Assessing Extracellular Vesicles in Human Biofluids Using Flow-Based Analyzers.

Extracellular vesicles (EVs) are increasingly being analyzed by flow cytometry. Yet their minuscule size and low refractive index cause the scatter intensity of most EVs to fall below the detection limit of most flow cytometers. A new class of devices, known as spectral flow analyzers, are becoming standards in cell phenotyping studies, largely due to their unique capacity to detect a vast panel of markers with higher sensitivity for light scatter detection. Another class of devices, known as nano-analyzers, provides high-resolution detection of sub-micron-sized particles. Here, the EV phenotyping performance between the Aurora (Cytek) spectral cell analyzer and the NanoFCM (nFCM) nanoflow analyzer are compared. These two devices are specifically chosen given their lead in becoming gold standards in their respective fields. Immune cell-derived EVs remain poorly characterized despite their clinical potential. Therefore, B- and T-cell line-derived EVs and donor-matched human biofluid-derived EVs from plasma, urine, and saliva are used in combination with a panel of established immune markers for this comparative study. A comparative evaluation of both cytometry platforms is performed, discussing their potential and suitability for different applications. It is found that nFCM can accurately i) analyze small EVs (40-200 nm) matching the size accuracy of electron microscopy; ii) measure the concentration of a single EV particle per volume; iii) identify underrepresented EV marker subsets; and iv) provide co-localization of EV surface markers. It can also be shown that human sample biofluids have unique EV marker signatures that can have future clinical relevance. Finally, nFCM and Aurora have their unique strength, preferred fashion of data acquisition, and visualization to fit different research interests.

Comparison of extracellular vesicle isolation processes for therapeutic applications.

While extracellular vesicles (EVs) continue to gain interest for therapeutic applications, their clinical translation is limited by a lack of optimal isolation methods. We sought to determine how universally applied isolation methods impact EV purity and yield. EVs were isolated by ultracentrifugation (UC), polyethylene glycol precipitation, Total Exosome Isolation Reagent, an aqueous two-phase system with and without repeat washes or size exclusion chromatography (SEC). EV-like particles could be detected for all isolation methods but varied in their purity and relative expression of surface markers (Alix, Annexin A2, CD9, CD63 and CD81). Assessments of sample purity were dependent on the specificity of characterisation method applied, with total particle counts and particle to protein (PtP) ratios often not aligning with quantitative measures of tetraspanin surface markers obtained using high-resolution nano-flow cytometry. While SEC resulted in the isolation of fewer particles with a relatively low PtP ratio (1.12 × 107 ± 1.43 × 106 vs highest recorded; ATPS/R 2.01 × 108 ± 1.15 × 109, p ⩽ 0.05), EVs isolated using this method displayed a comparatively high level of tetraspanin positivity (e.g. ExoELISA CD63⁺ particles; 1.36 × 1011 ± 1.18 × 1010 vs ATPS/R 2.58 × 1010 ± 1.92 × 109, p ⩽ 0.001). Results originating from an accompanying survey designed to evaluate pragmatic considerations surrounding method implementation (e.g. scalability and cost) identified that SEC and UC were favoured for overall efficiency. However, reservations were highlighted in the scalability of these methods, which could potentially hinder downstream therapeutic applications. In conclusion, variations in sample purity and yield were evident between isolation methods, while standard non-specific assessments of sample purity did not align with advanced quantitative high-resolution analysis of EV surface markers. Reproducible and specific assessments of EV purity will be critical for informing therapeutic studies.

Epigenetic Reprogramming via Synergistic Hypomethylation and Hypoxia Enhances the Therapeutic Efficacy of Mesenchymal Stem Cell Extracellular Vesicles for Bone Repair.

