Journal of extracellular biology最新文献

Natural killer (NK) cells are exploited in cellular therapies for cancer. While NK cell therapies are efficient against haematological cancers, it has been difficult to target solid tumours due to low tumour infiltration and a hostile tumour microenvironment. NK-cell derived extracellular vesicles (NK-EVs) target and kill cancer cells in vitro and represent an alternative treatment strategy for solid tumours. To exploit their potential, it is necessary to standardize NK-EV production protocols. Here, we have performed a comparative analysis of EVs from the human NK-92 cell line cultured in five serum-free commercial media optimized for growth of human NK cells and one serum-free medium for growth of lymphocytes. The effect of growing the NK-92 cells in static cell cultures versus shaking flasks was compared. EVs were purified via ultracentrifugation followed by size-exclusion chromatography. We found that there were no significant differences in EV yield from NK-92 cells grown under static or dynamic conditions. However, we found clear differences between the different culture media in terms of EV purity as assessed by the enrichment of the CD63 and CD81 markers in the isolates that translated into their capacity to induce apoptosis of the colon cancer cell line HCT 116. These findings will be instructive for the design of future production protocols for therapeutic NK-cell derived EVs.

Extracellular vesicles (EVs) are crucial for intercellular communication and are found in various biological fluids. The identification and immunophenotyping of such small particles continue to pose significant challenges. Here, we have developed a workflow for the optimisation of a next-generation panel for in-depth immunophenotyping of circulating plasma EVs using spectral flow cytometry. Our data collection followed a multistep optimisation phase for both instrument setup and 21-colour panel design, thus maximising fluorescent signal recovery. This spectral approach enabled the identification of novel EV subpopulations. Indeed, besides common EVs released by erythrocytes, platelets, leukocytes and endothelial cells, we observed rare and poorly known EV subsets carrying antigens related to cell activation or exhaustion. Notably, the unsupervised data analysis of major EV subsets revealed subpopulations expressing up to five surface antigens simultaneously. However, the majority of EVs expressed only a single surface antigen, suggesting they may not fully represent the phenotype of their parent cells. This is likely due to the small surface area or the biogenesis of EVs rather than antibody steric hindrance. Finally, we tested our workflow by analysing the plasma EV landscape in a cohort of systemic lupus erythematosus (SLE) patients. Interestingly, we observed a significant increase in CD54⁺ EVs, supporting the notion of elevated circulating ICAM under SLE conditions. To our knowledge, these are the first data highlighting the importance of a spectral flow cytometry approach in deciphering the heterogeneity of plasma EVs paving the way for the routine use of a high-dimensional immunophenotyping in EV research.

The ‛stickiness’ of extracellular vesicles (EVs) can pose challenges for EV processing and storage, but adhesive properties may also be exploited to immobilise EVs directly on surfaces for various measurement techniques, including super-resolution microscopy (SRM). Direct adhesion to surfaces may allow the examination of broader populations of EVs than molecular affinity approaches, which can also involve specialised, expensive affinity reagents. Here, we report on the interaction of EVs with borosilicate glass and quartz coverslips and on the effects of pre-coating coverslips with poly-L-lysine (PLL), a reagent commonly used to facilitate interactions between negatively charged surfaces of cells and amorphous surfaces. Additionally, we compared two mounting media conditions for SRM imaging and used immobilised EVs for a B-cell interaction test. Our findings suggest that borosilicate glass coverslips immobilise EVs better than quartz glass coverslips. We also found that PLL is not strictly required for EV retention but contributes to the uniform distribution of EVs on borosilicate glass coverslips. Overall, these findings suggest that standard lab materials like borosilicate glass coverslips, with or without PLL, can be effectively used for the immobilisation of EVs in specific imaging techniques.

