Exosome Yield Research Articles

Engineering exosomes derived from TNF-α preconditioned IPFP-MSCs enhance both yield and therapeutic efficacy for osteoarthritis

BackgroundThe pathogenesis of osteoarthritis (OA) involves the progressive degradation of articular cartilage. Exosomes derived from mesenchymal stem cells (MSC-EXOs) have been shown to mitigate joint pathological injury by attenuating cartilage destruction. Optimization the yield and therapeutic efficacy of exosomes derived from MSCs is crucial for promoting their clinical translation. The preconditioning of MSCs enhances the therapeutic potential of engineered exosomes, offering promising prospects for application by enabling controlled and quantifiable external stimulation. This study aims to address these issues by employing pro-inflammatory preconditioning of MSCs to enhance exosome production and augment their therapeutic efficacy for OA.MethodsThe exosomes were isolated from the supernatant of infrapatellar fat pad (IPFP)-MSCs preconditioned with a pro-inflammatory factor, TNF-α, and their production was subsequently quantified. The exosome secretion-related pathways in IPFP-MSCs were evaluated through high-throughput transcriptome sequencing analysis, q-PCR and western blot analysis before and after TNF-α preconditioning. Furthermore, exosomes derived from TNF-α preconditioned IPFP-MSCs (IPFP-MSC-EXOsTNF−α) were administered intra-articularly in an OA mouse model, and subsequent evaluations were conducted to assess joint pathology and gait alterations. The expression of proteins involved in the maintenance of cartilage homeostasis within the exosomes was determined through proteomic analysis.ResultsThe preconditioning with TNF-α significantly enhanced the exosome secretion of IPFP-MSCs compared to unpreconditioned MSCs. The potential mechanism involved the activation of the PI3K/AKT signaling pathway in IPFP-MSCs by TNF-α precondition, leading to an up-regulation of autophagy-related protein 16 like 1(ATG16L1) levels, which subsequently facilitated exosome secretion. The intra-articular administration of IPFP-MSC-EXOsTNF−α demonstrated superior efficacy in ameliorating pathological changes in the joints of OA mice. The preconditioning of TNF-α enhanced the up-regulation of low-density lipoprotein receptor-related protein 1 (LRP1) levels in IPFP-MSC-EXOsTNF−α, thereby exerting chondroprotective effects.ConclusionTNF-α preconditioning constitutes an effective and promising method for optimizing the therapeutic effects of IPFP-MSCs derived exosomes in the treatment of OA.Graphical

Hypoxia-preconditioned WJ-MSC spheroid-derived exosomes delivering miR-210 for renal cell restoration in hypoxia-reoxygenation injury

BackgroundRecent advancements in mesenchymal stem cell (MSC) technology have paved the way for innovative treatment options for various diseases. These stem cells play a crucial role in tissue regeneration and repair, releasing local anti-inflammatory and healing signals. However, challenges such as homing issues and tumorigenicity have led to exploring MSC-exosomes as a promising alternative. MSC-exosomes have shown therapeutic potential in conditions like renal ischemia-reperfusion injury, but low production yields hinder their clinical use.MethodsTo address this limitation, we examined hypoxic preconditioning of Wharton jelly-derived MSCs (WJ-MSCs) 3D-cultured in spheroids on isolated exosome yields and miR-21 expression. We then evaluated their capacity to load miR-210 into HEK-293 cells and mitigate ROS production, consequently enhancing their survival and migration under hypoxia-reoxygenation conditions.ResultsMiR-210 overexpression was significantly induced by optimized culture and preconditioning conditions, which also improved the production yield of exosomes from grown MSCs. The exosomes enriched with miR-210 demonstrated a protective effect by improving survival, reducing apoptosis and ROS accumulation in damaged renal cells, and ultimately promoting cell migration.ConclusionThe present study underscores the possibility of employing advanced techniques to maximize the therapeutic attributes of exosomes produced from WJ-MSC spheroid for improved recovery outcomes in ischemia-reperfusion injuries.

