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Extracellular vesicles (EVs) play key roles in diverse biological processes, transport biomolecules between cells and have been engineered for therapeutic applications. A useful EV bioengineering strategy is to express engineered proteins on the EV surface to confer targeting, bioactivity and other properties. Measuring how incorporation varies across a population of EVs is important for characterising such materials and understanding their function, yet it remains challenging to quantitatively characterise the absolute number of engineered proteins incorporated at single-EV resolution. To address these needs, we developed a HaloTag-based characterisation platform in which dyes or other synthetic species can be covalently and stoichiometrically attached to engineered proteins on the EV surface. To evaluate this system, we employed several orthogonal quantification methods, including flow cytometry and fluorescence microscopy, and found that HaloTag-mediated quantification is generally robust across EV analysis methods. We compared HaloTag-labelling to antibody-labelling of EVs using single vesicle flow cytometry, enabling us to measure the substantial degree to which antibody labelling can underestimate proteins present on an EV. Finally, we demonstrate the use of HaloTag to compare between protein designs for EV bioengineering. Overall, the HaloTag system is a useful EV characterisation tool which complements and expands existing methods.

ObjectiveExtracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) are promising avenues in regenerative medicine, offering unique immunomodulatory and regenerative properties with lower immunogenicity. This study delves into the distinctive features of EVs extracted from feline adipose-derived MSCs (ASCs) and placenta-derived MSCs (PMSCs). The tissues were collected from 11 female cats aged between 4 and 7 years old.SampleEVs extracted from MSCs from discarded fetal membranes from 7 female cats and SC adipose tissue from 11 cats.MethodsWe comprehensively explored morphological characteristics, mitochondrial density, surface markers, and pro- and anti-inflammatory mediators, uncovering notable differences between ASCs and PMSCs.ResultsMorphologically, ASCs exhibit a spindle-shaped form in contrast to the spherical morphology of PMSCs. Proliferation and clonogenic potential assessments reveal the faster proliferation and robust clonogenic nature of ASCs, suggesting their potential vital role in regenerative processes. Surface marker expression analysis indicates a significantly higher expression of multipotency-associated markers in ASCs, suggesting their superior proregenerative potential. Phenotyping of EVs demonstrates distinctive features, with CD9 expression suggesting varied EV secretion patterns. Notably, PMSCs exhibit superior CD81 expression, indicating their potential as preferred donors of mitochondria. Pro- and anti-inflammatory mediators analyzed at mRNA and microRNA levels reveal higher RNA content in EVs compared to source cells, emphasizing the potential of EVs in directing regenerative processes. Differential microRNA expression in EVs derived from ASCs hints at their regulatory roles in anti-inflammatory and immunometabolic processes.Clinical relevanceThis study lays a foundation for understanding the nuances between ASCs and PMSCs, which is crucial for harnessing the full therapeutic potential of MSCs and their EVs in tissue repair and regeneration.

Glioma stem‐like cell (GSC) inhibition is a promising strategy for glioblastoma therapy. However, it is hampered by two main obstacles: first, delivery barriers, including the blood–brain barrier (BBB) and deep tumor‐penetrating obstacles, hinder sufficient drug accumulation in GSCs that often reside in hypoxic niches deep in glioblastoma; second, hypoxia and the immunosuppressive tumor microenvironment (TME) limit the therapeutic responses of GSC inhibition strategies. An intercellular cell adhesion molecule 1 (ICAM‐1)‐expressing M1 macrophage‐derived small extracellular vesicles loaded with a complex of bovine serum albumin, MnO2, and the small‐molecule inhibitor PTC209 (IsEV‐BMP) is constructed to achieve sequential barrier‐breaking delivery and simultaneous TME modulation to boost GSC inhibition. ICAM‐1 facilitates endothelial transcytosis‐mediated BBB crossing, microglial transcytosis‐mediated deep tumor penetration, and internalization of IsEV‐BMPs in GSCs. Hypoxia alleviation inhibits the binding of hypoxia‐inducible factor 1α (HIF‐1α) to hypoxia‐responsive elements on the gene promoters of cancer stem cell‐related markers and CD47 in GSCs, thereby improving PTC209 sensitivity and immune phagocytosis of GSCs. IsEV‐BMP achieves simultaneous microglial polarity transition and immuno‐microenvironmental modulation during microglial transcytosis‐mediated deep tumor penetration. This study provides a strategy to overcome the sequential delivery barrier for effective glioblastoma treatment. [ABSTRACT FROM AUTHOR]

Naturally occurring cell-derived extracellular vesicles (EVs) have emerged as attractive nanocarriers for drug delivery. However, production of large quantities of EVs for clinical applications in a scalable manner remains a significant challenge. This study investigated at the single cell level how sonoporation, or membrane poration produced by ultrasound-induced microbubble cavitation, impacts EV production using mouse macrophage RAW 264.7 cells stably expressing CD63-GFP as a model system. Real-time fluorescence videomicroscopy detected rapid changes in CD63-GFP, a tetraspanin family member highly enriched in intraluminal vesicles tagged with GFP, to track changes in multivesicular bodies (MVBs), which are the cellular compartments where exosomes originate within the cells. Our results revealed distinct dynamic changes in CD63-GFP intensity and distribution in RAW 264.7 cells in terms of response time and duration depending on whether the cells were directly or indirectly impacted by sonoporation, suggesting reorganization of MVBs in response to direct and indirect mechanisms resulted from the mechanical impact of ultrasound pulse on the cells. Analysis of the supernatant from sonoporation-treated RAW 264.7 cells expressing CD63-GFP demonstrated a delayed and sustained increase in the production of CD63-GFP-positive EVs. These results show the robust and detailed effect of sonoporation and reveal insights into sonoporation-induced EV release useful for guiding the application of sonoporation to enhance large-scale EV production. [ABSTRACT FROM AUTHOR]

Background: In mucopolysaccharidosis type III (MPS III, also known as Sanfilippo syndrome), a pediatric neurodegenerative disorder, accumulation of abnormal glycosaminoglycans (GAGs) induces severe neuroinflammation by triggering the microglial pro-inflammatory cytokines production via a TLR4-dependent pathway. But the extent of the microglia contribution to the MPS III neuropathology remains unclear. Extracellular vesicles (EVs) mediate intercellular communication and are known to participate in the pathogenesis of adult neurodegenerative diseases. However, characterization of the molecular profiles of EVs released by MPS III microglia and their effects on neuronal functions have not been described. Methods: Here, we isolated EVs secreted by the microglial cells after treatment with GAGs purified from urines of Sanfilippo patients (sfGAGs-EVs) or from age-matched healthy subjects (nGAGs-EVs) to explore the EVs' proteins and small RNA profiles using LC–MS/MS and RNA sequencing. We next performed a functional assay by immunofluorescence following nGAGs- or sfGAGs-EVs uptake by WT primary cortical neurons and analyzed their extensions metrics after staining of βIII-tubulin and MAP2 by confocal microscopy. Results: Functional enrichment analysis for both proteomics and RNA sequencing data from sfGAGs-EVs revealed a specific content involved in neuroinflammation and neurodevelopment pathways. Treatment of cortical neurons with sfGAGs-EVs induced a disease-associated phenotype demonstrated by a lower total neurite surface area, an impaired somatodendritic compartment, and a higher number of immature dendritic spines. Conclusions: This study shows, for the first time, that GAGs from patients with Sanfilippo syndrome can induce microglial secretion of EVs that deliver a specific molecular message to recipient naive neurons, while promoting the neuroinflammation, and depriving neurons of neurodevelopmental factors. This work provides a framework for further studies of biomarkers to evaluate efficiency of emerging therapies. [ABSTRACT FROM AUTHOR]

Single-vesicle molecular profiling of cancer-associated extracellular vesicles (EVs) is increasingly being recognized as a powerful tool for cancer detection and monitoring. Mask and target dual imaging is a facile method to quantify the fraction of the molecularly targeted population of EVs in biofluids at the single-vesicle level. However, accurate and efficient dual imaging vesicle analysis has been challenging due to the interference of false signals on the mask images and the need to analyze a large number of images in clinical samples. In this work, we report a fully automatic dual imaging analysis method based on machine learning and use it with dual imaging single-vesicle technology (DISVT) to detect breast cancer at different stages. The convolutional neural network Resnet34 was used along with transfer learning to produce a suitable machine learning model that could accurately identify areas of interest in experimental data. A combination of experimental and synthetic data were used to train the model. Using DISVT and our machine learning-assisted image analysis platform, we determined the fractions of EpCAM-positive EVs and CD24-positive EVs over captured plasma EVs with CD81 marker in the blood plasma of pilot HER2-positive breast cancer patients and compared to those from healthy donors. The amount of both EpCAM-positive and CD24-positive EVs was found negligible for both healthy donors and Stage I patients. The amount of EpCAM-positive EVs (also CD81-positive) increased from 18% to 29% as the cancer progressed from Stage II to III. No significant increase was found with further progression to Stage IV. A similar trend was found for the CD24-positive EVs. Statistical analysis showed that both EpCAM and CD24 markers can detect HER2-positive breast cancer at Stages II, III, or IV. They can also differentiate individual cancer stages except those between Stage III and Stage IV. Due to the simplicity, high sensitivity, and high efficiency, the DISVT with the AI-assisted dual imaging analysis can be widely used for both basic research and clinical applications to quantitatively characterize molecularly targeted EV subtypes in biofluids. [ABSTRACT FROM AUTHOR]

