation analysis indicated close correlations among gut microbiota, microbial SCFAs and inflammation in MI. CONCLUSIONS: Our study highlighted that PMSC-Exos intervention alleviated MI via modulating gut microbiota and suppressing inflammation.

miR-223 in exosomes from bone marrow mesenchymal stem cells ameliorates rheumatoid arthritis via downregulation of NLRP3 expression in macrophages.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease with major clinical manifestations of human limb joint invasion, joint synovitis, and symmetrical lesions. In recent years, bone marrow mesenchymal stem cells (BMSCs) have been found to have low immunogenicity and immunomodulatory effects, which can regulate other types of cells through exosomes. However, the effect of BMSCs on immune response in the progression of RA has not been fully elucidated. AIMS: The current research aimed to investigate the therapeutic effect of microRNA (miR)-223 in exosomes secreted by BMSCs on immune response in the progression of RA. METHODS: Firstly, BMSCs were isolated and extracted, and then the influence of BMSCs on the level of inflammatory cytokines was detected by enzyme linked immunosorbent assay (ELISA). Exosomes from BMSCs were extracted and characterized. Some key autoimmune response genes and their protein products were detected in vivo and in vitro by real-time quantitative PCR, western blot and ELISA. Finally, the targeting relationship between miR-223 and NLR family pyrin domain-containing 3 (NLRP3) was predicted by bioanalytical software and verified by luciferase reporter assay and rescue experiments in vitro. RESULTS: Exosomes from BMSCs could inhibit the release of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and interleukin-18 (IL-18), and NLRP3 activation in macrophages and RA rats. In addition, we predicted online that miR-223 could target NLRP3 and provided a possible regulation pathway for the anti-inflammatory effects of BMSCs-secreted exosomes. Furthermore, we further confirmed that miR-223 could target and inhibit the expression of NLRP3. CONCLUSION: Taken together, these findings suggest that miR-223 carried by BMSCs-derived exosomes targets NLRP3 to regulate the activation of inflammasomes, which therefore can be served as a possible therapy for RA.

Exosome application in tumorigenesis: diagnosis and treatment of melanoma.

Melanoma is the most aggressive of skin cancer derived from genetic mutations in the melanocytes. Current therapeutic approaches include surgical resection, chemotherapy, photodynamic therapy, immunotherapy, biochemotherapy, and targeted therapy. However, the efficiency of these strategies may be decreased due to the development of diverse resistance mechanisms. Here, it has been proven that therapeutic monoclonal antibodies (mAbs) can improve the efficiency of melanoma therapies and also, cancer vaccines are another approach for the treatment of melanoma that has already improved clinical outcomes in these patients. The use of antibodies and gene vaccines provides a new perspective in melanoma treatment. Since the tumor microenvironment is another important factor for cancer progression and metastasis, in recent times, a mechanism has been identified to provide an opportunity for melanoma cells to communicate with remote cells. This mechanism is involved by a novel molecular structure, named extracellular vesicles (EVs). Depending on the functional status of origin cells, exosomes contain various cargos and different compositions. In this review, we presented recent progress of exosome applications in the treatment of melanoma. Different aspects of exosome therapy and ongoing efforts in this field will be discussed too.

ICAM-1-mediated adhesion is a prerequisite for exosome-induced T cell suppression.

Tumor-derived extracellular vesicles (TEVs) suppress the proliferation and cytotoxicity of CD8+ T cells, thereby contributing to tumor immune evasion. Here, we report that the adhesion molecule intercellular adhesion molecule 1 (ICAM-1) co-localizes with programmed death ligand 1 (PD-L1) on the exosomes; both ICAM-1 and PD-L1 are upregulated by interferon-γ. Exosomal ICAM-1 interacts with LFA-1, which is upregulated in activated T cells. Blocking ICAM-1 on TEVs reduces the interaction of TEVs with CD8+ T cells and attenuates PD-L1-mediated suppressive effects of TEVs. During this study, we have established an extracellular vesicle-target cell interaction detection through SorTagging (ETIDS) system to assess the interaction between a TEV ligand and its target cell receptor. Using this system, we demonstrate that the interaction of TEV PD-L1 with programmed cell death 1 (PD-1) on T cells is significantly reduced in the absence of ICAM-1. Our study demonstrates that ICAM-1-LFA-1-mediated adhesion between TEVs and T cells is a prerequisite for exosomal PD-L1-mediated immune suppression.