Exosomes derived from 3D-cultured MSCs alleviate knee osteoarthritis by promoting M2 macrophage polarization through miR-365a-5p and inhibiting TLR2/Myd88/NF-κB pathway

Exosomes (EXO) derived from mesenchymal stem cells (MSCs) are recently shown to have therapeutic effects in preclinical models of knee osteoarthritis (KOA). However, their potential for conversion into a clinical therapy are limited by several shortcomings such as low yield and suboptimal efficacy. This study developed 3D-EXO to improve exosome yield and efficacy through 3D-culturing of MSCs, with the aim of achieving overall better therapeutic effects compared to traditional 2D-cultured MSC-derived EXO (2D-EXO). In a rat KOA model, 3D-EXO led to better preservation of bone structure and protection from cartilage damage compared to 2D-EXO. Interestingly, in vitro experiments showed no significant differences between 3D-EXO and 2D-EXO in their direct effects on chondrocytes. To explain these findings, we looked into the possible effects of MSC-derived EXO on the immune microenvironment, specifically in modulating phenotypic expression in macrophages. In vitro investigations showed that 3D-EXO increased the expression of M2 phenotype markers in macrophages to a greater extent than 2D-EXO. RNA-seq analysis revealed that expression of miR-365a-5p was more upregulated in 3D-EXO than 2D-EXO. Through micro-RNA mimics transfection, we found that miR-365a-5p acted directly on TLR2 mRNA to inhibit TLR2 translation, indicating that 3D-EXO promoted M2 polarization of macrophages by inhibiting the TLR2/Myd88/NF-κB axis. The immunomodulatory effects of 3D-EXO were confirmed by the observation that the metabolic status of chondrocytes was significantly improved after co-culturing with 3D-EXO-treated macrophages. In summary, 3D-EXO can effectively promote M2 macrophage polarization through increased expression of miR-365a-5p, which in turn improves the metabolic state of chondrocytes and resulting in overall better in vivo therapeutic effects on KOA. The findings in this study imply the development of new therapies for KOA based on MSC-derived EXO.

IL-12 improves the anti-HCC efficacy of dendritic cells loaded with exosomes from overexpressing Rab27a tumor cells

Determining the appropriate source of antigens for optimal antigen presentation to T cells is a major challenge in designing dendritic cell (DC) -based therapeutic strategies against hepatocellular carcinoma (HCC). Tumor-derived exosomes (Tex) express a wide range of tumor antigens, making them a promising source of antigens for DC vaccines. As reported, the exosomes secreted by tumor cells can inhibit the antitumor function of immune cells. In this study, we transfected hepatocellular carcinoma cells with Rab27a to enhance the yield of exosomes, which were characterized using transmission electron microscopy and Western blot analysis. We found that Tex secreted by overexpressing Rab27a Hepatocellular carcinoma cell lines pulsed DC is beneficial for the differentiation and maturation of DCs but inhibits the secretion of the IL-12 cytokine. Consequently, we developed a complementary immunotherapy approach by using Tex as an antigen loaded onto DCs, in combination with the cytokine IL-12 to induce antigen-specific cytotoxic T lymphocytes （CTLs）. The results indicated that the combination of DC-Tex and IL-12 was more effective in stimulating T lymphocyte proliferation, releasing IFN-γ， and enhancing cytotoxicity compared to using exosomes or IL-12 alone. Additionally, the inclusion of IL-12 also compensated for the reduced IL-2 secretion by DCs caused by Tex. Moreover, in a BALB/c nude mice model of hepatocellular carcinoma, CTLs induced by DC-Tex combined with IL-12 maximized the tumor-specific T-cell immune effect and suppressed tumor growth. Thus, Tex provides a novel and promising source of antigens, with cytokines compensating for the shortcomings of Tex as a tumor antigen. This work helps to clarify the role of exosomes in tumor immunotherapy and may offer a safe and effective prospective strategy for the clinical application of exosome-based cellular immunotherapy.