Migrasomes are recently identified extracellular vesicles and organelles formed in conjunction with cell migration. They are situated at the rear of migrating cells, exhibit a circular or elliptical membrane-enclosed structure, and function as a new organelle. Migrasomes selectively sort intercellular components, mediating a cell migration-dependent release mechanism known as migracytosis and modulating cell–cell communication. Accumulated evidence clarifies migrasome formation processes and indicates their diverse functional roles. Migrasomes may also be potentially correlated with the occurrence, progression, and prognosis of certain diseases. Migrasomes' involvement in physiological and pathological processes highlights their potential for expanding our understanding of biological procedures and as a target in clinical therapy. However, the precise mechanisms and full extent of their involvement in immunity, barriers, and diseases remain unclear. This review aimed to provide a comprehensive overview of the roles of migrasomes in human immunity and barriers, in addition to providing insights into their impact on human diseases. Migrasomes, newly identified extracellular vesicles and organelles, form during cell migration and are located at the rear of migrating cells. These circular or elliptical structures mediate migracytosis, selectively sorting intercellular components and modulating cell–cell communication. Evidence suggests diverse functional roles for migrasomes, including potential links to disease occurrence, progression, and prognosis. Their involvement in physiological and pathological processes highlights their significance in understanding biological procedures and potential clinical therapies. However, their exact mechanisms in immunity, barriers, and diseases remain unclear. This review provides an overview of migrasomes' roles in human immunity and barriers, and their impact on diseases. [Display omitted] [ABSTRACT FROM AUTHOR]

Traumatic brain injury (TBI) is a prevalent and debilitating condition, which often leads to the development of post-traumatic epilepsy (PTE), a condition that yet lacks preventive strategies. Biperiden, an anticholinergic drug, is a promising candidate that has shown efficacy in murine models of PTE. MicroRNAs (miRNAs), small regulatory RNAs, can help in understanding the biological basis of PTE and act as TBI- and PTE-relevant biomarkers that can be detected peripherally, as they are present in extracellular vesicles (EVs) that cross the blood–brain barrier. This study aimed to investigate miRNAs in serum EVs from patients with TBI, and their association with biperiden treatment and PTE. Blood samples of 37 TBI patients were collected 10 days after trauma and treatment initiation in a double-blind clinical trial. A total of 18 patients received biperiden, with three subjects developing PTE, and 19 received placebo, with two developing PTE. Serum EVs were characterized by size distribution and protein profiling, followed by high-throughput sequencing of the EV miRNome. Differential expression analysis revealed no significant differences in miRNA expression between TBI patients with and without PTE. Interestingly, miR-9-5p displayed decreased expression in biperiden-treated patients compared to the placebo group. This miRNA regulates genes enriched in stress response pathways, including axonogenesis and neuronal death, relevant to both PTE and TBI. These findings indicate that biperiden may alter miR-9-5p expression in serum EVs, which may play a role in TBI resolution. [ABSTRACT FROM AUTHOR]

Embryo survival and pre‐implantation development depend on uterine luminal fluid, which is believed to play a role in early embryonic death and infertility in cows. Extracellular vesicles (EVs) in the uterine luminal fluid contain microRNAs (miRNAs), crucial mediators of intercellular communication. miRNAs regulate conceptus–maternal interactions and participate in embryonic development by suppressing gene expression. Therefore, we hypothesized that miRNAs in the intrauterine EVs of low‐fertility cows would hinder embryonic survival and development. EVs were collected from the bovine uterine luminal fluid of both normal‐ and low‐fertility cows 7 days post‐estrus. Small RNA‐sequencing analysis of miRNAs isolated from these EVs identified eight miRNAs that were highly expressed in normal‐fertility cows (normal‐fertility miRNAs) and eight with elevated expression in low‐fertility cows (low‐fertility miRNAs). These two sets of miRNAs were transfected into hatched blastocysts via lipofection. RNA‐seq following lipofection with low‐fertility miRNAs identified 424 differentially expressed genes (DEGs) relative to the control; in contrast, following lipofection with normal‐fertility miRNAs, seven DEGs were identified. Pathway analysis of the DEGs identified following lipofection with low‐fertility miRNAs revealed substantial enrichment of mitogen‐activated protein kinase (MAPK) signaling. Expression of activator protein 1 (AP1) and interferon‐tau (IFNT) mRNA was significantly lower in the low‐fertility miRNA transfection group than in the control. IFNT is essential for maternal pregnancy recognition. Therefore, miRNAs in intrauterine EVs from low‐fertility cows at 7 days post‐estrus may inhibit embryo development and suppress IFNT expression by altering MAPK signaling. [ABSTRACT FROM AUTHOR]

Recent studies have shown that extracellular vesicles (EVs) secreted by various parasites are capable of modulating the host's innate immune responses, such as by altering macrophage (Mϕ) phenotypes and functions. Studies have shown that Mϕ promote early host responses to amoebic infection by releasing proinflammatory cytokines that are crucial to combating amoebiasis. Here, we are reporting for the first time the effect of EVs released by Entamoeba histolytica (EhEVs) on human THP‐1 differentiated Mϕ (THP‐1 Mϕ). We show that the EhEVs are internalized by THP‐1 Mϕ which leads to differential regulation of various cytokines associated with both M1 and M2 Mϕ. We also saw that EhEV treatment thwarted Type 2 immune‐response‐related transcriptome pSTAT6 in the THP‐1 Mϕ. Furthermore, EhEVs stimulated Mϕ to reduce their energy demand by suppressing oxidative phosphorylation (OXPHOS) and adenosine triphosphate (ATP) production. Hence, the human parasite E. histolytica–derived EVs are capable of eliciting an immune response from Mϕ that may contribute to overall infection status. [ABSTRACT FROM AUTHOR]

With the severity of chronic kidney disease worldwide, strategies to recover renal function via tissue regeneration provide alternatives to kidney replacement therapy. To exclude side effects from direct cell transplantation, extracellular vesicles (EVs) are great substitutes representing paracrine cell signaling. To build three-dimensional structures for implantation into the 5/6 nephrectomy model by incorporating bioactive materials, including multifunctional EVs (mEVs), porous PMEZE/mEV scaffolds were developed in combination with edaravone (EDV; E) and mEV based on PMEZ scaffolds with PLGA (P), MH-RA (M), ECM (E), ZnO-ALA (Z). The oxygen free radical scavenger EDV was incorporated to induce tubular regeneration. mEVs were engineered to serve regenerative activities with a combination of two EVs from SDF-1α overexpressed tonsil-derived mesenchymal stem cells (sEVs) and intermediate mesoderm (IM) cells during differentiation into kidney progenitor cells (dEVs). mEVs displayed beneficial effects on regeneration by facilitating migration and inducing differentiation of surrounding stem cells, and EDV improved kidney function by regulating the GDNF/RET pathway and their downstream genes. The promotion of MSC recruitment was confirmed with sEV particles number dependently, and the regulation of the GDNF/RET pathway by the effect of EDV and its enhanced effect by mEVs were elucidated using in vitro analysis. The regeneration of tubules was additionally demonstrated through the increased expression of aquaporin-1 (AQP-1) and cadherin-16 (CDH16) for proximal tubules, and calbindin and PAX2 for distal tubules in the renal defect model. With these, structural regeneration and functional recovery were achieved with kidney regeneration in the 5/6 nephrectomy mice model. [ABSTRACT FROM AUTHOR]

Background: Primary ovarian insufficiency (POI) manifests with hormonal imbalances, menstrual irregularities, follicle loss, and infertility. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are emerging as a promising treatment for POI. This systematic review aims to assess the effects of MSC-EVs on follicle number, hormonal profile, and fertility in POI animal models. Methods: A systematic search of PubMed, Scopus, and Web of Science databases up to December 14th, 2023 was conducted. Two reviewers independently conducted screening, risk of bias assessment, and data extraction. Meta-analysis was performed to analyze treatment versus control outcomes using a random effects model. Publication bias was assessed using Egger's regression test and sensitivity analysis was assessed using the leave-one-out method. Subgroup analyses and meta-regressions were conducted based on EV source, induction model, type of animal, study quality, administration route, administration frequency and route, and dose. Results: a total of 29 studies were included. MSC-EVs treatment significantly increased total follicle count (SMD, (95CI), p-value; 3.56, (0.91, 6.21), < 0.001), including primordial (SMD, (95CI), p-value; 2.86, (1.60, 4.12), < 0.001), primary (SMD, (95CI), p-value; 3.17, (2.28, 4.06), < 0.001), mature (SMD, (95CI), p-value; 2.26, (1.02, 3.50), < 0.001), and antral follicles (SMD, (95CI), p-value; 2.44, (1.21, 3.67), < 0.001). Administration frequency and route did not affect this outcome, but EV source affected primordial, primary, secondary and antral follicle count. Additionally, MSC-EVs treatment elevated anti-müllerian hormone (SMD, (95CI); 3.36, (2.14, 4.58)) and estradiol (SMD, (95CI); 3.19, (2.20, 4.17)) levels while reducing follicle stimulating hormone levels (SMD, (95CI); -2.68, (-4.42, -0.94)). Unlike EV source, which had a significant impact on all three hormones, administration frequency, route, and EV dose did not affect this outcome. Moreover, treatment increased offspring number (SMD, (95CI); 3.70, (2.17, 5.23)) and pregnancy odds (OR, (95CI); 10.25, (4.29, 24.46)) compared to controls. Publication bias and a high level of heterogeneity was evident in all analyses, except for the analysis of the pregnancy odds. However, sensitivity analysis indicated that all of the analyses were stable. Conclusion: MSC-EVs therapy shows promise for POI treatment, potentially facilitating clinical translation. However, Further research is warranted to optimize methodology and assess side effects. [ABSTRACT FROM AUTHOR]