Fibroblast-like Synoviocytes-derived Exosomal PCGEM1 Accelerates IL-1ß-induced Apoptosis and Cartilage Matrix Degradation by miR-142-5p/RUNX2 in Chondrocytes.

BACKGROUND: Long non-coding RNA (lncRNA) prostate cancer gene expression marker 1 (PCGEM1) has been revealed to participate in the pathogenesis of osteoarthritis (OA). However, the molecular mechanism of PCGEM1 regulating OA progression has not been fully elucidated. METHODS: Fibroblast-like synoviocytes (FLSs) were isolated from synovium tissues of OA patients (OA-FLSs) and trauma donors (Normal-FLSs). The size and morphology of the isolated exosomes were analyzed by transmission electron microscopy and nanoparticle tracking analysis. Protein levels were analyzed by western blotting. Expression levels of PCGEM1, microRNA-142-5p (miR-142-5p), runt-related transcription factor 2 (RUNX2) mRNA, and OA related genes were assessed by qRT-PCR. Cell proliferation, viability, and apoptosis were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide or flow cytometry assays. The relationship between miR-142-5p and PCGEM1 or RUNX2 was verified by dual-luciferase reporter and/or RNA pull down assays. RESULTS: PCGEM1 was overexpressed in OA cartilages and exosomes from OA-FLSs. Exosomal PCGEM1 from OA-FLSs facilitated IL-1ß-induced apoptosis and cartilage matrix degradation in chondrocytes. MiR-142-5p was downregulated while RUNX2 was upregulated in OA cartilages. Exosomal PCGEM1 from OA-FLSs regulated RUNX2 expression by sponging miR-142-5p in IL-1ß-induced chondrocytes. MiR-142-5p inhibitor offset exosomal PCGEM1 knockdown-mediated effects on the apoptosis and cartilage matrix degradation of IL-1ß-induced chondrocytes. RUNX2 overexpression counteracted the suppressive effect of miR-142-5p mimic on apoptosis and cartilage matrix degradation of IL-1ß-induced chondrocytes. CONCLUSION: Exosomal PCGEM1 from OA-FLSs facilitated IL-1ß-induced apoptosis and cartilage matrix degradation in chondrocytes by sequestering miR-142-5p and upregulating RUNX2, which offered new insights into the pathogenesis of OA.

Attenuation of hepatic ischemia­reperfusion injury by adipose stem cell­derived exosome treatment via ERK1/2 and GSK­3ß signaling pathways.

Exosomes are an emerging therapeutic tool for the treatment of tissue injuries. In the present study, the protective effect of isolated exosomes from adipose­derived stem cells (ADSCs­exo) against hepatic ischemia­reperfusion (I/R) injury was explored. Hepatic I/R injury was achieved by inducing ischemia for 60 min followed by reperfusion for 2 and 6 h. Pre­treatment with ADSCs­exo revealed a significant reduction in necrosis and apoptosis in liver tissue induced by I/R injury. Hypoxic oxidative stress was managed by exosome­mediated reduced reactive oxygen species and increased superoxide dismutase that in turn protected mitochondrial damage and apoptosis. Reduction in inflammatory mediators such as IL­1ß and TNF­α was also observed and protection of hepatocytes from I/R injury was evidenced by a significant decrease in biochemical markers of liver damage (alanine transaminase, aspartate transaminase and lactate dehydrogenase). Exosomal prostaglandin E2 (PGE2)­mediated ERK1/2 and GSK­3ß phosphorylation were revealed to increase Bcl­2 and decrease Bax expression with mitochondrial permeability transition pore­inhibition which may be considered a prime mechanism of exosome­mediated hepatoprotection. In conclusion, our results indicated that ADSCs­exo pre­treatment is effective in protecting liver I/R injury.