Establishing Salvia miltiorrhiza-Derived Exosome-like Nanoparticles and Elucidating Their Role in Angiogenesis.

Exosomes are multifunctional, cell-derived nanoscale membrane vesicles. Exosomes derived from certain mammalian cells have been developed as angiogenesis promoters for the treatment of myocardial ischemia-reperfusion injury, as they possess the capability to enhance endothelial cell proliferation, migration, and angiogenesis. However, the low yield of exosomes derived from mammalian cells limits their clinical applications. Therefore, we chose to extract exosome-like nanoparticles from the traditional Chinese medicine Salvia miltiorrhiza, which has been shown to promote angiogenesis. Salvia miltiorrhiza-derived exosome-like nanoparticles offer advantages, such as being economical, easily obtainable, and high-yielding, and have an ideal particle size, Zeta potential, exosome-like morphology, and stability. Salvia miltiorrhiza-derived exosome-like nanoparticles can enhance the cell viability of Human Umbilical Vein Endothelial Cells and can promote cell migration and improve the neovascularization of the cardiac tissues of myocardial ischemia-reperfusion injury, indicating their potential as angiogenesis promoters for the treatment of myocardial ischemia-reperfusion injury.

Effect of extracellular matrices on production and potency of mesenchymal stem cell-derived exosomes.

Mesenchymal stem cell (MSC) derived exosomes have emerged as potential acellular therapeutics for various tissue regenerative applications. However, successful clinical translation of exosome-based therapy is limited by lack of a structured production platform. Thus, in this study, the effect of decellularized extracellular matrix (dECM) was assessed on the production and potency of exosomes secreted by bone marrow-derived human MSCs. The results indicate that there was a ∼2-fold increase in MSC-exosome production when MSCs were cultured on dECM compared to TCP. Further, our study revealed that dECM generation induced by ascorbic acid (AA) up to 100µgmL-1 highly increased exosome yield thereby indicating a potential scale up method for MSC exosome production. The bioactivity of exosomes was investigated by their ability to improve the healing of wounded human skin explants. Wound closure was enhanced in the presence of exosomes isolated from MSCs cultured on ascorbic acid-induced dECM compared to TCP generated MSC-exosomes. In summary, this study suggests a promising solution to a major bottleneck in large-scale production of MSC exosomes for cell-free therapy.

Pathogenic and therapeutic role of exosomes in neurodegenerative disorders.

Neurodegenerative disorders affect millions of people worldwide, and the prevalence of these disorders is only projected to rise as the number of people over 65 will drastically increase in the coming years. While therapies exist to aid in symptomatic relief, effective treatments that can stop or reverse the progress of each neurodegenerative disease are lacking. Recently, research on the role of extracellular vesicles as disease markers and therapeutics has been intensively studied. Exosomes, 30-150 nm in diameter, are one type of extracellular vesicles facilitating cell-to-cell communication. Exosomes are thought to play a role in disease propagation in a variety of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Accordingly, the exosomes derived from the patients are an invaluable source of disease biomarkers. On the other hand, exosomes, especially those derived from stem cells, could serve as a therapeutic for these disorders, as seen by a rapid increase in clinical trials investigating the therapeutic efficacy of exosomes in different neurological diseases. This review summarizes the pathological burden and therapeutic approach of exosomes in neurodegenerative disorders. We also highlight how heat shock increases the yield of exosomes while still maintaining their therapeutic efficacy. Finally, this review concludes with outstanding questions that remain to be addressed in exosomal research.