Background: Parascaris spp. represent a significant threat to equine health worldwide, particularly in foals. The long-term survival of parasites in the host necessitates persistent modulation of the host immune response. Intercellular communication achieved through the exchange of molecules via extracellular vesicles (EVs) released from the parasite could be a crucial factor in this regard. This study aimed to isolate and characterize EVs released by adult male and female Parascaris worms and conduct a proteomic analysis to identify sex-specific proteins and potential immunomodulatory factors. Methods: Live adult Parascaris worms were collected, and EVs were isolated from spent culture media using differential ultracentrifugation. Nanoparticle tracking analysis and transmission electron microscopy confirmed the size, concentration, and morphology of the isolated EVs. Proteins within the isolated EVs were analyzed using mass spectrometry-based proteomics (LC–MS/MS). Results: Proteomic analysis revealed a total of 113 proteins in Parascaris EVs, with several proteins showing homology to known helminth exosome proteins and exhibiting immunomodulatory functions. Sex-specific differences in EV protein composition were observed, with a distinct abundance of C-type lectins in female EVs, suggesting potential sex-specific roles or regulation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed metabolic pathways shared between male and female Parascaris EVs, as well as differences in signal transduction, and cell growth and death pathways, indicating sex-specific variations. Conclusions: These findings imply that Parascaris EVs and their protein cargo are complex. This data potentially opens avenues for discovering innovative approaches to managing and understanding helminth infection. [ABSTRACT FROM AUTHOR]

Background: Ovarian follicular fluid (FF) is a dynamic environment that changes with the seasons, affecting follicle development, ovulation, and oocyte quality. Cells in the follicles release tiny particles called extracellular vesicles (EVs) containing vital regulatory molecules, such as microRNAs (miRNAs). These miRNAs are pivotal in facilitating communication within the follicles through diverse signaling and information transfer forms. EV-coupled miRNA signaling is implicated to be associated with ovarian function, follicle and oocyte growth and response to various environmental insults. Herein, we investigated how seasonal variations directly influence the ovulatory and anovulatory states of ovarian follicles and how are they associated with follicular fluid EV-coupled miRNA dynamics in horses. Results: Ultrasonographic monitoring and follicular fluid aspiration of preovulatory follicles in horses during the anovulatory (spring: non-breeding) and ovulatory (spring, summer, and fall: breeding) seasons and subsequent EV isolation and miRNA profiling identified significant variation in EV-miRNA cargo content. We identified 97 miRNAs with differential expression among the groups and specific clusters of miRNAs involved in the spring transition (miR-149, -200b, -206, -221, -328, and -615) and peak breeding period (including miR-143, -192, -451, -302b, -100, and let-7c). Bioinformatic analyses showed enrichments in various biological functions, e.g., transcription factor activity, transcription and transcription regulation, nucleic acid binding, sequence-specific DNA binding, p53 signaling, and post-translational modifications. Cluster analyses revealed distinct sets of significantly up- and down-regulated miRNAs associated with spring anovulatory (Cluster 1) and summer ovulation–the peak breeding season (Clusters 4 and 6). Conclusions: The findings from the current study shed light on the dynamics of FF-EV-coupled miRNAs in relation to equine ovulatory and anovulatory seasons, and their roles in understanding the mechanisms involved in seasonal shifts and ovulation during the breeding season warrant further investigation. [ABSTRACT FROM AUTHOR]

Ischemic heart disease stands as the primary cause of mortality among diabetic patients. Despite advancements in vascular reconstruction and thrombolytic therapy that restore myocardial blood flow, these patients often experience poor cardiac function recovery and a higher mortality rate. This makes myocardial ischemia/reperfusion (I/R) injury a significant therapeutic challenge. Researches indicate that, under diabetic or obese conditions, adipocytes release extracellular vesicles (EVs) containing a variety of biomolecules, including RNA, proteins, adipocytokines, and mitochondria. These EVs play a pivotal role in maintaining systemic metabolic homeostasis. Importantly, adipocyte - derived EVs facilitate communication with diabetic hearts and play a regulatory role in myocardial I/R injury. This review summarizes recent studies on the modulatory effects of adipocyte ⁃derived EVs on diabetic myocardial I/R injury, highlighting potential underlying mechanisms. [ABSTRACT FROM AUTHOR]

The characterization of single extracellular vesicle (EV) has been an emerging tool for the early detection of various diseases despite there being challenges regarding how to interpret data with different protocols or instruments. In this work, standard EV particles were characterized for single CD9+, single CD81+ or double CD9+/CD81+ tetraspanin molecule positivity with two single EV analytic technologies in order to optimize their EV sample preparation after antibody labelling and analysis methods: NanoImager for direct stochastic optical reconstruction microscopy (dSTORM)‐based EV imaging and characterization, and Flow NanoAnalyzer for flow‐based EV quantification and characterization. False positives from antibody aggregates were found during dSTORM‐based NanoImager imaging. Analysis of particle radius with lognormal fittings of probability density histogram enabled the removal of antibody aggregates and corrected EV quantification. Furthermore, different machine learning models were trained to differentiate antibody aggregates from EV particles and correct EV quantification with increased double CD9+/CD81+ population. With Flow NanoAnalyzer, EV samples were prepared with different dilution or fractionation methods, which increased the detection rate of CD9+/CD81+ EV population. Comparing the EV phenotype percentages measured by two instruments, differences in double positive and single positive particles existed after percentage correction, which might be due to the different detection limit of each instrument. Our study reveals that the characterization of individual EVs for tetraspanin positivity varies between two platforms—the NanoImager and the Flow NanoAnalyzer—depending on the EV sample preparation methods used after antibody labelling. Additionally, we applied machine learning models to correct for false positive particles identified in imaging‐based results by fitting size distribution data. [ABSTRACT FROM AUTHOR]

The human phosphatase and tensin homolog (PTEN) is a tumor suppressor. A slight deficiency in PTEN might cause cancer susceptibility and progression. Infection by the liver fluke Clonorchis sinensis could lead to persistent loss of PTEN in cholangiocarcinoma. However, the mechanism of PTEN loss and its malignant effect on cholangiocarcinoma have not yet been elucidated. Extracellular vesicles secreted by Clonorchis sinensis (CS-EVs) are rich in microRNAs (miRNAs) and can mediate communication between hosts and parasites. Herein, we delved into the miRNAs present in CS-EVs, specifically those that potentially target PTEN and modulate the progression of cholangiocarcinoma via ferroptosis mechanisms. CS-EVs were extracted by differential ultra-centrifugation for high-throughput sequencing of miRNA. Lentiviral vectors were used to construct stably transfected cell lines. Erastin was used to construct ferroptosis induction models. Finally, 36 miRNAs were identified from CS-EVs. Among them, csi-miR-96-5p inhibited PTEN expression according to the predictions and dual luciferase assay. The CCK-8 assay, xenograft tumor assays and transwell assay showed that csi-miR-96-5p overexpression and PTEN knockout significantly increased the proliferation and migration of cholangiocarcinoma cells and co-transfection of PTEN significantly reversed the effect. In the presence of erastin, the cell proliferation and migration ability of the negative transfection control group were significantly impaired, although they did not significantly change with transfection of csi-miR-96-5p and PTEN knockout, indicating that they obtained ferroptosis resistance. Mechanistically, csi-miR-96-5p and PTEN knockout significantly inhibited ferroptosis through a decrease in ferrous ion (Fe2+) and malondialdehyde (MDA), and an increase in glutathione reductase (GSH), Solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4). In conclusion, loss of PTEN promoted the progression of cholangiocarcinoma via the ferroptosis pathway and csi-miR-96-5p delivered by CS-EVs may mediate this process. [ABSTRACT FROM AUTHOR]

Despite the heterogeneity associated with extracellular vesicle (EV) biogenesis pathways and the physiological state of the EV parental cells, standardization of EV manufacturing workflows could contribute to increase intra- and inter-batch reproducibility. Reaching effective EV doses for clinical use in cardiac regenerative settings requires large-scale platforms for EV production and isolation and/or strategies that maximize EV secretion per cell. While bioreactors – such as stirred-tank or vertical-wheel bioreactors – are envisioned as scalable platforms to sustain cell secretion of EVs, membrane-based processes can allow continuous and large-scale isolation of EVs. Both single-vesicle analysis and omics approaches can be relevant tools to dissect the mechanism of action of EVs in cardiac repair. For the past two decades researchers have linked extracellular vesicle (EV)-mediated mechanisms to various physiological and pathological processes in the heart, such as immune response regulation, fibrosis, angiogenesis, and the survival and growth of cardiomyocytes. Although use of EVs has gathered momentum in the cardiac field, several obstacles in both upstream and downstream processes during EV manufacture need to be addressed before clinical success can be achieved. Low EV yields obtained in small-scale cultures deter clinical translation, as mass production is a prerequisite to meet therapeutic doses. Moreover, standardizing EV manufacture is critical given the inherent heterogeneity of EVs and the constraints of current isolation techniques. In this review, we discuss the critical steps for the large-scale manufacturing of high-potency EVs for cardiac therapies. [ABSTRACT FROM AUTHOR]