Exosomal microRNA-107 reverses chemotherapeutic drug resistance of gastric cancer cells through HMGA2/mTOR/P-gp pathway.

BACKGROUND: RNA cargo in exosomes, especially microRNAs (miRNAs), play an important role in the chemotherapy drug resistance of human cancers. However, the role and mechanism of exosomal miR-107 on multidrug resistance of gastric cancer cells was still not clear. In this study, we sought to explore whether exosomal miR-107 could reverse the resistance of gastric cancer cells to the chemotherapy drugs. METHODS: We extracted exosomes from sensitive (SGC-7901, MGC-803) and resistant (SGC-7901/5-FU) gastric cancer cells by ultracentrifugation and the isolated exosomes were identified using transmission electron microscopy (TEM) and dynamic light scattering analysis (DLS). The expression of miR-107 and high mobility group A2 (HMGA2) were detected by real-time quantitative PCR (RT-qPCR). MTT assay was used to investigate the effect of exosomes on gastric cancer cells growth in vitro. The uptake of exosomes by recipient cells were observed using a fluorescence microscope. The predicted target relationship between miR-107 and HMGA2 was verified by gauss-luciferase reporter assay. The expression of HMGA2, p-mTOR/mTOR, P-gp and other exosomal indicated marker proteins was detected by western blot. RESULTS: Our results indicated that the isolated exosomes were typically cup-like lipid bilayer membranes structure. SGC-7901/5-FU cells were cross-resistant to chemotherapy drug cisplatin (CDDP), and the sensitive cells-secreted exosomes drastically reversed the resistance of the resistant GC cells to the chemotherapeutic drugs, which was verified by exosomal inhibitor GW4896. Mechanistically, the reversal effect was mainly mediated by exosome-secreted miR-107 through downregulating the expression of target molecular HMGA2 and inhibiting HMGA2/mTOR/P-gp pathway, which were supported by results from luciferase reporter assay and rescue assay. CONCLUSIONS: These findings demonstrated that exosome-transmitted miR-107 significantly enhanced the sensitivity of resistant gastric cancer cells to chemotherapeutic agents by mediating the HMGA2/mTOR/P-gp axis and exosomal miR-107 may be a novel target in gastric cancers treatment.

Human umbilical vein endothelial cells derived-exosomes promote osteosarcoma cell stemness by activating Notch signaling pathway.

Osteosarcoma is one of the most common primary malignant tumors of bone in adolescents. Human umbilical vein endothelial cells (HUVECs) derived exosomes are associated with osteosarcoma cell stemness. Little is known about the function of HUVECs-exosomes in osteosarcoma cell stemness. This work aimed to investigate the mechanism of action of HUVECs-exosomes in regulating stem cell-like phenotypes of osteosarcoma cells. HUVECs were treated with GW4869 (exosome inhibitor). Human osteosarcoma cells (U2OS and 143B) were treated with HUVECs supernatant, HUVECs-exosomes with or without RO4929097 (γ secretase inhibitor, used to block Notch signaling pathway). We found that HUVECs supernatant and HUVECs-exosomes enhanced the proportions of STRO-1+CD117+ cells and the expression of stem cell-related proteins Oct4 and Sox2. Both HUVECs supernatant and HUVECs-exosomes promoted the sarcosphere formation efficiency of U2OS and 143B cells. These stem-like phenotypes of U2OS and 143B cells conferred by HUVECs-exosomes were repressed by GW4869. Moreover, HUVECs-exosomes promoted the expression of Notch1, Hes1 and Hey1 in the U2OS and 143B cells. RO4929097 treatment reversed the impact of HUVECs-exosomes on Notch1, Hes1, and Hey1 expression by inhibiting Notch1 signaling pathway. In conclusion, this work demonstrated that HUVECs-exosomes promoted cell stemness in osteosarcoma through activating Notch signaling pathway. Thus, our data reveal the mechanism of HUVECs-exosomes in regulating cell stemness of osteosarcoma, and provide a theoretical basis for osteosarcoma treatment by exosomes.

Milk Exosome-Derived MicroRNA-2478 Suppresses Melanogenesis through the Akt-GSK3ß Pathway.