Investigate the efficacy of size exclusion chromatography for the isolation of extracellular vesicles from C. elegans

Isolation of Extracellular Vesicles (EVs) has been done extensively in the past using ultracentrifugation, a recent shift has been observed towards precipitation, and exosome isolation kits. These methods often co-elute contaminants of similar size and density which makes their detection and downstream applications quite challenging. As well as the EV yield is also compromised in some methodologies due to aggregate formation. In recent reports, size-exclusion chromatography (SEC) is replacing density gradient-based ultracentrifugation as the gold standard of exosome isolation. It outperforms in yield, purity and does not account for any physical damage to the EVs.We have standardized the methodology for an efficient pure yield of homogenous exosomes of size even smaller than 75 nm in Caenorhabditis elegans homogenate. The paper entails the application and optimization of EV isolation by SEC based on previous studies by optimizing bed size and type of sepharose column employed. We propose that this method is economically feasible in comparison with currently available approaches. A comparative study was conducted to investigate the performance of CL-6B in relation to CL-2B and further, this was combined with ultracentrifugation for higher efficacy. The methodology could be introduced in a clinical setting due to its therapeutic potential and scope. The eluted EVs were studied by flow cytometry, nanotracking and characterized for size and morphology.

Thermostable Basic Fibroblast Growth Factor Enhances the Production and Activity of Human Wharton's Jelly Mesenchymal Stem Cell-Derived Extracellular Vesicles.

Wharton's jelly-derived mesenchymal stem cell (WJ-MSC)-derived exosomes contain a diverse cargo and exhibit remarkable biological activity, rendering them suitable for regenerative and immune-modulating functions. However, the quantity of secretion is insufficient. A large body of prior work has investigated the use of various growth factors to enhance MSC-derived exosome production. In this study, we evaluated the utilization of thermostable basic fibroblast growth factor (TS-bFGF) with MSC culture and exosome production. MSCs cultured with TS-bFGF displayed superior proliferation, as evidenced by cell cycle analysis, compared with wild-type bFGF (WT-bFGF). Stemness was assessed through mRNA expression level and colony-forming unit (CFU) assays. Furthermore, nanoparticle tracking analysis (NTA) measurements revealed that MSCs cultured with TS-bFGF produced a greater quantity of exosomes, particularly under three-dimensional culture conditions. These produced exosomes demonstrated substantial anti-inflammatory and wound-healing effects, as confirmed by nitric oxide (NO) assays and scratch assays. Taken together, we demonstrate that utilization of TS-bFGF for WJ-MSC-derived exosome production not only increases exosome yield but also enhances the potential for various applications in inflammation regulation and wound healing.

3D-exosomes laden multifunctional hydrogel enhances diabetic wound healing via accelerated angiogenesis

Impaired vascular networks, local insufficiency of neovascularization, and tissue inflammation caused by the accumulation of reactive oxygen species (ROS) and bacterial infections contribute to the delayed healing of wounds in patients with diabetes. Thus, an urgent need is to develop effective diabetic wound treatments that promote angiogenesis, inhibit bacterial infections, and reduce oxidative damage and tissue inflammation. Exosome therapy promotes angiogenesis and wound healing. However, traditional exosomes have limitations such as low yield and rapid release. Compared with two-dimensional (2D) cell culture, three-dimensional (3D) cell culture results in a higher yield of exosomes and better healing effects. In this study, we designed a multifunctional hydrogel with 3D-exosome-sustained release features to improve diabetic wound healing. Chitosan-grafted-dihydrocaffeic acid (CS-DA) and benzaldehyde-terminated Pluronic®F127 (PF127-CHO) were combined using a dynamic Schiff base bond to form a dynamic hydrogel network and simultaneously fused tannic acid (TA) and 3D adipose-derived mesenchymal stem cells-derived exosomes (3D ADSCs-Exos, referred to in this study as 3D-Exo). The phosphoric acid groups of 3D-Exo combine with the polyphenol groups of DA/TA through reversible interactions, enabling the sustained release of 3D-Exo. The CS-DA/PF/TA/3D-Exo hydrogel exhibited tissue-adhesive, self-healing, antibacterial, anti-inflammatory, and antioxidant properties. The CS-DA/PF/TA/3D-Exo hydrogel significantly accelerated the recovery of diabetic wounds by promoting angiogenesis and collagen deposition in vivo. Furthermore, rapid hemostasis was achieved owing to the wet tissue adhesion and platelet activation of the hydrogel. In general, this multifunctional hydrogel could be used as a wound dressing for diabetic wound management.

