Bacterial extracellular vesicles (BEVs) are naturally occurring functional structures that play critical roles in bacterial life processes. These vesicles, commonly known as outer membrane vesicles (OMVs), were first found to be released by Gram-negative bacteria; however, it has since been confirmed that Gram-positive bacteria also secrete BEVs. As research advances, BEVs are increasingly utilized in diverse applications, including vaccine development and drug delivery. Nevertheless, the effective employment of BEVs in these contexts requires the acquisition of vesicles with consistent properties and functions through appropriate culture, isolation, and purification methods. This review examines the advantages and disadvantages of various purification techniques alongside the heterogeneity they may introduce. We utilize the heterogeneity of BEVs as a framework to critically analyze the barriers to their application and the factors influencing their characteristics. Additionally, we constructively propose solutions to enhance the consistency of BEVs, thereby facilitating their further development and application.

Aim: Cells in the human body release extracellular vesicles (EVs) into fluids, such as plasma, urine, and cerebrospinal fluid. EVs express tetraspanin family proteins (e.g., CD63, CD9, and CD81) and cell-specific antigens on their surface as common and specific markers, respectively. In this study, we hypothesized that the profile of blood cell-derived circulating EVs could reveal both common and specific pathophysiology in atherogenic diseases.

Methods: Using surface plasmon resonance imaging (SPRi), we analyzed EVs surface molecules and identified circulating EVs in healthy controls (n = 18), patients with type 2 diabetes mellitus (T2DM; n = 71), and those with hypertension (HT; n = 47).

Results: Patients with T2DM and HT exhibited distinct EV profiles: (i) CD9, CD110, CD20, activin receptor type-2A (AcvRIIA), Duffy antigen receptor for chemokine, and CD44 positive EVs were upregulated in T2DM; (ii) CD9, Maackia amurensis agglutinin lectin binding molecules (MBM), CD20, AcvRIIA, and CD44 positive EVs were upregulated in HT. By analyzing an appropriate set of three antigens or using dimensional reduction clustering, we were able to clearly differentiate between T2DM, HT, and control groups. In some patients, disease severity correlated with CD44 and CD20 in T2DM and MBM and AcvRIIA in HT.

Conclusion: Our findings demonstrate that profiling of circulating EVs via the SPRi method offers a novel approach for diagnosing and monitoring human diseases.

Triple-negative breast cancer (TNBC) is one of the most aggressive and challenging subtypes for treatment, due to the lack of hormone receptors and the human epidermal growth factor receptor 2 (HER2) protein. The identification of new molecular targets is important for the development of targeted and specific therapies for TNBC patients. MicroRNAs (miRNAs) have emerged as promising molecular targets, being involved in cellular processes such as cell survival, apoptosis, differentiation, carcinogenesis, and metastasis. Extracellular vesicles (EVs) have gained prominence in areas such as drug delivery, immune modulation, biomarkers for diagnosis and prognosis, and therapeutics, due to their use as vehicles for the delivery of miRNAs, regulation of gene expression, and development of combined therapeutic strategies. In particular, mesenchymal stem cell-derived EVs (MSC-derived EVs) can transfer proteins, mRNAs/miRNAs, or DNA molecules and are being considered safer treatment options due to their inability to directly form tumors and contain lower amounts of membrane proteins such as MHC molecules. Numerous studies have highlighted the role of miRNAs in EVs in TNBC tumorigenesis, with a focus on diagnosis, prognosis, treatment selection, and monitoring. However, the development of therapies with EVs, especially MSC-derived EVs, is still in its infancy. Therefore, the aim of this review is to address new therapeutic strategies based on the delivery of miRNAs through EVs, with a focus on MSC-derived EVs, for the treatment of TNBC as an innovative therapy in oncology.

Aim: A PCR- and sequencing-free mutation detection assay facilitates cancer diagnosis and reduces over-reliance on specialized equipment. This benefit was highlighted during the pandemic when high demand for viral nucleic acid testing often sidelined mutation analysis. This shift led to substantial challenges for patients on targeted therapy in tracking mutations. Here, we report a 30-min DNA mutation detection technique using Cas12a-loaded liposomes in a microplate reader, a fundamental laboratory tool.

Methods: CRISPR-Cas12a complex and fluorescence-quenching (FQ) probes are introduced into tumor-derived extracellular vesicles (EV) through membrane fusion. When CRISPR-RNA hybridizes with the DNA target, activated Cas12a can trans-cleave FQ probes, resulting in fluorescence signals for the quantification of DNA mutation.

