Journal of Extracellular Vesicles最新文献

Intercellular communication via extracellular vesicles (EVs) has been identified as a vital component of a steadily expanding number of physiological and pathological processes. To accommodate these roles, EVs have highly heterogeneous molecular compositions. Given that surface molecules on EVs determine their interactions with their environment, EV functionality likely differs between subpopulations with varying surface compositions. However, it has been technically challenging to examine such functional heterogeneity due to a lack of non-destructive methods to separate EV subpopulations based on their surface markers. Here, we used the Design-of-Experiments (DoE) methodology to optimize a protocol, which we name ‘EV-Elute’, to elute intact EVs from commercially available Protein G-coated magnetic beads. We captured EVs from various cell types on these beads using antibodies against CD9, CD63, CD81 and a custom-made protein binding phosphatidylserine (PS). When applying EV-Elute, over 70% of bound EVs could be recovered from the beads in a pH- and incubation-time-dependent fashion. EV subpopulations showed intact integrity by electron microscopy and Proteinase K protection assays and showed uptake patterns similar to whole EV isolates in co-cultures of peripheral blood mononuclear cells (PBMCs) and endothelial cells. However, in Cas9/sgRNA delivery assays, CD63⁺ EVs showed a lower capacity to functionally deliver cargo as compared to CD9⁺, CD81⁺ and PS⁺ EVs. Taken together, we developed a novel, easy-to-use platform to isolate and functionally compare surface marker-defined EV subpopulations. This platform does not require specialized equipment or reagents and is universally applicable to any capturing antibody and EV source. Hence, EV-Elute can open new opportunities to study EV functionality at the subpopulation level.

T-cell haematological malignancies progress rapidly and have a high mortality rate and effective treatments are still lacking. Here, we developed a drug delivery system utilizing 293T cell-derived extracellular vesicles (EVs) modified with an anti-CD7 single-chain variable fragment (αCD7/EVs). Given the challenges of chemotherapy resistance in patients with T-cell malignancy, we selected cytochrome C (CytC) and Bcl2 siRNA (siBcl2) as therapeutic agents and loaded them into αCD7/EVs (αCD7/EVs/CytC/siBcl2). We found that αCD7/EVs efficiently targeted and were internalized by human T-ALL Molt-4 cells. In addition, the interaction between αCD7 and CD7 switched the EV entry pathway in Molt-4 cells from macropinocytosis-dependent endocytosis to clathrin-mediated endocytosis, thereby reducing EV-lysosome colocalization, ultimately improving CytC delivery efficiency and increasing the cytotoxicity of nascent EVs from EV-treated Molt-4 cells. Notably, αCD7/EVs/CytC/siBcl2 demonstrated similar efficacy against both Molt-4 and chemotherapy-resistant Molt-4 cells (CR-Molt-4). Furthermore, αCD7/EVs/CytC/siBcl2 exhibited high safety, low immunogenicity and minimal impact on human T cells. Therefore, αCD7/EVs/CytC/siBcl2 are promising therapeutic approaches for treating CD7⁺ T-cell malignancies.

Due to their intercellular communication properties and involvement in a wide range of biological processes, extracellular vesicles (EVs) are increasingly being studied and exploited for different applications. Nevertheless, their complex nature and heterogeneity, as well as the challenges related to their purification and characterization procedures, require a cautious assessment of the qualitative and quantitative parameters that need to be monitored. This translates into a multitude of choices and putative solutions that any EV researcher must confront in both research and translational environments. In this respect, decision-making tools may help assess various options, weigh pros and cons, and ultimately arrive at a thought-out decision that considers both the best fit-to-source and fit-to-scope EV application(s). Here, we present a multi-criteria EV decision-making grid (EV-DMG) as a novel, efficient, customizable, and easy-to-use tool to support EV research and innovation. By identifying and weighing key assessment criteria for comparing distinct EV-based preparations and related processes, our EV-DMG may assist any EV community member in making informed, traceable, and reproducible decisions regarding the management of EV sources or samples. Ultimately, this EV-DMG may guide the adoption of the most suitable EV production and analytical pipelines for targeting a defined aim or application.

Palviainen, M., Puutio, J., Østergaard, R. H., Eble, J. A., Maaninka, K., Butt, U., Ndika, J., Kari, O. K., Kamali-Moghaddam, M., Kjaer-Sorensen, K., Oxvig, C., Aransay, A. M., Falcon-Perez, J. M., Federico, A., Greco, D., Laitinen, S., Hayashi, Y., & Siljander, P. R.-M. (2024). Beyond basic characterization and omics: Immunomodulatory roles of platelet-derived extracellular vesicles unveiled by functional testing. Journal of Extracellular Vesicles, 13, e12513. https://doi.org/10.1002/jev2.12513

In Figure 1(f), the 14 hpi column showed the same image in the first row and the second row, whereas it should instead show a high contrast image of the first row following a different colour scheme as correctly shown in the other three columns (1, 4 and 7 hpi). This has been corrected by replacing the image in question. This does not affect the scientific content or the conclusion, since the results have already been fully presented in the first row of images, whereas the second row shows the same results with visual enhancement to highlight two of the three colours for clarity. There was also a typo in the label for the second row “(High constrast)”, which should have read “(High contrast)”.

In Figure 1(a), in the bright-field image, the scale bar was not fully shown. This has also been corrected by adjusting its position.

We apologize for these errors.