Exosomes participate in intercellular communication by transferring molecules from donor to recipient cells. Exosomes are found in various body fluids, including blood, urine, cerebrospinal fluid and milk. Milk exosomes contain many endogenous microRNA molecules. MicroRNAs are small noncoding RNAs and have important roles in biological processes. The specific biological functions of milk exosomes are not well understood. In this study, we investigated the effects of milk exosomes on melanin production in melanoma cells and melanocytes. We found that milk exosomes decreased melanin contents, tyrosinase activity and the expression of melanogenesis-related genes in melanoma cells and melanocytes. Bovine-specific miR-2478 in exosomes inhibited melanin production. We found that Rap1a is a direct target gene of miR-2478 in melanoma cells and melanocytes. MiR-2478 overexpression decreased Rap1a expression, which led to downregulated melanin production and expression of melanogenesis-related genes. Inhibition of Rap1a expression decreased melanogenesis through the Akt-GSK3ß signal pathway. These results support the role of miR-2478 derived from milk exosomes as a regulator of melanogenesis through direct targeting of Rap1a. These results show that milk exosomes could be useful cosmeceutical ingredients to improve whitening.

Exosomes derived from microRNA-129-5p-modified tumor cells selectively enhanced suppressive effect in malignant behaviors of homologous colon cancer cells.

Exosome-encapsulated microRNAs (miRNAs) are novel diagnostic and predictive markers in colon cancer. Hence, the study of serum exosomal miRNAs in patients with colon cancer may help its diagnosis and treatment. PKH26-labeled exosomal uptake analysis identified whether exosomes transfer miRNA-129-5p to target cells. Transmission electron microscopy and dynamic light scattering analysis were applied to determine exosome morphology and size distribution. The Cell Counting Kit-8, wound healing assay and Transwell assays were used to detect cell proliferation, migration, and invasion after treatment with engineered exosomes. Moreover, the Western blotting was used to quantify the expression of proteins involved in cell apoptosis. In our study, hepatocellular liver carcinoma, cervical cancer and colon cancer cells were selected as the target cells of miRNA-129-5p exosomes. Exosomes containing miRNA-129-5p were found to be significantly more easily absorbed by colon cancer cells, presenting a stronger inhibitory effect on colon cancer cell proliferation. MiRNA-129-5p exosomes induced apoptosis in colon cancer cells while inhibiting their proliferation, migration, and invasion. In conclusion, exosomes derived from miRNA-129-5p-modified tumor cells selectively inhibited colon cancer progression, shedding new insights to therapeutic efficacy of this cancer.

A Comparative Study on the Effect of Exosomes Secreted by Mesenchymal Stem Cells Derived from Adipose and Bone Marrow Tissues in the Treatment of Osteoarthritis-Induced Mouse Model.

BACKGROUND: Exosomes as extracellular vesicles (EVs) are nanoscale intercellular messengers secreted from cells to deliver biological signals. Today, exosomes have become a new field of research in regenerative medicine and are considered as potential therapies to control inflammation and wound healing and enhance and improve healing in many diseases. Given the global burden of osteoarthritis (OA) as the fastest-growing health condition and one of the major causes of physical disability in the aging population, research to establish EVs as therapeutic products can meet the basic clinical needs in the management of osteoarthritis and provide a therapeutic solution. OBJECTIVES: The present study is aimed at evaluating the regenerative potentials of the exosomes secreted from adipose and bone marrow tissue-derived mesenchymal stem cells (AD- and BM-MSCs) in ameliorating the symptoms of OA. METHOD: In this experimental study, AD- and BM-MSCs were isolated and cultured in the laboratory until passage 3. Finally, these cells' secreted exosomes were isolated from their conditioned medium. Ciprofloxacin-induced OA mouse models underwent intra-articular injection of exosomes from AD-MSCs and BM-MSCs. Finally, the expression levels of collagen I and II, sox9, and aggrecan genes using real-time PCR, histological analysis, and immunohistochemical (IHC) studies were performed. RESULTS: Real-time PCR data showed that although the expression level of collagen type II was lower in both exosome-treated groups than the normal, but it was significantly increased in comparison with the sham and OA, with higher expression in BM-Exo rather than AD-Exo group. Similarly, the histological staining and IHC results have provided almost identical data, emphasizing on better therapeutic effect of BM-MSCs-exosome than AD-MSCs-exosome. CONCLUSION: BM-MSCs secreted exosomes in comparison with AD-MSCs could be considered as a better therapeutic option to improve osteoarthritis and exhibit potential as a disease-modifying osteoarthritis cell-free product.