Harvesting stem cell exosomes from herringbone microfluidic bioreactor for wound healing

Stem cell exosomes have attracted increasing attention in effective wound treatment, while their mass production are still challenging. In this paper, we proposed a novel herringbone microfluidic bioreactor with cell microcarriers integration for batch-preparation of stem cell exosomes. The microfluidic device was designed with parallel herringbone microgrooves on the top and a microcolumn array at the bottom, which provided a controllable fluidic environment for stem cells culturing on the gelatin methacryloyl (GelMA) microcarriers and for exosomes secretion from these cells. Thus, it was demonstrated that the yield of exosomes by this bioreactor was much higher compared with traditional flask cell culture. Besides, bone mesenchymal stem cells (BMSCs) exosomes harvested from the bioreactor under fluidic stimulation expressed an excellent promoting effect on wound healing. These features indicated that the present microfluidic chip integrated with microcarriers possesses great potential for harvesting exosomes for different biomedical applications.

An injectable, activated neutrophil-derived exosome mimetics/extracellular matrix hybrid hydrogel with antibacterial activity and wound healing promotion effect for diabetic wound therapy

Chronic diabetic wounds are primarily caused by infection, inflammation, and angiogenesis-related disorders. An ideal approach for treating chronic diabetic wounds is by combining anti-infection strategies, immune microenvironment regulation, and angiogenesis promotion. Vascular endothelial growth factor (VEGF) can promote the proliferation and migration of vascular endothelial cells, thereby promoting angiogenesis. However, the low stability and inability to target lesions limit its application. Polymorphonuclear neutrophil-derived exosomes (PMNExo) exhibit good delivery properties and can be used for the therapeutic delivery of VEGF. Furthermore, they retain the antibacterial ability of polymorphonuclear neutrophils (PMNs). Nonetheless, low PMNExo generation impedes its therapeutic applications. In this study, we prepared exosome mimetics (EM) from PMNs using the extrusion process; as a result, exosome yield significantly improved. To increase the residence of exosomes, an extracellular matrix (ECM) hydrogel, a thermosensitive material that can function as an in situ gel in vivo, was used as an exosome carrier. The active peptides in the ECM regulated the immune microenvironment of the wound. In summary, we loaded ECM with VEGF-encapsulated activated neutrophil exosome mimetics (aPMNEM) to develop VEGF–aPMNEM–ECM hybrid hydrogel for treating chronic wounds. The hydrogel accelerates the regeneration of chronic diabetic wounds. Our study provides a prospective therapy platform involving cytokines for treating different diseases.Graphical

Abstract P3021: Circulating Exosomal MicroRNAs As Biomarkers For Pulmonary Embolism

Pulmonary Embolism (PE) is the third leading cause of death worldwide. Current treatment for PE, including thrombolytics and anticoagulants, have serious side effects. Protein and RNA biomarkers presently used to diagnose PE do not have the necessary specificity or sensitivity. Therefore, discovery of predictive biomarkers for therapeutic effects and adverse outcomes is imperative. MicroRNAs (miRNAs) are known to regulate gene expression networks and their expression levels reflect the physiological state of their cell of origin. By investigating these circulating miRNAs, we can gain valuable insight into the state of the damaged cells or tissue. Here, we innovated a novel methodology to isolate exosomes from patient platelet-free plasma that provides better yield of both exosomes and their RNA content. We are the first to stratify the PE patient samples according to clinical severity (low-risk, sub-massive, massive PE) and analyze the correlations of the exosomal miRNA biomarkers with PE severity. We found that exosomes from PE patients are more heterogeneous, lower in concentration and larger in size compared to healthy controls. Further, exosomes from PE patient samples pack more RNA than healthy controls. With the results from ongoing miRNA sequencing, we will be able to identify miRNAs and other short non-coding RNAs as potential diagnostic biomarkers that best correlate with PE severity. Validation and predictive studies using qRT-PCR will push the frontiers on circulating small RNA biomarkers in PE and other cardiovascular diseases. Finally, network analysis on the potential RNA biomarkers will provide information for future studies on disease mechanisms and therapeutics development.