Various molecules in plasma extracellular vesicles (EVs) are expected to be applied to minimally invasive diagnosis; however, the high concentration of lipoproteins in plasma, which are similar in size, density and content to EVs, hampers analysis on plasma EVs. To overcome this, we explored an effective isolation method for plasma EVs that excludes lipoproteins by applying precipitation methods that are conventionally used to separate lipoproteins. Human plasma was mixed with heparin and MnCl₂, phosphotungstic acid and MgCl₂, or polyethylene glycol (PEG), and the expression level of CD9, Apo B and Apo A-I in both the supernatant and pellet was measured by enzyme-linked immunosorbent assay. Morphology was observed by transmission electron microscopy to assess EV yield and lipoprotein contamination. The combination of heparin and MnCl₂, or phosphotungstic acid and MgCl₂, could not separate plasma EVs and lipoproteins. PEG precipitated EVs and lipoproteins differently, and EVs were specifically precipitated by PEG (3%) to some extent. In comparison with differential ultracentrifugation (UC), size-exclusion chromatography, density gradient centrifugation and precipitation with PEG (8%) followed by UC, PEG (3%) was not inferior in efficiency but was superior in terms of time and cost. The precipitation method using PEG (3%) may contribute to the application of plasma EVs in disease diagnosis.

Plant-derived substances are widely used as cosmeceutical and food materials owing to their beneficial properties that promote human health, such as antioxidant, nutritional supply and regenerative potential. In particular, nanovesicles (NVs) from plants contain various biomolecules, including signal proteins, nucleic acids, and metabolites, that participate in cross-kingdom communication. In this study, we isolated NVs from Artemisia princeps (APNVs) based on differential centrifugation and further purification via tangential flow filtration (TFF). Evaluation of the effects of these NVs on the cellular proliferation of fibroblasts clearly indicated their anti-ageing potential for the skin. Specifically, exposure of human dermal fibroblast cells to low concentrations of APNVs (100–200 ng/mL) accelerated cell proliferation over a 7-day period. Treatment with APNVs decreased the senescence level of dermal fibroblast cells, as evidenced by senescence-associated β-galactosidase activity connected with cellular ageing. In the anti-ageing efficacy assessment, inhibition of MMP-1 activity in nanovesicle-treated cells was higher than that induced by the positive control epigallocatechin-3-gallate (EGCG). To validate the inhibitory effect of APNVs on anti-ageing in human skin, three-dimensional, reconstituted human keratinocytes and dermal fibroblasts were cultured with 1000 ng/mL APNVs. Notably, procollagen type I expression was increased in the culture medium following APNVs treatment. Our collective results suggest that APNVs accelerate type I procollagen production through inhibition of MMP-1. In view of the significant anti-ageing potential of APNVs, we recommend their implementation as an active substance in pharmaceutical and functional cosmeceutical products.

Neuronal circuitry remodelling, which comprises excitatory and inhibitory neurons, is critical for improving neurological outcomes after a stroke. Preclinical studies have shown that small extracellular vesicles (sEVs) have a therapeutic effect on stroke recovery. However, it is highly challenging to use sEVs to specifically target individual neuronal populations to enhance neuronal circuitry remodelling after stroke. In the present study, using a chemogenetic approach to specifically activate peri-infarct cortical interneurons in combination with the administration of sEVs derived from cerebral endothelial cells (CEC-sEVs), we showed that the CEC-sEVs were preferentially taken up by the activated neurons, leading to significant improvement of functional outcome after stroke, which was associated with augmentation of peri-infarct cortical axonal/dendritic outgrowth and of axonal remodelling of the corticospinal tract. The ultrastructural and Western blot analyses revealed that neurons with internalization of CEC-sEVs exhibited significantly reduced numbers of damaged mitochondria and proteins that mediate dysfunctional mitochondria, respectively. Together, these data indicate that the augmented uptake of CEC-sEVs by activated peri-infarct cortical interneurons facilitates neuronal circuitry remodelling and functional recovery after stroke, which has the potential to be a novel therapy for improving stroke recovery.

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) have shown significant therapeutic potential across a wide range of clinical conditions, complementing the progress of MSC-based therapies, some of which have already received regulatory approval. However, the high cost of these therapies has limited their accessibility, creating an urgent need to explore manufacturing strategies that reduce the cost of goods and selling prices. This study presents the design and simulation of a scalable manufacturing platform for the co-production of clinical-grade MSC and MSC-EVs using SuperPro Designer. Various production scenarios were evaluated to maximise manufacturing capacity while analysing their impact on economic performance. Our findings demonstrate that for MSC-EVs doses containing 10¹⁰ and 10¹¹ particles, selling prices range from 166 to 309€ and from 1659 to 3082€, respectively. For clinical doses of MSC, selling prices vary between 965 and 42,673€ depending on dose size and production scale. Importantly, the co-production approach enables cost-sharing between products, contributing to significantly lower prices compared to individual production. Overall, the proposed platform achieved an attractive payback time of 3 years and a return on investment of 36%. By increasing the number of staggered production units, further price reductions and improved economic metrics could be attained. In conclusion, this study highlights the potential of the proposed manufacturing platform to deliver cost-effective, clinical-grade MSC and MSC-EVs products, advancing the field of regenerative medicine and enhancing the accessibility of these innovative treatments.