Apoptosis is the most prominent mode of programmed cell death and is necessary for the maintenance of tissue homeostasis. During cell apoptosis, a distinctive population of extracellular vesicles is generated, termed apoptotic vesicles (apoVs). ApoVs inherit a variety of biological molecules such as proteins, RNAs, nuclear components, lipids, and gasotransmitters from their parent cells. ApoVs have shown promising therapeutic potential for inflammation, tumors, immune disorders, and tissue regeneration. In addition, apoVs can be used as drug carriers, vaccine development, and disease diagnosis. Recently, apoVs have been used in clinical trials to treat a variety of diseases, such as temporomandibular joint osteoarthritis and the regeneration of functional alveolar bone. Here, we review the history of apoV research, current preclinical and clinical studies, and the potential issues of apoV application. [ABSTRACT FROM AUTHOR]

Non-muscle-invasive bladder cancer (NMIBC) prognosis varies significantly due to the biological and clinical heterogeneity. High-risk stage T1-G3, comprising 15–20% of NMIBCs, involves the lamina propria and is associated with higher rates of recurrence, progression, and cancer-specific mortality. In the present study, we have evaluated the enumeration of tumour-derived extracellular vesicles (tdEVs) and circulating tumour cells (CTCs) in high-risk NMIBC patients and their correlation with survival outcomes such as time to progression (TTP), and cancer-specific survival (CSS). Eighty-three high-risk T1-G3 NMIBC patients treated between September 2010 and January 2013 were included. Blood samples were collected before a transurethral resection of the bladder (TURB) and analysed using the CellSearch® system. The presence of at least one CTC was associated with a shorter TTP and CSS. Extending follow-up to 120 months and incorporating automated tdEV evaluation using ACCEPT software demonstrated that tdEV count may additionally stratify patient risk. Combining tdEVs and CTCs improves risk stratification for NMIBC progression, suggesting that tdEVs could be valuable biomarkers for prognosis and disease monitoring. Further research is needed to confirm these findings and establish the clinical significance of tdEVs in early-stage cancers. [ABSTRACT FROM AUTHOR]

Background: Cancer stem cells (CSCs) represent a potential mechanism contributing to tumorigenesis, metastasis, recurrence, and drug resistance. The objective of this study is to investigate the status quo and advancements in CSC research utilizing bibliometric analysis. Methods: Publications related to CSCs from 2010 to 2022 were collected from the Web of Science Core Collection database. Various analytical tools including CiteSpace, VOSviewer, Scimago Graphica, and GraphPad Prism were used to visualize aspects such as co-authorship, co-occurrence, and co-citation within CSC research to provide an objective depiction of the contemporary status and developmental trajectory of the CSC field. Results: A total of 22,116 publications were included from 1942 journals written by 95,992 authors. Notably, China emerged as the country with the highest number of publications, whereas the United States exerted the most significant influence within the field. MD Anderson Cancer Center emerged as the institution making the most comprehensive contributions. Wicha M.S. emerged as the most prolific and influential researcher. Among journals, Cancers emerged as a focal point for CSC research, consistently publishing a wealth of high-quality papers. Furthermore, it was observed that most journals tended to approach CSC research from molecular, biological, and immunological perspectives. The research into CSCs encompassed a broad array of topics, including isolation and enrichment techniques, biomarkers, biological characteristics, cancer therapy strategies, and underlying biological regulatory mechanisms. Notably, exploration of the tumor microenvironment and extracellular vesicles emerged as burgeoning research frontiers for CSCs. Conclusion: The research on CSCs has garnered growing interest. A trend toward multidisciplinary homogeneity is emerging within the realm of CSCs. Further investigation could potentially center on the patients of extracellular vesicles and the tumor microenvironment in relation to CSCs. [ABSTRACT FROM AUTHOR]

Exosomes are extracellular vesicles involved in intercellular signaling, carrying various cargo from microRNAs to metabolites and proteins. They are released by practically all cells and are highly heterogenous due to their origin and content. Several groups of exosomes are known to be involved in various pathological conditions including autoimmune, neurodegenerative, and infectious diseases as well as cancer, and therefore a substantial understanding of their biogenesis and release is crucial. Polarized cells display an array of specific functions originated from differentiated membrane trafficking systems and could lead to hints in untangling the complex process of exosomes. Indeed, recent advances have successfully revealed specific regulation pathways for releasing different subsets of exosomes from different sides of polarized epithelial cells, underscoring the importance of polarized cells in the field. Here we review current evidence on exosome biogenesis and release, especially in polarized cells, highlight the challenges that need to be combatted, and discuss potential applications related to exosomes of polarized-cell origin. [ABSTRACT FROM AUTHOR]

The delivery of CRISPR/Cas systems holds immense potential for revolutionizing cancer treatment, with recent advancements focusing on extracellular vesicles (EVs) and viral vectors. EVs, particularly exosomes, offer promising opportunities for targeted therapy due to their natural cargo transport capabilities. Engineered EVs have shown efficacy in delivering CRISPR/Cas components to tumor cells, resulting in inhibited cancer cell proliferation and enhanced chemotherapy sensitivity. However, challenges such as off-target effects and immune responses remain significant hurdles. Viral vectors, including adeno-associated viruses (AAVs) and adenoviral vectors (AdVs), represent robust delivery platforms for CRISPR/Cas systems. AAVs, known for their safety profile, have already been employed in clinical trials for gene therapy, demonstrating their potential in cancer treatment. AdVs, capable of infecting both dividing and non-dividing cells, offer versatility in CRISPR/Cas delivery for disease modeling and drug discovery. Despite their efficacy, viral vectors present several challenges, including immune responses and off-target effects. Future directions entail refining delivery systems to enhance specificity and minimize adverse effects, heralding personalized and effective CRISPR/Cas-mediated cancer therapies. This article underscores the importance of optimized delivery mechanisms in realizing the full therapeutic potential of CRISPR/Cas technology in oncology. As the field progresses, addressing these challenges will be pivotal for translating CRISPR/Casmediated cancer treatments from bench to bedside. [ABSTRACT FROM AUTHOR]

BACKGROUND: Despite a series of clinical treatment measures, the treatment of pulmonary fibrosis still faces challenges. In recent years, mesenchymal stem cells and their extracellular vesicles have attracted extensive attention as an emerging therapeutic strategy and are considered to be a promising means of treating pulmonary fibrosis. OBJECTIVE: To systematically review the application of mesenchymal stem cells and their extracellular vesicles in the treatment of pulmonary fibrosis, to comprehensively understand their therapeutic mechanism, efficacy evaluation and problems, and provide reference and guidance for further research and clinical application in the future. METHODS: Using Chinese and English search terms “mesenchymal stem cells”, “ mesenchymal stem cell extracellular vesicles”, “pulmonary fibrosis”, we searched the CNKI and PubMed electronic journal databases. By means of manual reading and eliminating duplicate articles, 112 articles were selected, but 58 Chinese and English articles were finally included for summary. RESULTS AND CONCLUSION: (1) Mesenchymal stem cells and their extracellular vesicles have shown great potential in the treatment of pulmonary fibrosis, such as regulating inflammatory responses, inhibiting fibroblast proliferation, and promoting damaged tissue repair. Preliminary results from clinical trials have also shown some effects of the treatment, including improved lung function and quality of life in patients. (2) However, mesenchymal stem cells and extracellular vesicles in the treatment of pulmonary fibrosis still face some challenges. During treatment, technical challenges such as cell migration and intrachistological localization need to be addressed for it to accurately reach the damaged lung tissue. Furthermore, its long-term safety also needs to be further studied and improved. For translational medicine development, standardized procedures such as cell collection, cell isolation, cell culture, cell harvesting, and cell identification need to be refined. (3) Despite these challenges, through the joint efforts of scientific researchers and medical personnel, these problems are expected to be gradually solved. In the future, we can further improve treatment outcomes by optimizing treatment regimens and exploring individualized treatments. At the same time, in-depth research on the therapeutic mechanism of stem cells and their extracellular vesicles is expected to develop more efficient and safe therapeutic strategies. [ABSTRACT FROM AUTHOR]