Isolation of Bovine Milk Exosome Using Electrophoretic Oscillation Assisted Tangential Flow Filtration with Antifouling of Micro-ultrafiltration Membrane Filters

Tangent flow-driven ultrafiltration (TF-UF) is an efficient isolation process of milk exosomes without morphological deformation. However, the TF-UF approach with micro-ultrafiltration SiNx membrane filters suffers from the clogging and fouling of micro-ultrafiltration membrane filter pores with large bioparticles. Thus, it is limited in the long term, continuous isolation of large quantities of exosomes. In this work, we introduced electrophoretic oscillation (EPO) in the TF-UF approach to remove pore clogging and fouling of with micro-ultrafiltration SiNx membrane filters by large bioparticles. As a result, the combined EPO-assisted TF (EPOTF) filtration can isolate large quantities of bovine milk exosomes without deformation. Furthermore, several morphological and biological analyses confirmed that the EPOTF filtration approach could isolate the milk exosomes in high concentrations with high purity and intact morphology. In addition, the uptake test of fluorescent-labeled exosomes by the keratinocyte cells visualized the biological function of purified exosomes. Hence, compared to the TF-UF process, the EPOTF filtration produced a higher yield of bovine milk exosomes without stopping the filtering process for over 200 h. Therefore, this isolation process enables scalable and continuous production of morphologically intact exosomes from bovine milk, suggesting that high-quality exosome purification is possible for future applications such as drug nanocarriers, diagnosis, and treatments.

Three-dimensional-cultured MSC-derived exosome with hydrogel for cerebral ischemia repair

Microglia-mediated neuroinflammatory response, one of the most essential pathological processes of cerebral ischemia-reperfusion (I/R) injury, is acknowledged as the main factors leading to poor prognosis of cerebral ischemia. Exosome derived from mesenchymal stem cell (MSC-Exo) exhibits neuroprotective functions by reducing cerebral ischemia-induced neuroinflammatory response and promoting angiogenesis. However, MSC-Exo has disadvantages such as insufficient targeting capability and low production, which limits their clinical applications. Here, we fabricated gelatin methacryloyl (GelMA) hydrogel for three-dimensional (3D) culture of MSCs. It is indicated that 3D environment could simulate the biological niches of MSCs, thereby significantly increasing the cell stemness of MSCs and improving the yield of MSCs-derived exosomes (3D-Exo). In this study, we utilized the modified Longa method to induce middle cerebral artery occlusion (MCAO) model. Additionally, in vitro and in vivo studies were conducted to interrogate the mechanism of the stronger neuroprotective effect of 3D-Exo. Furthermore, the administration of 3D-Exo in MCAO model could promote neovascularization in infarct region and result in a significant suppression of inflammatory response. This study proposed an exosome-based targeting delivery system for cerebral ischemia and provided a promising strategy for efficient and large-scale production of MSC-Exo.

Treatment with Mesenchymal Stem Cell-Derived Nanovesicle-Containing Gelatin Methacryloyl Hydrogels Alleviates Osteoarthritis by Modulating Chondrogenesis and Macrophage Polarization.