Results: This method enables the detection of EGFR L858R mutation in EV DNA within 30 min. Laborious extraction, purification, and other preparation steps for EV DNA are eliminated. The need for advanced data processing is also dispensed with. In a cohort study involving 10 healthy donors and 30 patients with advanced non-small cell lung cancer (NSCLC), the assay achieved a sensitivity of 86.7%, a specificity of 90%, and an accuracy of 87.5%.

Conclusion: The limit of detection of our Cas12 assay was ~ 8 × 10⁵ EVs, corresponding to a mutation allele frequency (MAF) of ~ 10%. The MAF in late-stage cancers varies widely but often falls within 5%-50%. Therefore, without amplification of targets, this Cas12 assay can detect mutations in patients with advanced lung cancer. Future advancements in multiplex and high-throughput mutation detection using this assay will streamline self-diagnosis and treatment monitoring at home.

Aim: Microglial activation plays a pivotal role in the pathogenesis of retinal ganglion cell (RGC) degeneration resulting from optic nerve crush (ONC). Small extracellular vesicles (sEVs) secreted by mesenchymal stem cells (MSCs) have the potential to prevent retinal degeneration by modulating microglial activation. In this study, we elucidated the specific effects of sEVs derived from IFN-γ-primed MSCs on the phenotypic transition of microglia and the associated pathways in ONC mice.

Methods: The ONC mice model was established and administered intravitreal injection with the sEVs derived from native MSCs (native sEVs) and the sEVs derived from MSCs primed with IFN-γ (IFNγ-sEVs). Their respective effects on the survival of the retinal ganglion cells (RGCs) and the transition of microglia phenotypes were determined through visual function testing and immunohistochemical staining. Combined with mRNA seq and microRNA seq techniques, we elucidated the mechanism of modulation of microglia phenotypic transformation by sEVs derived from MSCs primed by IFNγ.

Results: It demonstrated that IFNγ-sEVs exhibited superior protective effects against RGC loss and reduced inflammatory responses in the ONC retina compared to native sEVs. Both types of sEVs promoted microglia activation to disease-associated microglia (DAM) phenotype, while IFNγ-sEVs especially suppressed interferon-responsive microglia (IRM) activation during RGCs degeneration. Subsequent miRNA sequencing suggested that miR-423-5p, which exhibited the most significant differential expression between the two sEVs types and elevated expression in IFNγ-sEVs, inhibited the expression of IRM and ribosomal genes.

Conclusion: These findings suggest that IFN-γ-preconditioned MSCs may enhance sEVs of neuroprotection on RGCs by suppressing IRM activation through the secretion of sEVs containing specific microRNAs in ONC mice.

The role of extracellular vesicles (EVs) in mediating chemoresistance has gained significant attention due to their ability to transfer bioactive molecules between drug-resistant and drug-sensitive cells. In particular, they have been demonstrated to play an active part in breast cancer chemoresistance by the horizontal transfer of genetic and protein material. This review highlights the role of EVs, particularly their miRNA cargo, in driving drug resistance in breast cancer. EVs derived from chemoresistant cells carry miRNAs and lncRNAs, which are known to modulate gene networks involved in cell proliferation and survival. These cargo molecules suppress apoptosis by targeting pro-apoptotic genes like PTEN and BIM, promote epithelial-mesenchymal transition (EMT) through the regulation of pathways such as TGF-β and Wnt/b-catenin, and contribute to tumor growth and resistance by enhancing angiogenesis and modulating the tumor microenvironment. Beyond RNA-mediated effects, EVs also transfer functional proteins, including P-glycoprotein and Hsp70, which impact cellular metabolism and survival pathways. Our findings underscore the significance of EVs in breast cancer chemoresistance, suggesting their potential involvement as possible prognostic factors to predict therapy response and as therapeutic targets in combination with usual therapy.