Mesenchymal stem cells (MSCs) are a promising cell population for regenerative medicine applications, where paracrine signalling through the extracellular vesicles (EVs) regulates bone tissue homeostasis and development. MSCs are known to reside in low oxygen tension, which promotes osteogenic differentiation via hypoxia-inducible factor-1α activation. Epigenetic reprogramming has emerged as a promising bioengineering strategy to enhance MSC differentiation. Particularly, the process of hypomethylation may enhance osteogenesis through gene activation. Therefore, this study aimed to investigate the synergistic effects of inducing hypomethylation and hypoxia on improving the therapeutic efficacy of EVs derived from human bone marrow MSCs (hBMSCs). The effects of the hypoxia mimetic agent deferoxamine (DFO) and the DNA methyltransferase inhibitor 5-azacytidine (AZT) on hBMSC viability was assessed by quantifying the DNA content. The epigenetic functionality was evaluated by assessing histone acetylation and histone methylation. hBMSC mineralisation was determined by quantifying alkaline phosphate activity, collagen production and calcium deposition. EVs were procured from AZT, DFO or AZT/DFO-treated hBMSCs over a two-week period, with EV size and concentration defined using transmission electron microscopy, nanoflow cytometry and dynamic light scattering. The effects of AZT-EVs, DFO-EVs or AZT/DFO-EVs on the epigenetic functionality and mineralisation of hBMSCs were evaluated. Moreover, the effects of hBMSC-EVs on human umbilical cord vein endothelial cells (HUVECs) angiogenesis was assessed by quantifying pro-angiogenic cytokine release. DFO and AZT caused a time-dose dependent reduction in hBMSC viability. Pre-treatment with AZT, DFO or AZT/DFO augmented the epigenetic functionality of the MSCs through increases in histone acetylation and hypomethylation. AZT, DFO and AZT/DFO pre-treatment significantly enhanced extracellular matrix collagen production and mineralisation in hBMSCs. EVs derived from AZT/DFO-preconditioned hBMSCs (AZT/DFO-EVs) enhanced the hBMSC proliferation, histone acetylation and hypomethylation when compared to EVs derived from AZT-treated, DFO-treated and untreated hBMSCs. Importantly, AZT/DFO-EVs significantly increased osteogenic differentiation and mineralisation of a secondary hBMSC population. Furthermore, AZT/DFO-EVs enhanced the pro-angiogenic cytokine release of HUVECs. Taken together, our findings demonstrate the considerable utility of synergistically inducing hypomethylation and hypoxia to improve the therapeutic efficacy of the MSC-EVs as a cell-free approach for bone regeneration.

TOP-EVs: Technology of Protein delivery through Extracellular Vesicles is a versatile platform for intracellular protein delivery.

Extracellular vesicles (EVs) have emerged as biocompatible drug delivery vehicles due to their native ability to deliver bioactive cargo to recipient cells. However, the application of EVs as a therapeutic delivery vehicle is hampered by effective methods for endogenously loading target proteins inside EVs and unloading proteins after delivery to recipient cells. Most EV-based engineered loading methods have a limited delivery efficiency owing to their inefficient endosomal escape or cargo release from the intraluminal attachment from the EV membrane. Here, we describe the 'Technology Of Protein delivery through Extracellular Vesicles' (TOP-EVs) as a tool for efficient intracellular delivery of target proteins mediated via EVs. The vesicular stomatitis virus glycoprotein and the rapamycin-heterodimerization of the FKBP12/T82L mutant FRB proteins were both important for the effective protein delivery through TOP-EVs. We showed that TOP-EVs could efficiently deliver Cre recombinase and CRISPR/Cas9 ribonucleoprotein complex in vitro. Moreover, our results demonstrated that the capacity of TOP-EVs to deliver intracellular proteins in recipient cells was not an artifact of plasmid contamination or direct plasmid loading into EVs. Finally, we showed that TOP-EVs could successfully mediate intracellular protein delivery in the liver in vivo. Taken together, TOP-EVs are a versatile platform for efficient intracellular protein delivery in vitro and in vivo, which can be applied to advance the development of protein-based therapeutics.

Platelet Activation in Ovarian Cancer Ascites: Assessment of GPIIb/IIIa and PF4 in Small Extracellular Vesicles by Nano-Flow Cytometry Analysis.

In ovarian cancer, ascites represent the microenvironment in which the platelets extravasate to play their role in the disease progression. We aimed to develop an assay to measure ascites' platelet activation. We enriched small extracellular vesicles (EVs) (40-200 nm) from ascites of high-grade epithelial ovarian cancer patients (n = 12) using precipitation with polyethylene glycol, and we conducted single-particle phenotyping analysis by nano-flow cytometry after labelling and ultra-centrifugation. Atomic force microscopy single-particle nanomechanical analysis showed heterogeneous distributions in the size of the precipitated particles and their mechanical stiffness. Samples were fluorescently labelled with antibodies specific to the platelet markers GPIIb/IIIa and PF4, showing 2.6 to 18.16% of all particles stained positive for the biomarkers and, simultaneously, the EV membrane labelling. Single-particle phenotyping analysis allowed us to quantify the total number of non-EV particles, the number of small-EVs and the number of platelet-derived small-EVs, providing a platelet activation assessment independent of the ascites volume. The percentage of platelet-derived small-EVs was positively correlated with platelet distribution width to platelet count in sera (PDW/PLT). Overall, we presented a high-throughput method that can be helpful in future studies to determine the correlation between the extent of platelet activation in ascites and disease status.

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry.