Osteoarthritis is a degenerative disorder that can severely affect joints, and new treatment strategies are urgently needed. Administration of mesenchymal stem cell (MSC)-derived exosomes is a promising therapeutic strategy in osteoarthritis treatment. However, the poor yield of exosomes is an obstacle to the use of this modality in the clinic. Herein, a promising strategy is developed to fabricate high-yield exosome-mimicking MSC-derived nanovesicles (MSC-NVs) with enhanced regenerative and anti-inflammatory capabilities. MSC-NVs are prepared using an extrusion approach and are found to increase chondrocyte and human bone marrow MSC differentiation, proliferation, and migration, in addition to inducing M2 macrophage polarization. Furthermore, gelatin methacryloyl (GelMA) hydrogelsloaded with MSC-NVs (GelMA-NVs) are formulated, which exhibitsustained release of MSC-NVs and are shownto be biocompatible with excellent mechanical properties. In a mouse osteoarthritis model constructed by surgical destabilization of the medial meniscus (DMM), GelMA-NVseffectively ameliorate osteoarthritisseverity, reduce the secretion of catabolic factors, and enhance matrix synthesis. Furthermore, GelMA-NVs induce M2 macrophage polarization and inflammatory response inhibition in vivo. The findings demonstrate that GelMA-NVshold promise for osteoarthritis treatment through modulation of chondrogenesis and macrophage polarization.

An optimized exosome production strategy for enhanced yield while without sacrificing cargo loading efficiency

BackgroundExosome mediated mRNA delivery is a promising strategy for the treatment of multiple diseases. However, the low yield of exosomes is a bottleneck for clinical translation. In this study, we boosted exosome production via simultaneously reducing the expression of genes inhibiting exosome biogenesis and supplementing the culture medium with red cell membrane components.ResultsAmong the candidate genes, knocking down of Rab4 was identified to have the highest efficacy in promoting exosome biogenesis while without any obvious cytotoxicity. Additionally, supplementing red cell membrane particles (RCMPs) in the culture medium further promoted exosome production. Combination of Rab4 knockdown and RCMP supplement increased exosome yield up to 14-fold. As a proof-of-concept study, low-density lipoprotein receptor (Ldlr) mRNA was forced expressed in the exosome donor cells and passively encapsulated into the exosomes during biogenesis with this strategy. Though exosome production per cell increased, the booster strategy didn’t alter the loading efficiency of therapeutic Ldlr mRNA per exosome. Consistently, the therapeutic exosomes derived by the strategy alleviated liver steatosis and atherosclerosis in Ldlr−/− mice, similar as the exosomes produced by routine methods.ConclusionsTogether, the proposed exosome booster strategy conquers the low yield bottleneck to some extent and would certainly facilitate the clinical translation of exosomes.

Exosome mimetics derived from bone marrow mesenchymal stem cells ablate neuroblastoma tumor in vitro and in vivo

PurposeTo develop exosome-mimetics derived from bone marrow mesenchymal stem cells (EM) as a novel nanoscale drug delivery system(nanoDDS) with improved tumor targeting activity, therapeutic effect, and biosafety, and to evaluate the therapeutic effect of doxorubicin loaded EM (EM-Dox) on neuroblastoma (NB) in vitro and in vivo. MethodsEM was prepared by serial extrusion of bone marrow mesenchymal stem cells (BMSCs), ammonium sulfate gradient method was used to promote the active loading of doxorubicin, and EM-Dox was obtained after removal of free doxorubicin by dialysis. The obtained EM and EM-Dox were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), Western Blot assay(WB), and the yield of exosomes and EM was further compared. Confocal fluorescent microscopy was used to verify the uptake of EM-Dox and free doxorubicin (Free-Dox) by NB cells. CCK-8 assay, cell cycle assay, and cell apoptosis assay were used to evaluate the antitumor effect of EM-Dox on NB cells in vitro. In addition, the targeted therapeutic effect and biosafety of EM-Dox against NB were evaluated in tumor-bearing nude mice. ResultsTEM, NTA, and WB verified that both EM and EM-Dox feature highly similar morphology, size and marker protein expression in comparison with naturally occurred exosomes, but the particle size of EM-Dox increased slightly after loading doxorubicin. The protein yield and particle yield of EM-Dox were 16.8 and 26.3-folds higher than those of exosomes, respectively. Confocal fluorescent microscopy showed that EM and doxorubicin had a definite co-localization. EM-Dox was readily internalized in two well-established human NB cell lines. The intracellular content of doxorubicin in cells treated with EM-Dox was significantly higher than that treated with Free-Dox. CCK-8 assay and flow cytometry confirmed that EM-Dox could inhibit NB cell proliferation, induce G2/M phase cell cycle arrest, and promote NB cell apoptosis in vitro. In vivo bioluminescence imaging results demonstrated that EM-Dox effectively targets NB tumors in vivo. Compared with Free-Dox, EM-Dox had a significantly increased inhibitory effect against NB tumor proliferation and progression in vivo, without inducing any myocardial injury. ConclusionsEM-Dox showed significantly increased anti-tumor activity in comparison with free doxorubicin in vitro and in vivo, and scalable EMs may represent a new class of NanoDDS that can potentially replace naturally occurred exosomes in preclinical or clinical translations.