BACKGROUND: De novo adipose regeneration induced by small extracellular vesicles has become a promising method for repairing soft tissue defects. However, due to different animal models and small extracellular vesicle application dosages, it is difficult to quantitatively compare the therapeutic effect of small extracellular vesicles from various sources on adipose regeneration. OBJECTIVE: To compare the regenerative effects of small extracellular vesicles derived from stem cells and small extracellular vesicles from tissue. METHODS: Small extracellular vesicles derived from adipose-derived stem cells and from adipose tissue were isolated by ultracentrifugation. The particle number, particle size, morphology, and protein expression of small extracellular vesicles were identified by nanoparticle tracking analysis, transmission electron microscopy and western blot assay. A quantitative and evaluative subcutaneous model for adipose regeneration in C57 mice was established using a customized silicone tube. The regenerative effects of induced de novo adipose were compared by cell counting and hematoxylin-eosin staining. RESULTS AND CONCLUSION: (1) Small extracellular vesicles derived from adipose-derived stem cells and from adipose tissue were isolated by ultracentrifugation. Both small extracellular vesicles were round-shape in transmission electron microscopy with particle size between 50-200 nm, and abundant with the small extracellular vesicles marker protein CD81, CD63 and TSG101. (2) An equal number of small extracellular vesicles were mixed with matrigel in customized silicone tubes, implanted subcutaneously in the back of mice to establish a cell-free and quantifiable adipose regeneration model. (3) On days 3 and 7 after implantation, the results of cell counting and hematoxylin-eosin staining showed that both small extracellular vesicle groups recruited more host cells than the blank group, and the small extracellular vesicles derived from adipose tissue group were superior to the small extracellular vesicles derived from adipose-derived stem cell group. (4) 4 weeks after implantation, hematoxylin-eosin staining of the contents in silicone tubes showed that small extracellular vesicles induced de novo adipose regeneration in vivo, and the small extracellular vesicles derived from adipose tissue group were superior to the small extracellular vesicles derived from adipose-derived stem cell group. The above results indicated that small extracellular vesicles derived from tissues have a superior effect on inducing de novo adipose regeneration compared to small extracellular vesicles derived from stem cells. [ABSTRACT FROM AUTHOR]

BACKGROUND: It has been shown that neural stem cells can differentiate into neurons, astrocytes, and oligodendrocytes. Mesenchymal stem cells-derived extracellular vesicles have also been shown to cross the blood-brain barrier to reach sites of central nervous injury and promote neural repair. However, it is not clear whether neuron-derived extracellular vesicles promote the differentiation of neural stem cells in a direction that is beneficial for neurogenesis. OBJECTIVE: To investigate whether neuron-derived extracellular vesicles facilitate neural stem cell differentiation towards neurogenesis. METHODS: Neurons and neural stem cells were extracted from neonatal SD rat cerebral cortex by trypsin digestion. Cell supernatants of neurons were collected. Neuron-derived extracellular vesicles were extracted. Neural stem cells cultured for 10 days were co-cultured with neuron-derived extracellular vesicles or PBS for 7 days. Immunoblotting, immunofluorescence, and RT-qPCR were used to detect proteins specifically expressed by neurons, neural stem cells, oligodendrocytes, and astrocytes. RESULTS AND CONCLUSION: The neural stem cells co-cultured with neuron-derived extracellular vesicles showed high expression of neuron-specific proteins and oligodendrocyte-specific proteins including β3-tubulin, neurofilament 200 and myelin basic protein, and low expression of astrocyte-specific protein glial fibrillary acidic protein. These results suggest that neuron-derived extracellular vesicles can promote the differentiation of neural stem cells into neurons and oligodendrocytes and prevent the differentiation of neural stem cells into astrocytes. [ABSTRACT FROM AUTHOR]

Background: Bacterial extracellular vesicles (EVs) are pivotal mediators of intercellular communication and influence host cell biology, thereby contributing to the pathogenesis of infections. Despite their significance, the precise effects of bacterial EVs on the host cells remain poorly understood. This study aimed to elucidate ultrastructural changes in host cells upon infection with EVs derived from a pathogenic bacterium, Staphylococcus aureus (S. aureus). Results: Using super-resolution fluorescence microscopy and high-voltage electron microscopy, we investigated the nanoscale alterations in mitochondria, endoplasmic reticulum (ER), Golgi apparatus, lysosomes, and microtubules of skin cells infected with bacterial EVs. Our results revealed significant mitochondrial fission, loss of cristae, transformation of the ER from tubular to sheet-like structures, and fragmentation of the Golgi apparatus in cells infected with S. aureus EVs, in contrast to the negligible effects observed following S. epidermidis EV infection, probably due to the pathogenic factors in S. aureus EV, including protein A and enterotoxin. These findings indicate that bacterial EVs, particularly those from pathogenic strains, induce profound ultrastructural changes of host cells that can disrupt cellular homeostasis and contribute to infection pathogenesis. Conclusions: This study advances the understanding of bacterial EV-host cell interactions and contributes to the development of new diagnostic and therapeutic strategies for bacterial infections. [ABSTRACT FROM AUTHOR]

Intercellular communication is a cell‐type and stimulus‐dependent event driven not only by soluble factors but also by extracellular vesicles (EVs). EVs include vesicles of different size and origin that contain a myriad of molecules. Among them, small EVs (sEV; <200 nm) have been shown to modulate not just regional cell responses but also distant organ behavior. In cancer, distant organ modulation by sEVs has been associated to disease dissemination, which is one of the main concerns in melanoma. Description of broadly conserved alterations in sEV‐contained molecules represents a strategy to identify key modifications in cellular communication as well as new disease biomarkers. Here, we characterize proteomes of cutaneous melanocyte and melanoma‐derived sEVs to deepen on the landscape of normal and disease‐related cell communication. Results reveal the presence of unique protein signatures for melanocytes and melanoma cells that reflect cellular transformation‐related profound modifications. Melanocyte‐derived sEVs are enriched in oxidative metabolism (e.g., aconitase 2, ACO2) or pigmentation (e.g., tyrosinase, TYR) related proteins while melanoma‐derived sEVs reflect a generalized decrease in mature melanocytic markers (e.g., melanoma antigen recognized by T‐cells 1, MART‐1, also known as MLANA) and an increase in epithelial to mesenchymal transition (EMT)‐related adhesion molecules such as tenascin C (TNC). [ABSTRACT FROM AUTHOR]

Brain‐derived extracellular vesicles (EVs) serve a prominent role in maintaining homeostasis and contributing to pathology in health and disease. This review establishes a crucial link between physiological processes leading to EV biogenesis and their impacts on disease. EVs are involved in the clearance and transport of proteins and nucleic acids, responding to changes in cellular processes associated with neurodegeneration, including autophagic disruption, organellar dysfunction, aging, and other cell stresses. In neurodegenerative disorders (e.g., Alzheimer's disease, Parkinson's disease, etc.), EVs contribute to the spread of pathological proteins like amyloid β, tau, ɑ‐synuclein, prions, and TDP‐43, exacerbating neurodegeneration and accelerating disease progression. Despite evidence for both neuropathological and neuroprotective effects of EVs, the mechanistic switch between their physiological and pathological functions remains elusive, warranting further research into their involvement in neurodegenerative disease. Moreover, owing to their innate ability to traverse the blood–brain barrier and their ubiquitous nature, EVs emerge as promising candidates for novel diagnostic and therapeutic strategies. The review uniquely positions itself at the intersection of EV cell biology, neurophysiology, and neuropathology, offering insights into the diverse biological roles of EVs in health and disease. [ABSTRACT FROM AUTHOR]

Respiratory diseases, including chronic obstructive pulmonary disease (COPD), asthma, lung cancer, and coronavirus pneumonia, present a major global health challenge. Current diagnostic and therapeutic options for these diseases are limited, necessitating the urgent development of novel biomarkers and therapeutic strategies. In recent years, microRNAs (miRNAs) within extracellular vesicles (EVs) have received considerable attention due to their crucial role in intercellular communication and disease progression. EVs are membrane-bound structures released by cells into the extracellular environment, encapsulating a variety of biomolecules such as DNA, RNA, lipids, and proteins. Specifically, miRNAs within EVs, known as EV-miRNAs, facilitate intercellular communication by regulating gene expression. The expression levels of these miRNAs can reflect distinct disease states and significantly influence immune cell function, chronic airway inflammation, airway remodeling, cell proliferation, angiogenesis, epithelial-mesenchymal transition, and other pathological processes. Consequently, EV-miRNAs have a profound impact on the onset, progression, and therapeutic responses of respiratory diseases, with great potential for disease management. Synthesizing the current understanding of EV-miRNAs in respiratory diseases such as COPD, asthma, lung cancer, and novel coronavirus pneumonia, this review aims to explore the potential of EV-miRNAs as biomarkers and therapeutic targets and examine their prospects in the diagnosis and treatment of these respiratory diseases. [ABSTRACT FROM AUTHOR]