Artificial intelligence (AI) is revolutionizing scientific research by facilitating a paradigm shift in data analysis and discovery. This transformation is characterized by a fundamental change in scientific methods and concepts due to AI’s ability to process vast datasets with unprecedented speed and accuracy. In breast cancer research, AI aids in early detection, prognosis, and personalized treatment strategies. Liquid biopsy, a noninvasive tool for detecting circulating tumor traits, could ideally benefit from AI’s analytical capabilities, enhancing the detection of minimal residual disease and improving treatment monitoring. Extracellular vesicles (EVs), which are key elements in cell communication and cancer progression, could be analyzed with AI to identify disease-specific biomarkers. AI combined with EV analysis promises an enhancement in diagnosis precision, aiding in early detection and treatment monitoring. Studies show that AI can differentiate cancer types and predict drug efficacy, exemplifying its potential in personalized medicine. Overall, the integration of AI in biomedical research and clinical practice promises significant changes and advancements in diagnostics, personalized medicine-based approaches, and our understanding of complex diseases like cancer.

Aim: Exosomes derived from adipose-derived stem cells (ASCs) in mice have been reported to influence immune regulation. Yet, the potential immunological effects of ASCs-derived exosomes and their interaction with lymphocytes during transplant immunity remain understudied.

Methods: ASCs from BALB/c mice, along with their conditioned culture medium, were collected for the extraction, isolation, and comprehensive characterization of exosomes. Splenic cell suspensions were isolated from BALB/c mice and subsequently processed for downstream analyses. Lymphocytes were isolated via gradient centrifugation and stimulated in vitro with the purified exosomes to assess their functional responses. Lymphocyte proliferation was quantified using the CCK8 assay, and the relative frequencies of CD4+ T cells, CD8+ T cells, Treg cells, NK (natural killer) cells, macrophages, B cells, dendritic cells (DCs), and Th17 cells were determined through flow cytometric analysis. Before establishing the skin transplantation model, the mice were administered PBS, 0.5 × 10⁸ exosomes, 1 × 10⁸ exosomes, 1.5 × 10⁸ exosomes, or ASCs via intravenous injection through the tail vein. Seven days after transplantation, the spleens, drainage lymph nodes, and blood samples were harvested for lymphocyte isolation and further downstream analyses.

Results: Exosomes derived from ASCs significantly increased the CD4+/CD8+ ratio and Treg cell levels, without inducing any notable changes in Th17 cell content or CTLA-4 protein expression in CD4+ T cells. Compared to the PBS-treated group, both ASC and exosome treatment groups demonstrated an enhanced CD4+/CD8+ ratio, increased Treg cell content, and elevated CTLA-4 protein expression in spleen tissue following skin transplantation, while Th17 cell levels remained unaffected. Compared to the ASC treatment group, the exosome group exhibited a higher CD4+/CD8+ ratio and Treg cell levels, alongside a reduced proportion of PD-1+ Treg cells and lower CTLA-4 protein expression in CD3+CD4+ T cells. No significant differences were observed in the proportions of NK cells, macrophages, B cells, and DCs in the spleens across all treatment groups. In peripheral blood, an increased proportion of CD3+ T cells, macrophages, and DCs was detected, accompanied by a reduced proportion of NK cells and B cells. In the draining lymph nodes, no significant changes were observed in the proportions of CD3+ T cells and B cells, while macrophages, NK cells, and DCs showed elevated proportions. In the exosome-treated group, mouse grafts exhibited a disorganized and thinner granular layer, accompanied by focal regions of inflammatory cell infiltration. Both exosome and ASC treatments significantly extended the survival of skin grafts.

Conclusion: Exosomes derived from ASCs promote lymphocyte proliferation and modulate their phenotypic profiles in mouse skin graft models, effectively extending graft survival.

The signaling gas hydrogen sulfide (H₂S) has recently been implicated in the regulation of bone remodeling and the maintenance of periodontal health. Exploring the underlying mechanisms for this regulation holds promise for the development of new treatment strategies to block bone resorption and stimulate bone regeneration. A recent study by Zhou et al. (Bioactive Materials, 2024) showed that treatment with H₂S stimulated changes in the extracellular vesicles (EVs) released by M2 macrophages, enhancing their capacity to promote the osteogenic differentiation of mesenchymal stem cells in vitro. The H₂S-stimulated EVs, given together with mesenchymal stem cells (MSCs), also promoted bone regeneration in vivo in a mouse calvarial critical-size defect model. This activity was linked to augmented expression of moesin, a membrane-cytoskeletal linkage protein, which was found at increased levels in EVs from cells stimulated by H₂S. The study identifies a new strategy for generating EVs that are pro-osteogenic. It also uncovers a surprising role for moesin in stimulating osteogenesis in MSCs.