Several reports have demonstrated that CD147, an N-glycosylated protein that is exchanged by cells in soluble form or through small extracellular vesicles (sEVs), can promote cancer progression. However, its activity related to EVs in colorectal cancer (CRC) is still not fully understood. Previously, we showed that sEV secretion during CRC stem cell (CR-CSCs) differentiation is partially controlled by CD147, and that CD147-expressing sEVs (sEVs-CD147) activate a signalling cascade in recipient cells, inducing molecular invasive features in CR-CSCs. In the present study, we demonstrated that sEVs-CD147 increase the expression of myofibroblast and activation markers in cancer-associated fibroblasts (CAF). In sEVs-CD147-activated CAF, aerobic glycolysis was also triggered by the β-catenin signalling pathway and induced lactate release. These effects were associated with NFKβ upregulation and NO secretion that caused increased cytokines production and VEGF release, respectively. Furthermore, co-culture with CAF promoted CR-CSC invasivity in vitro and tumour growth in vivo. Spatial proteomics analysis confirmed in vivo the activation of fibroblasts and the modulation of their metabolic features, within their biological context, after their conditioning with CD147-expressing sEVs. Our findings indicate that sEV-packaged CD147 is involved in CAF activation, thus promoting tumour progression via stroma metabolism modification.

Extracellular vesicles (EVs) are secreted nanoparticles composed of a lipid bilayer that carry lipid, protein, and nucleic acid cargo between cells as a mode of intercellular communication. Although EVs can promote tissue repair in mammals, their roles in animals with greater regenerative capacity are not well understood. Planarian flatworms are capable of whole-body regeneration due to pluripotent somatic stem cells called neoblasts that proliferate in response to injury. Here, using transmission electron microscopy, nanoparticle tracking analysis, and protein content examination, we showed that EVs enriched from the tissues of the planarian Schmidtea mediterranea had similar morphology and size as other eukaryotic EVs, and that these EVs carried orthologs of the conserved EV biogenesis regulators ALIX and TSG101. PKH67-labeled EVs were taken up efficiently by planarian cells, including S/G2 neoblasts, G1 neoblasts/early progeny, and differentiated cells. When injected into living planarians, EVs from regenerating tissue fragments enhanced the upregulation of neoblast-enriched and proliferation-related transcripts. In addition, EV injection increased the number of F-ara-EdU-labelled cells by 49% as compared to buffer injection only. Our findings demonstrate that regenerating planarians produce EVs that promote stem cell proliferation, and suggest the planarian as an amenable in vivo model for the study of EV function during regeneration.

As a life history strategy, some semelparous organisms, such as the ayu fish (Plecoglossus altivelis), reproduce only once in their lifetime and then die. They invest heavily in their single reproductive event, producing many offspring. However, the regulatory mechanisms that trigger mortality after reproduction are not well understood. Exosomes serve as an essential pathway for intercellular communication, with internal microRNA (miRNA) playing a crucial role in regulating physiological activities within the organism. This study aimed to elucidate the function of exosomal miRNA during the semelparous reproduction period in P. altivelis. Exosomes were successfully extracted from P. altivelis plasma during reproduction, and abundant miRNA molecules were discovered through small RNA sequencing. The miRNA expression patterns in ayu fish during reproduction exhibited notable differences between females and males. Furthermore, it was observed that key cellular processes and signalling pathways associated with intercellular transmission and intracellular stress are regulated by miRNA expression, and these changes in regulation may be responsible for post-breeding mortality. This study enhances our understanding of the function of exosomal miRNA during semelparous reproduction in ayu fish and provides further insight into the intrinsic mechanisms of ageing and mortality in organisms.