We aim to explore the potential mechanism of bone marrow mesenchymal stem cells–derived extracellular vesicles (BMSCs-Exo) in improving spinal cord injury (SCI). Thirty male 12-week specific pathogen-free (SPF) Sprague–Dawley (SD) rats were used to construct SCI model in vivo. Ten male 12-week SPF SD rats were used to extract BMSCs. The Basso, Beattie, Bresnahan (BBB) score was used to evaluate the motor function of rats. Real-time fluorescence quantitative PCR (RT-PCR), western blot (WB), and double luciferase assay were used to explore the regulation between rno-miR-208a-3p and Cdkn1a (p21) in BMSCs. Primary spinal cord neurons were treated with lipopolysaccharide (100 ng/mL) for 30 min to mimic SCI in vitro. Compared with the model group (14 scores), BMSCs-Exo increased BBB score (19 scores) in SCI rats. Compared with the sham group, Cdkn1a was upregulated, whereas rno-miR-208a-3p was downregulated in the model group. However, compared with the model group, Cdkn1a was downregulated, whereas rno-miR-208a-3p was upregulated in the BMSCs-Exo group. In addition, rno-miR-208a-3p inhibited the expression of Cdkn1a via direct binding way. BMSCs-Exo-rno-miR-208a-3p promoted the proliferation of primary spinal neurons via inhibiting apoptosis in vitro. Moreover, BMSCs-Exo-rno-miR-208a-3p promoted cyclin D1, CDK6, and Bcl-2 and inhibited Bax expression in a cell model of SCI. In conclusion, BMSCs-Exo-carried rno-miR-208a-3p significantly protects rats from SCI via regulating the Cdkn1a pathway. [ABSTRACT FROM AUTHOR]

In adverse environments, fine dust is linked to a variety of health disorders, including cancers, cardiovascular, neurological, renal, reproductive, motor, systemic, and respiratory diseases. Although PM10 is associated with oral inflammation and cancer, there is limited research on biomaterials that prevent damage caused by fine dust. In this study, we evaluated the effects of biomaterials using microRNA profiling, flow cytometry, conventional PCR, immunocytochemistry, Alizarin O staining, and ELISA. Compared to SBE (Scutellaria baicalensis extract), the preventive effectiveness of SBEIEs (SBE-induced exosomes) against fine dust was approximately two times higher. Furthermore, SBEIEs promoted cellular differentiation of periodontal ligament stem cells (PDLSCs) into osteoblasts, periodontal ligament cells (PDLCs), and pulp progenitor cells (PPCs), enhancing immune modulation for oral health against fine dust. In terms of immune modulation, SBEIEs activated the secretion of cytokines such as IL-10, LL-37, and TGF-β in T cells, B cells, and macrophages, while attenuating the secretion of MCP-1 in macrophages. MicroRNA profiling revealed that significantly modulated miRNAs in SBEIEs influenced four biochemical categories: apoptosis, cellular differentiation, immune activation, and anti-inflammation. These findings suggest that SBEIEs are an optimal biomaterial for developing oral health care products. Additionally, this study proposes functional microRNA candidates for the development of pharmaceutical liposomes. [ABSTRACT FROM AUTHOR]

Pre‐implantation embryonic development occurs in the oviduct during the first few days of pregnancy. The presence of oviductal extracellular vesicles (oEVs, also called oviductosomes) is crucial for pre‐implantation embryonic development in vivo as oEVs often contain molecular transmitters such as proteins. Therefore, evaluating oEV cargo during early pregnancy could provide insights into factors required for proper early embryonic development that are missing in the current in vitro embryo culture setting. In this study, we isolated oEVs from the oviductal fluid at estrus and different stages of early embryonic development. The 2306–3066 proteins in oEVs identified at the different time points revealed 58–60 common EV markers identified in exosome databases. Oviductal extracellular vesicle proteins from pregnant samples significantly differed from those in non‐pregnant samples. In addition, superovulation changes the protein contents in oEVs compared to natural ovulation at estrus. Importantly, we have identified that embryo‐protectant proteins such as high‐mobility protein group B1 and serine (or cysteine) peptidase inhibitor were only enriched in the presence of embryos. We also visualized the physical interaction of EVs and the zona pellucida of 4‐ to 8‐cell stage embryos using transmission electron microscopy as well as in vivo live imaging of epithelial cell‐derived GFP‐tagged CD9 mouse model. All protein data in this study are readily available to the scientific community in a searchable format at https://genes.winuthayanon.com/winuthayanon/oviduct%5fev%5fproteins/. In conclusion, we identified oEVs proteins that could be tested to determine whether they can improve embryonic developmental outcomes in vivo and in vitro setting. [ABSTRACT FROM AUTHOR]

Androgenetic alopecia (AGA) is a non-fatal disease prevalent worldwide. However, mixed efficacy has been observed among different therapies for hair regrowth in AGA patients. Thus, a nano-platform with synergistic treatments based on a hybrid extracellular vesicle encapsulating gold nanoparticles (AuNPs) and finasteride (Hybrid/Au@Fi) was constructed through membrane fusion between hair follicle stem cell (HFSC)-derived extracellular vesicles and liposomes. These hybrid vesicles (HVs) not only fuel hair regrowth by providing cellular signals in extracellular vesicles, but also improve storage stability, follicle retention, and drug encapsulation efficiency (EE%) for finasteride inhibiting 5α-reductase, and nano-size AuNPs that simulate low-level laser therapy (LLLT) with similar photothermal effects in vitro. The EE% of finasteride in these HVs reached 45.33%. The dual administration of these extracellular vesicles and finasteride showed a strong synergistic effect on HFSCs in vitro. In an AGA mouse model, once-daily topical Hybrid/Au@Fi (115.07 ± 0.32 nm, −7.50 ± 1.68 mV) gel led to a faster transition of hair follicles (HFs) from the catagen to the anagen, increased hair regrowth coverage, and higher quality of regrowth hair, compared to once-daily 5% minoxidil treatment. Compared to topical minoxidil, the multifaceted synergistic therapy of Hybrid/Au@Fi through topical administration offers a new option for intractable AGA patients with low side effects. [Display omitted] • These hybrid vesicles improved storage stability for cargos, follicle retention, and drug encapsulation efficiency (EE%) for finasteride. • The Hybrid/Au@Fi (115.07 ± 0.32 nm, −7.50 ± 1.68 mV) facilitated simulated in vitro layered dermal papilla cell (DPC) migration and angiogenesis with favorable cell dynamic behavior and no cytotoxicity. • In an AGA mouse model, once-daily topical application of Hybrid/Au@Fi gel exhibited a faster transition of hair follicles from the catagen to the anagen, higher hair regrowth coverage, and improved quality of regrowth hair, compared to once-daily 5% minoxidil treatment. [ABSTRACT FROM AUTHOR]

There is a growing interest in using extracellular vesicles (EVs) for therapeutic applications. EVs are composed of cytoplasmic proteins and nucleic acids and an external lipid bilayer containing transmembrane proteins on their surfaces. EVs can alter the state of the target cells by interacting with the receptor ligand of the target cell or by being internalised by the target cell. Blood cells are the primary source of EVs, and 1 μL of plasma contains approximately 1.5 × 107 EVs. Owing to their easy acquisition and the avoidance of cell amplification in vitro , using blood cells as a source of therapeutic EVs has promising clinical application prospects. This review summarises the characteristics and biological functions of EVs derived from different blood cell types (platelets, erythrocytes, and leukocytes) and analyses the prospects and challenges of using them for clinical therapeutic applications. In summary, blood cell-derived EVs can regulate different cell types such as immune cells (macrophages, T cells, and dendritic cells), stem cells, and somatic cells, and play a role in intercellular communication, immune regulation, and cell proliferation. Overall, blood cell-derived EVs have the potential for use in vascular diseases, inflammatory diseases, degenerative diseases, and injuries. To promote the clinical translation of blood cell-derived EVs, researchers need to perform further studies on EVs in terms of scalable and reproducible isolation technology, quality control, safety, stability and storage, regulatory issues, cost-effectiveness, and long-term efficacy. [Display omitted] • Blood contains EVs derived from platelets, erythrocytes, and leukocytes. • Blood cell-derived EVs regulate intercellular communication and immune responses. • Blood cell-derived EVs have therapeutic potential in various diseases. [ABSTRACT FROM AUTHOR]

Plant-derived extracellular vesicles (EVs), containing a myriad of bioactive proteins, microRNAs, lipids, and secondary metabolites, have recently become the focus of rising interest due to their important roles in various applications. The widely accepted method for isolating plant EVs is differential ultracentrifugation plus density gradient centrifugation. However, the combination of differential ultracentrifugation and density gradient centrifugation for the isolation of plant EVs is time-consuming and labor-intensive. Hence, there is a need for more efficient methods to perform the separation of plant EVs. In this study, EVs were separated from Arabidopsis thaliana leaves by a cost-effective polyethylene glycol (PEG)-based precipitation approach. The mean size of purified Arabidopsis thaliana EVs determined by dynamic light scattering was 266 nm, which is consistent with nanoparticle tracking analysis. The size was also confirmed via transmission electron microscopy with morphology of a cup-shaped appearance which is the typical mammalian exosome's morphology. Additionally, Western blotting of the purified Arabidopsis thaliana EVs, using commercially available mammalian exosomal kits, displayed surface marker tetraspanin proteins (CD9, CD63, and CD81), and endosomal sorting complexes required for transport (ESCRT)-associated proteins (TSG101 and ALIX). This demonstrates that the purified Arabidopsis thaliana EVs reveal the typical proteins reported in mammalian exosomes. [ABSTRACT FROM AUTHOR]