CD63 is a tetraspanin initially associated with late endosomes and contributes to numerous functions at the cell level, such as intracellular endosomal and lysosomal trafficking, adhesion, and motility. CD63 also plays a key role in the biogenesis and release of exosomes, i.e., small extracellular vesicles (EVs) of endosomal origin, facilitating the formation of multivesicular bodies (MVBs), the coordination with the endosomal sorting complexes required for transport (ESCRT) machinery, the selection of cargoes carried by future exosomes, and the fusion of MVBs with the plasma membrane for exosome release. In a recent publication in Nature Cell Biology, Guillaume van Niel’s team provides arguments in favor of another EV-linked function for CD63, namely the regulation of cholesterol storage and release by small EVs of endogenous origin. Complemented by two other publications from the teams of Keisuke Ito and Xabier Ostreikoetxea, which respectively describe the role of (i) mitochondrial metabolism on CD63 function and (ii) the link between the reduced CD63⁺ small EVs and dyslipidemia, these arguments highlight the key role of CD63 in the regulation of cholesterol homeostasis through exosomes and more widely small EVs in physiological and pathological conditions. Future research on CD63 may thus redefine our approach to cellular lipid management and therapeutic lipid delivery.

Cytokines are released by cells in response to infections and tissue damage. A recent paper by Jiao et al. demonstrates that circulating monocytes release the cytokines tumor necrosis factor and Interleukin-6 encapsulated in large extracellular vesicles called migrasomes, from which the cytokines are secreted locally in a sustained fashion.

Mast cells (MCs) play a crucial role in immune responses by storing and releasing inflammatory mediators from secretory granules (SGs). The biogenesis, maturation, and fusion of these granules with the plasma membrane regulate inflammation, immune cell recruitment, and tissue homeostasis. However, the exact mechanism underlying this process remains unclear. Recent studies have identified a novel mechanism of SG fusion involving amphisomes, hybrid organelles formed by the fusion of late endosomes and autophagosomes. This process not only facilitates SG enlargement but also promotes the release of exosomes, small vesicles crucial for intercellular communication and immune modulation. In particular, Omari et al. delve into the molecular machinery governing amphisome formation and SG fusion, focusing on key players such as Rab5, PTPN9, CD63, and phosphoinositides (PIs). They propose a dynamic model wherein amphisomes act as intermediates in SG maturation, promoting homotypic fusion events that regulate SG content and size. A critical aspect of this process is the lipid signaling cascade, particularly involving PI4K and CD63, which coordinates SG fusion and exosome release. These findings challenge the conventional view of SGs as static storage compartments, positioning them as dynamic hubs of vesicle trafficking and secretion. By elucidating the role of amphisomes and lipid signaling in SG biology, this study offers a significant shift in understanding and introduces new concepts that could drive future research. This commentary, while endorsing the authors’ key conclusions, also highlights important questions regarding the functional implications of these novel mechanisms and their potential therapeutic applications.

A recent study introduces Stem Cell-derived Exosome Nebulization Therapy (SCENT), a novel, non-invasive strategy that delivers lung spheroid cell-derived exosomes via inhalation to promote cardiac repair after myocardial infarction. This approach improves cardiac function, reduces injury, and demonstrates translational potential in both small and large animal models, offering a promising avenue for cell-free, inhalable regenerative therapies.

Small extracellular vesicles (sEVs) derived from mesenchymal stromal cells (MSCs) hold substantial promise for therapeutic applications, including immune modulation and tissue regeneration. However, challenges such as batch-to-batch variability, donor material diversity, and the lack of standardized potency testing remain significant barriers to clinical translation. The recent U.S. Food and Drug Administration (FDA) approval of Ryoncil (remestemcel-L) for steroid-refractory acute graft-versus-host disease (aGvHD) in pediatric patients represents a crucial milestone for MSC-based therapies, offering also valuable insights for the development of MSC-EV therapies. This approval highlights the critical need to address variability and standardization issues in MSC products. Strategies like immortalizing MSCs and expanding them clonally can improve scalability, consistency, and overcome limitations inherent to cellular MSC therapies. With the FDA’s decision signaling significant progress, optimizing MSC expansion protocols and refining potency testing methods will be crucial for advancing MSC-EVs as a viable therapeutic option, overcoming current challenges, and expanding clinical applications.