Crosstalk between Oxidative Stress and Exosomes.

Mammals have several organs comprising various cells with different functions. Furthermore, eukaryotic cells are compartmentalized into functionally distinct organelles. Thus, for good organismal health, exosomes, which play an important role in cell-to-cell communication, interact closely with oxidative stress. Oxidative stress, which is recognized as a type of intracellular second signal, is aggravated by reactive species. As a subtype of reactive species, reactive oxygen species (ROS) can be produced on the extracellular face of the plasma membrane by NADPH oxidases, via the mitochondrial electron transport chain, in peroxisomes, and in the lumen of the endoplasmic reticulum. The scavenging of ROS is mainly dependent on peroxiredoxins, including GSH peroxidases, peroxiredoxins 3 and 5, and thioredoxin reductase. Intracellular ROS increase the number of intracellular multivesicular bodies (MVBs) by restraining their degradation in lysosomes, thereby enhancing the release of exosomes under the synergy of the depletion of exofacial GSH, which can be regulated by oxidative stress. In contrast, higher ROS levels can decrease the yield of exosomes by activating cellular autophagy to degrade MVBs. Moreover, exosomes can transfer the characteristics of parent cells to recipient cells. Here, we review the interaction between oxidative stress and exosomes in the hope of providing insights into their interplay.

Comprehensive proteomic analysis of exosome mimetic vesicles and exosomes derived from human umbilical cord mesenchymal stem cells

BackgroundExosomes derived from mesenchymal stem cells (MSCs) have shown to have effective application prospects in the medical field, but exosome yield is very low. The production of exosome mimetic vesicles (EMVs) by continuous cell extrusion leads to more EMVs than exosomes, but whether the protein compositions of MSC-derived EMVs (MSC-EMVs) and exosomes (MSC-exosomes) are substantially different remains unknown. The purpose of this study was to conduct a comprehensive proteomic analysis of MSC-EMVs and MSC-exosomes and to simply explore the effects of exosomes and EMVs on wound healing ability. This study provides a theoretical basis for the application of EMVs and exosomes.MethodsIn this study, EMVs from human umbilical cord MSCs (hUC MSCs) were isolated by continuous extrusion, and exosomes were identified after hUC MSC ultracentrifugation. A proteomic analysis was performed, and 2315 proteins were identified. The effects of EMVs and exosomes on the proliferation, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) were evaluated by cell counting kit-8, scratch wound, transwell and tubule formation assays. A mouse mode was used to evaluate the effects of EMVs and exosomes on wound healing.ResultsBioinformatics analyses revealed that 1669 proteins in both hUC MSC-EMVs and hUC MSC-exosomes play roles in retrograde vesicle-mediated transport and vesicle budding from the membrane. The 382 proteins unique to exosomes participate in extracellular matrix organization and extracellular structural organization, and the 264 proteins unique to EMVs target the cell membrane. EMVs and exosomes can promote wound healing and angiogenesis in mice and promote the proliferation, migration and angiogenesis of HUVECs.ConclusionsThis study presents a comprehensive proteomic analysis of hUC MSC-derived exosomes and EMVs generated by different methods. The tissue repair function of EMVs and exosomes was herein verified by wound healing experiments, and these results reveal their potential applications in different fields based on analyses of their shared and unique proteins.