Exosomes from plants or animals are a cheap, available, and promising option in medicine, which can be used for the detection or treatment of various diseases. This study aims to evaluate the antitoxic and antioxidant properties of Extracellular vesicle (EVs) extracted from chicken embryo blood using a fibroblast cell line (NIH/3T3). EVs from chick embryos were extracted in this experimental investigation using the sedimentation method and examined using dynamic light scattering (DLS) and field emission electron microscopy (FE-SEM). The protein concentration and overall antioxidant capacity of the EVs were determined using bicinchoninic acid (BCA) and antioxidant capacity (FRAP). EVs were added to NIH/3T3 cells at varying concentrations (1, 2, and 10 mg/ml), and the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay test was used to measure cell survival. The size of the isolated EVs was confirmed to be less than 100 nm by electron microscopy and DLS. The quantity of protein in these EVs was 3200 µg/ml, and their total antioxidant capacities were 3130.17, 1914.122, and 976.9 μMol/L. The MTT test findings demonstrated that NIH/3T3 cells survived treatment with EVs (P ≤ 0.001) compared to the control group. Antioxidant-rich and protein-rich exosomes in chicken embryos may be valuable in managing oxidative stress. [ABSTRACT FROM AUTHOR]

Oxaliplatin, a platinum-based chemotherapeutic agent, frequently causes acute and chronic peripheral sensory neuropathy, for which no effective treatment has been established. In particular, chronic neuropathy can persist for years even after treatment completion, thus worsening patients' quality of life. To avoid the development of intractable adverse effects, a predictive biomarker early in treatment is awaited. In this study, we explored extracellular long non-coding RNAs (lncRNAs) released from primary sensory neurons as biomarker candidates for oxaliplatin-induced peripheral neuropathy. Because many human-specific lncRNA genes exist, we induced peripheral sensory neurons from human induced pluripotent stem cells. Oxaliplatin treatment changed the levels of many lncRNAs in extracellular vesicles (EVs) released from cultured primary sensory neurons. Among them, the levels of release of lncRNAs that were considered to be selectively expressed in dorsal root ganglia were correlated with those of lncRNAs in plasma EV obtained from healthy individuals. Several lncRNAs in plasma EVs early after the initiation of treatment showed greater changes in patients who did not develop chronic neuropathy that persisted for more than 1 year than in those who did. Therefore, these extracellular lncRNAs in plasma EVs may represent predictive biomarkers for the development of chronic peripheral neuropathy induced by oxaliplatin. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cell‐derived secretome represents an emerging acellular therapeutic which possess significant opportunity for clinical applications due to its anti‐inflammatory, immunomodulatory, and wound healing properties. However, maintaining therapeutic efficacy and ensuring stability of cell‐based products is challenging, requiring a robust delivery method. Therefore, we designed a hydrogel‐based scaffold loaded with CK Cell Technologies' proprietary Mesenchymal stem cell‐secretome for controlled release treatment of acute and chronic wounds. We incorporated both conditioned media (CM) and extracellular vesicles (EVs) into gelatin methacryloyl (GelMA) hydrogels and demonstrated how we can tune the diffusive release of the EVs from them. To demonstrate viability of the approach, we developed a wound healing scratch assay where we see in situ release of CM and EVs promote enhanced migration of human dermal fibroblasts (hDFs). We see the colocalization of these EVs in the fibroblasts using fluorescent microscopy. Finally, as a surrogate for in vivo neovascularization, we conducted an in vitro tube formation assay for the MSC‐secretome using matrigel‐embedded human microvascular endothelial cells. By adding CM and EVs, we observe an increase in tubulogenesis. Collectively, our data demonstrates by tuning the GelMA properties, we can influence the controlled release of the MSC‐secretome for a wound dressing and bandage application for chronic and acute wounds. [ABSTRACT FROM AUTHOR]

The significance of anterior cruciate ligament (ACL) remnants during reconstruction remains unclear. Co‐culturing ACL remnant cells and bone marrow stromal cells (BMSCs) may reduce apoptosis and enhance hamstring tendon activity. This study investigated whether extracellular vesicles (EVs), which facilitate cell–cell interactions, act as the active components, improving graft maturation in this co‐culture. The effects of EVs on cell viability, proliferation, migration and gene expression in the rabbit ACL remnant cells and BMSCs were assessed using control (BMSC‐only culture), co‐culture (ACL remnant cells and BMSCs, CM) and co‐culture without EVs (CM ∆ EVs) media. EVs were isolated from control (BMSC‐EV) and co‐culture (CM‐EV) media and characterized. CM significantly enhanced the proliferation, migration and expression of transforming growth factor (TGF‐β)‐, vascular endothelial growth factor (VEGF)‐, collagen synthesis‐ and tenogenesis‐related genes. However, CM‐induced effects were reversed by the CM ∆ EVs treatment. CM‐EV treatment exhibited higher potential to enhance proliferation, migration and gene expression in the ACL remnant cells and BMSCs than BMSC‐EV and non‐EV treatments. In conclusion, EVs, secreted under the coexistence of ACL remnant cells and BMSCs, primarily increase the cell viability, proliferation, migration and gene expression of collagen synthesis‐, TGF‐β‐, VEGF‐ and tenogenesis‐related genes in both cell types. [ABSTRACT FROM AUTHOR]

Exosomes are a subpopulation of the heterogenous pool of extracellular vesicles that are secreted to the extracellular space. Exosomes have been purported to play a role in intercellular communication and have demonstrated utility as biomarkers for a variety of diseases. Despite broad interest in exosome biology, the conditions that regulate their secretion are incompletely understood. The goal of this procedure is to biochemically reconstitute exosome secretion in Streptolysin O (SLO)-permeabilized mammalian cells. This protocol describes the reconstitution of lyophilized SLO, preparation of cytosol and SLO-permeabilized cells, assembly of the biochemical reconstitution reaction, and quantification of exosome secretion using a sensitive luminescence-based assay. This biochemical reconstitution reaction can be utilized to characterize the molecular mechanisms by which different gene products regulate exosome secretion. Key features This protocol establishes a functional in vitro system to reconstitute exosome secretion in permeabilized mammalian cells upon addition of cytosol, ATP, GTP, and calcium (Ca2+).

Exosomes are small extracellular vesicles (sEVs) of ~30-150 nm in diameter that are enriched in exosome marker proteins and play important roles in health and disease. To address large unanswered questions regarding exosome biology in vivo, we created the Exomap1 transgenic mouse, which in response to Cre recombinase expresses the most highly enriched exosomal marker protein known, human CD81, fused to mNeonGreen (HsCD81mNG), and prior to Cre expresses a mitochondrial red fluorescent protein. Validation of the exomap1 mouse with eight distinct Cre drivers demonstrated that HsCD81mNG was expressed only in response to Cre, that murine cells correctly localized HsCD81mNG to the plasma membrane, and that this led to the secretion of HsCD81mNG in EVs that had the size (~70-80 nm), topology, and composition of exosomes. Furthermore, cell type-specific activation of the exomap1 transgene allowed us to use quantitative single molecule localization microscopy to calculate the cell type-specific contribution to biofluid exosome populations. Specifically, we show that neurons contribute ~1% to plasma and cerebrospinal fluid exosome populations whereas hepatocytes contribute ~15% to plasma exosome populations, numbers that reflect the known vascular permeabilities of brain and liver. These observations validate the use of Exomap1 mouse models for in vivo studies of exosome biology.

Abstract Naturally occurring cell-derived extracellular vesicles (EVs) have emerged as attractive nanocarriers for drug delivery. However, production of large quantities of EVs for clinical applications in a scalable manner remains a significant challenge. This study investigated at the single cell level how sonoporation, or membrane poration produced by ultrasound-induced microbubble cavitation, impacts EV production using mouse macrophage RAW 264.7 cells stably expressing CD63-GFP as a model system. Real-time fluorescence videomicroscopy detected rapid changes in CD63-GFP, a tetraspanin family member highly enriched in intraluminal vesicles tagged with GFP, to track changes in multivesicular bodies (MVBs), which are the cellular compartments where exosomes originate within the cells. Our results revealed distinct dynamic changes in CD63-GFP intensity and distribution in RAW 264.7 cells in terms of response time and duration depending on whether the cells were directly or indirectly impacted by sonoporation, suggesting reorganization of MVBs in response to direct and indirect mechanisms resulted from the mechanical impact of ultrasound pulse on the cells. Analysis of the supernatant from sonoporation-treated RAW 264.7 cells expressing CD63-GFP demonstrated a delayed and sustained increase in the production of CD63-GFP-positive EVs. These results show the robust and detailed effect of sonoporation and reveal insights into sonoporation-induced EV release useful for guiding the application of sonoporation to enhance large-scale EV production.