In recent years, extracellular vesicles (EVs) have emerged as novel key players in plant–microbe interactions. While it is immensely useful to draw on the established “minimal information for studies of extracellular vesicles” (MISEV) guidelines and precedents in mammalian systems, working with plants and their associated microbes poses specific challenges. To navigate researchers through these obstacles, we offer detailed step-by-step suggestions for those embarking on EV research in the context of plant–microbe interactions. The advice is based on recent publications and our collective experience from the diverse plant and microbe systems studied in a dedicated research consortium. We provide considerations for experimental design, optimization, quality control, and recommendations on how to increase yield, purity, and reproducibility of EV isolation. With this perspective article, we aim not only to assist researchers in our field but also to promote discussions on plant and microbe EVs in the broader EV community.

With an evolving understanding and new discoveries in extracellular vesicle (EV) biology and their implications in health and disease, the significant diagnostic and therapeutic potential of EVs for vision research has gained recognition. In 2021, the National Eye Institute (NEI) unveiled its Strategic Plan titled ‘Vision for the Future (2021–2025),’ which listed EV research as a priority within the domain of Regenerative Medicine, a pivotal area outlined in the Plan. In alignment with this prioritization, NEI organized a workshop inviting twenty experts from within and beyond the visual system. The workshop aimed to review current knowledge in EV research and explore gaps, needs and opportunities for EV research in the eye, including EV biology and applications of EVs in diagnosis, therapy and prognosis within the visual system. This perspective encapsulates the workshop's deliberations, highlighting the current landscape and potential implications of EV research in advancing eye health and addressing visual diseases.

Small extracellular vesicles and nanoparticles (sEVPs) are cell-secreted entities with potential as diagnostic biomarkers and therapeutic vehicles. However, significant intrinsic sEVP heterogeneity impedes analysis and understanding of their composition and functions. We employ multidimensional fluorescent labelling on sEVPs, leveraging the robustness of a newly developed membrane probe—conjugated oligoelectrolytes (COEs), and conduct total internal reflection fluorescence (TIRF) microscopy on sEVP arrays. These arrays comprise single sEVPs anchored to a soft material functionalized surface with little bias. We then develop an enhanced algorithm for colocalization analysis of the multiple labels on individual sEVPs and perform deep profiling of particle content. We categorize sEVPs derived from the same cell type into seven distinct subpopulations—some vesicular whereas others non-vesicular, and we demonstrate that sEVPs from four cell types exhibit quantitatively distinguishable subpopulation distributions. Furthermore, we gain insights into specific particle features within each subpopulation, including CD63 counts, relative particle size, relative concentration of cargoes, and correlations among different cargoes. This high-content analysis reveals common cargo sorting features in sEVP subpopulations across different cell types and suggests new statistics within the sEVP inherent heterogeneity that could differentiate sEVPs from two types of cancer cells and two types of normal cells. Collectively, our study presents a robust single-sEVP characterization platform, combining high-content imaging with comprehensive analysis. This platform is poised to advance sEVP-based theranostic assays and facilitate exploration into disease-associated sEVP biogenesis and sEVP-mediated intercellular communication.

The study introduces a charge-based fractionation method for fractionating plasma-derived extracellular vesicles (EVs) into sub-populations aimed at the improved purification from free plasma proteins to enhance the diagnostic potential of EV sub-populations for specific pathophysiological states. Here, we present a novel approach for EV fractionation that leverages EVs’ inherent surface charges, differentiating them from other plasma components and, thus, reducing the sample complexity and increasing the purity of EVs. The developed method was optimized and thoroughly evaluated using proteomic analysis, transmission electron microscopy, nanoparticle tracking, and western blotting of isolated EVs from healthy donors. Subsequently, we pilot-tested the developed technique for its applicability to real-world specimens using a small set of clinical prostate cancer samples and matched controls. The presented technique demonstrates the effective isolation and fractionation of EV sub-populations based on their surface charge, which may potentially help enhance EV-based diagnostics, biomarker discovery, and basic biology research. The method is designed to be straightforward, scalable, easy-to-use, and it does not require specialized skills or equipment.

The application of extracellular vesicles (EVs) as vehicles for anti-Parkinson's agents represents a significant advance, yet their clinical translation is hampered by challenges in efficient brain delivery and complex blood-brain barrier (BBB) targeting strategies. In this study, we engineered dopamine onto the surface of adipose-derived stem cell EVs (Dopa-EVs) utilizing a facile, two-step cross-linking approach. This engineering enhanced neuronal uptake of the EVs in primary neurons and neuroblastoma cells, a process shown to be competitively inhibited by dopamine pretreatment and dopamine receptor antibodies. Notably, Dopa-EVs demonstrated increased brain accumulation in mouse Parkinson's disease (PD) models. Therapeutically, Dopa-EVs administration led to the rescue of dopaminergic neuronal loss and amelioration of behavioural deficits in both 6-hydroxydopamine (6-OHDA) and α-Syn PFF-induced PD models. Furthermore, we observed that Dopa-EVs stimulated autophagy evidenced by the upregulation of Beclin-1 and LC3-II. These findings collectively indicate that surface modification of EVs with dopamine presents a potent strategy for targeting dopaminergic neurons in the brain. The remarkable therapeutic potential of Dopa-EVs, demonstrated in PD models, positions them as a highly promising candidate for PD treatment, offering a significant advance over current therapeutic modalities.