Cancer-associated fibroblasts-derived exosomes promote lung cancer progression by OIP5-AS1/ miR-142-5p/ PD-L1 axis.

Cancer-associated fibroblasts (CAFs) are the most important stromal cells in the tumor microenvironment (TEM) and have been reported to regulate various cancer development. Exosomes are considered important elements involved in intercellular communication and TME regulation, while the potential function of CAFs in lung cancer immunosuppressive microenvironments remains unknown. CAFs-derived exosomes (CAFs-exo) and normal fibroblasts (NFs)-derived exosomes (NFs-exo) were isolated by ultra-centrifugation and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blot analysis. A549 cells were co-cultured with peripheral blood mononuclear cells (PBMCs). Flow cytometry assay was performed to detect the killing role of PBMCs on A549 cells. Bioinformatics and luciferase reporter assays were used to analyze the relationship among microRNA (miRNA), long non-coding RNA (lncRNA) and target gene. BALB/c mice were used to construct the lung cancer model by subcutaneous injection. Programmed death ligand 1 (PD-L1) was up-regulated in lung cancer tissues and cells. PD-L1 also up-regulated in CAFs cell medium-mediated A549 cells. CAFs decreased PBMCs induced-cell apoptosis through increasing PD-L1 in A549 cells. Moreover, CAFs transferred exosomes to lung cancer cells to suppress the killing effect of PBMCs through up-regulating PD-L1. Using microarray assays, opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) level was highly expressed in CAFs-exos. After treatment by CAFs-exos, miR-142-5p level was significantly down-regulated in A549 cells. OIP5-AS1 served as a sponge to target miR-142-5p and negatively regulated miR-142-5p expression in lung cancer cells. In addition, PD-L1 was a direct target of miR-142-5p. CAFs derived exosomal OIP5-AS1 reduced PBMCs induced-cell apoptosis and promoted tumor growth through decreasing miR-142-5p and up-regulating PD-L1. CAFs-derived exosomes suppressed the role of PBMCs induced-killing of lung cancer cells and promoted lung cancer progression by OIP5-AS1/ miR-142-5p/ PD-L1 axis, which provided a potential opportunity for diagnosis and treatment of lung cancer.

Pathogenic mechanisms of preeclampsia with severe features implied by the plasma exosomal mirna profile.

Preeclampsia is a complication of pregnancy characterized by high blood pressure and organ damage after 20 gestational weeks. It is associated with high maternal and fetal morbidity and mortality. However, at present, there is no effective prevention or treatment for this condition. Previous studies have revealed that plasma exosomal mirnas from pregnant women with preeclampsia could serve as biomarkers of pathogenic factors. However, the roles of plasma exosomal mirnas in preeclampsia with severe features (sPE), which is associated with poorer pregnancy outcomes, remain unknown. Thus, the aims of this study were to characterize plasma exosomal miRNAs in sPE and explore the related pathogenic mechanisms using bioinformatic analysis. Plasma exosomes were isolated using a mirVana RNA isolation kit. the exosomal miRNAs were detected using high-throughput sequencing and the mirnas related to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and gene ontology (GO) terms were analyzed using the clusterprofiler package of R. Fifteen miRNAs exhibited increased expression and fourteen miRNAs exhibited reduced expression in plasma exosomes from women with sPE as compared to normal pregnant women. Further, gene set enrichment analysis revealed that the differentially expressed plasma exosomal miRNAs were related to the stress response and cell junction regulation, among others. In summary, this study is the first to identify the differentially expressed plasma exosomal miRNAs in sPE. These findings highlight promising pathogenesis mechanisms underlying preeclampsia.