Abstract Background In mucopolysaccharidosis type III (MPS III, also known as Sanfilippo syndrome), a pediatric neurodegenerative disorder, accumulation of abnormal glycosaminoglycans (GAGs) induces severe neuroinflammation by triggering the microglial pro-inflammatory cytokines production via a TLR4-dependent pathway. But the extent of the microglia contribution to the MPS III neuropathology remains unclear. Extracellular vesicles (EVs) mediate intercellular communication and are known to participate in the pathogenesis of adult neurodegenerative diseases. However, characterization of the molecular profiles of EVs released by MPS III microglia and their effects on neuronal functions have not been described. Methods Here, we isolated EVs secreted by the microglial cells after treatment with GAGs purified from urines of Sanfilippo patients (sfGAGs-EVs) or from age-matched healthy subjects (nGAGs-EVs) to explore the EVs’ proteins and small RNA profiles using LC–MS/MS and RNA sequencing. We next performed a functional assay by immunofluorescence following nGAGs- or sfGAGs-EVs uptake by WT primary cortical neurons and analyzed their extensions metrics after staining of βIII-tubulin and MAP2 by confocal microscopy. Results Functional enrichment analysis for both proteomics and RNA sequencing data from sfGAGs-EVs revealed a specific content involved in neuroinflammation and neurodevelopment pathways. Treatment of cortical neurons with sfGAGs-EVs induced a disease-associated phenotype demonstrated by a lower total neurite surface area, an impaired somatodendritic compartment, and a higher number of immature dendritic spines. Conclusions This study shows, for the first time, that GAGs from patients with Sanfilippo syndrome can induce microglial secretion of EVs that deliver a specific molecular message to recipient naive neurons, while promoting the neuroinflammation, and depriving neurons of neurodevelopmental factors. This work provides a framework for further studies of biomarkers to evaluate efficiency of emerging therapies. Graphical Abstract

Abstract With the severity of chronic kidney disease worldwide, strategies to recover renal function via tissue regeneration provide alternatives to kidney replacement therapy. To exclude side effects from direct cell transplantation, extracellular vesicles (EVs) are great substitutes representing paracrine cell signaling. To build three-dimensional structures for implantation into the 5/6 nephrectomy model by incorporating bioactive materials, including multifunctional EVs (mEVs), porous PMEZE/mEV scaffolds were developed in combination with edaravone (EDV; E) and mEV based on PMEZ scaffolds with PLGA (P), MH-RA (M), ECM (E), ZnO-ALA (Z). The oxygen free radical scavenger EDV was incorporated to induce tubular regeneration. mEVs were engineered to serve regenerative activities with a combination of two EVs from SDF-1α overexpressed tonsil-derived mesenchymal stem cells (sEVs) and intermediate mesoderm (IM) cells during differentiation into kidney progenitor cells (dEVs). mEVs displayed beneficial effects on regeneration by facilitating migration and inducing differentiation of surrounding stem cells, and EDV improved kidney function by regulating the GDNF/RET pathway and their downstream genes. The promotion of MSC recruitment was confirmed with sEV particles number dependently, and the regulation of the GDNF/RET pathway by the effect of EDV and its enhanced effect by mEVs were elucidated using in vitro analysis. The regeneration of tubules was additionally demonstrated through the increased expression of aquaporin-1 (AQP-1) and cadherin-16 (CDH16) for proximal tubules, and calbindin and PAX2 for distal tubules in the renal defect model. With these, structural regeneration and functional recovery were achieved with kidney regeneration in the 5/6 nephrectomy mice model. Graphical abstract

Abstract Background Primary ovarian insufficiency (POI) manifests with hormonal imbalances, menstrual irregularities, follicle loss, and infertility. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are emerging as a promising treatment for POI. This systematic review aims to assess the effects of MSC-EVs on follicle number, hormonal profile, and fertility in POI animal models. Methods A systematic search of PubMed, Scopus, and Web of Science databases up to December 14th, 2023 was conducted. Two reviewers independently conducted screening, risk of bias assessment, and data extraction. Meta-analysis was performed to analyze treatment versus control outcomes using a random effects model. Publication bias was assessed using Egger’s regression test and sensitivity analysis was assessed using the leave-one-out method. Subgroup analyses and meta-regressions were conducted based on EV source, induction model, type of animal, study quality, administration route, administration frequency and route, and dose. Results a total of 29 studies were included. MSC-EVs treatment significantly increased total follicle count (SMD, (95CI), p-value; 3.56, (0.91, 6.21),

Abstract Background Parascaris spp. represent a significant threat to equine health worldwide, particularly in foals. The long-term survival of parasites in the host necessitates persistent modulation of the host immune response. Intercellular communication achieved through the exchange of molecules via extracellular vesicles (EVs) released from the parasite could be a crucial factor in this regard. This study aimed to isolate and characterize EVs released by adult male and female Parascaris worms and conduct a proteomic analysis to identify sex-specific proteins and potential immunomodulatory factors. Methods Live adult Parascaris worms were collected, and EVs were isolated from spent culture media using differential ultracentrifugation. Nanoparticle tracking analysis and transmission electron microscopy confirmed the size, concentration, and morphology of the isolated EVs. Proteins within the isolated EVs were analyzed using mass spectrometry-based proteomics (LC–MS/MS). Results Proteomic analysis revealed a total of 113 proteins in Parascaris EVs, with several proteins showing homology to known helminth exosome proteins and exhibiting immunomodulatory functions. Sex-specific differences in EV protein composition were observed, with a distinct abundance of C-type lectins in female EVs, suggesting potential sex-specific roles or regulation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed metabolic pathways shared between male and female Parascaris EVs, as well as differences in signal transduction, and cell growth and death pathways, indicating sex-specific variations. Conclusions These findings imply that Parascaris EVs and their protein cargo are complex. This data potentially opens avenues for discovering innovative approaches to managing and understanding helminth infection. Graphical Abstract

Abstract Background Ovarian follicular fluid (FF) is a dynamic environment that changes with the seasons, affecting follicle development, ovulation, and oocyte quality. Cells in the follicles release tiny particles called extracellular vesicles (EVs) containing vital regulatory molecules, such as microRNAs (miRNAs). These miRNAs are pivotal in facilitating communication within the follicles through diverse signaling and information transfer forms. EV-coupled miRNA signaling is implicated to be associated with ovarian function, follicle and oocyte growth and response to various environmental insults. Herein, we investigated how seasonal variations directly influence the ovulatory and anovulatory states of ovarian follicles and how are they associated with follicular fluid EV-coupled miRNA dynamics in horses. Results Ultrasonographic monitoring and follicular fluid aspiration of preovulatory follicles in horses during the anovulatory (spring: non-breeding) and ovulatory (spring, summer, and fall: breeding) seasons and subsequent EV isolation and miRNA profiling identified significant variation in EV-miRNA cargo content. We identified 97 miRNAs with differential expression among the groups and specific clusters of miRNAs involved in the spring transition (miR-149, -200b, -206, -221, -328, and -615) and peak breeding period (including miR-143, -192, -451, -302b, -100, and let-7c). Bioinformatic analyses showed enrichments in various biological functions, e.g., transcription factor activity, transcription and transcription regulation, nucleic acid binding, sequence-specific DNA binding, p53 signaling, and post-translational modifications. Cluster analyses revealed distinct sets of significantly up- and down-regulated miRNAs associated with spring anovulatory (Cluster 1) and summer ovulation–the peak breeding season (Clusters 4 and 6). Conclusions The findings from the current study shed light on the dynamics of FF-EV-coupled miRNAs in relation to equine ovulatory and anovulatory seasons, and their roles in understanding the mechanisms involved in seasonal shifts and ovulation during the breeding season warrant further investigation.

Abstract Background Bacterial extracellular vesicles (EVs) are pivotal mediators of intercellular communication and influence host cell biology, thereby contributing to the pathogenesis of infections. Despite their significance, the precise effects of bacterial EVs on the host cells remain poorly understood. This study aimed to elucidate ultrastructural changes in host cells upon infection with EVs derived from a pathogenic bacterium, Staphylococcus aureus (S. aureus). Results Using super-resolution fluorescence microscopy and high-voltage electron microscopy, we investigated the nanoscale alterations in mitochondria, endoplasmic reticulum (ER), Golgi apparatus, lysosomes, and microtubules of skin cells infected with bacterial EVs. Our results revealed significant mitochondrial fission, loss of cristae, transformation of the ER from tubular to sheet-like structures, and fragmentation of the Golgi apparatus in cells infected with S. aureus EVs, in contrast to the negligible effects observed following S. epidermidis EV infection, probably due to the pathogenic factors in S. aureus EV, including protein A and enterotoxin. These findings indicate that bacterial EVs, particularly those from pathogenic strains, induce profound ultrastructural changes of host cells that can disrupt cellular homeostasis and contribute to infection pathogenesis. Conclusions This study advances the understanding of bacterial EV-host cell interactions and contributes to the development of new diagnostic and therapeutic strategies for bacterial infections.

Mitochondria are multitasking organelles involved in maintaining the cell homoeostasis. Beyond its well-established role in cellular bioenergetics, mitochondria also function as signal organelles to propagate various cellular outcomes. However, mitochondria have a self-destructive arsenal of factors driving the development of diseases caused by mitochondrial dysfunction. Extracellular vesicles (EVs), a heterogeneous group of membranous nano-sized vesicles, are present in a variety of bodily fluids. EVs serve as mediators for intercellular interaction. Exosomes are a class of small EVs (30–100 nm) released by most cells. Exosomes carry various cargo including microRNAs (miRNAs), a class of short noncoding RNAs. Recent studies have closely associated exosomal miRNAs with various human diseases, including diseases caused by mitochondrial dysfunction, which are a group of complex multifactorial diseases and have not been comprehensively described. In this review, we first briefly introduce the characteristics of EVs. Then, we focus on possible mechanisms regarding exosome-mitochondria interaction through integrating signalling networks. Moreover, we summarize recent advances in the knowledge of the role of exosomal miRNAs in various diseases, describing how mitochondria are changed in disease status. Finally, we propose future research directions to provide a novel therapeutic strategy that could slow the disease progress mediated by mitochondrial dysfunction.