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and first-generation particle analysis to comprehensive multi-parameter measurements such as nano-flow cytometry (nFCM). nFCM is a form of flow cytometry that utilizes instrumentation specifically designed for nano-particle analysis, allowing for thousands of EVs to be characterized per minute both with and without the use of staining techniques. High resolution side scatter (SS) detection allows for size and concentration to be determined for all biological particles larger than 45 nm, while simultaneous fluorescence (FL) detection identifies the presence of labeled markers and targets of interest. Labeled subpopulations can then be described in quantitative units of particles/mL or as a percentage of the total particles identified by side scatter. Here, EVs derived from conditioned cell culture media (CCM) are labeled with both a lipid dye, to identify particles with a membrane, and antibodies specific for CD9, CD63, and CD81 as common EV markers. Measurements of comparison material, a concentration standard and a size standard of silica nanospheres, as well as labeled sample material are analyzed in a 1-minute analysis. The software is then used to measure the concentration and size distribution profile of all particles, independent of labeling, before determining the particles that are positive for each of the labels. Simultaneous SS and FL detection can be utilized flexibly with many different EV sources and labeling targets, both external and internal, describing EV samples in a comprehensive and quantitative manner.

T-cell trans-synaptic vesicles are distinct and carry greater effector content than constitutive extracellular vesicles.

The immunological synapse is a molecular hub that facilitates the delivery of three activation signals, namely antigen, costimulation/corepression and cytokines, from antigen-presenting cells (APC) to T cells. T cells release a fourth class of signaling entities, trans-synaptic vesicles (tSV), to mediate bidirectional communication. Here we present bead-supported lipid bilayers (BSLB) as versatile synthetic APCs to capture, characterize and advance the understanding of tSV biogenesis. Specifically, the integration of juxtacrine signals, such as CD40 and antigen, results in the adaptive tailoring and release of tSV, which differ in size, yields and immune receptor cargo compared with steadily released extracellular vesicles (EVs). Focusing on CD40L+ tSV as model effectors, we show that PD-L1 trans-presentation together with TSG101, ADAM10 and CD81 are key in determining CD40L vesicular release. Lastly, we find greater RNA-binding protein and microRNA content in tSV compared with EVs, supporting the specialized role of tSV as intercellular messengers.

Controlled Release of Epigenetically-Enhanced Extracellular Vesicles from a GelMA/Nanoclay Composite Hydrogel to Promote Bone Repair.

Extracellular vesicles (EVs) have garnered growing attention as promising acellular tools for bone repair. Although EVs' potential for bone regeneration has been shown, issues associated with their therapeutic potency and short half-life in vivo hinders their clinical utility. Epigenetic reprogramming with the histone deacetylase inhibitor Trichostatin A (TSA) has been reported to promote the osteoinductive potency of osteoblast-derived EVs. Gelatin methacryloyl (GelMA) hydrogels functionalised with the synthetic nanoclay laponite (LAP) have been shown to effectively bind, stabilise, and improve the retention of bioactive factors. This study investigated the potential of utilising a GelMA-LAP hydrogel to improve local retention and control delivery of epigenetically enhanced osteoblast-derived EVs as a novel bone repair strategy. LAP was found to elicit a dose-dependent increase in GelMA compressive modulus and shear-thinning properties. Incorporation of the nanoclay was also found to enhance shape fidelity when 3D printed compared to LAP-free gels. Interestingly, GelMA hydrogels containing LAP displayed increased mineralisation capacity (1.41-fold) (p ≤ 0.01) over 14 days. EV release kinetics from these nanocomposite systems were also strongly influenced by LAP concentration with significantly more vesicles being released from GelMA constructs as detected by a CD63 ELISA (p ≤ 0.001). EVs derived from TSA-treated osteoblasts (TSA-EVs) enhanced proliferation (1.09-fold), migration (1.83-fold), histone acetylation (1.32-fold) and mineralisation (1.87-fold) of human bone marrow stromal cells (hBMSCs) when released from the GelMA-LAP hydrogel compared to the untreated EV gels (p ≤ 0.01). Importantly, the TSA-EV functionalised GelMA-LAP hydrogel significantly promoted encapsulated hBMSCs extracellular matrix collagen production (≥1.3-fold) and mineralisation (≥1.78-fold) in a dose-dependent manner compared to untreated EV constructs (p ≤ 0.001). Taken together, these findings demonstrate the potential of combining epigenetically enhanced osteoblast-derived EVs with a nanocomposite photocurable hydrogel to promote the therapeutic efficacy of acellular vesicle approaches for bone regeneration.

Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution.

Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRß transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50-170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications.

Neural stem cells traffic functional mitochondria via extracellular vesicles.

Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.

Measuring particle concentration of multimodal synthetic reference materials and extracellular vesicles with orthogonal techniques: Who is up to the challenge?

The measurement of physicochemical properties of polydisperse complex biological samples, for example, extracellular vesicles, is critical to assess their quality, for example, resulting from their production and isolation methods. The community is gradually becoming aware of the need to combine multiple orthogonal techniques to perform a robust characterization of complex biological samples. Three pillars of critical quality attribute characterization of EVs are sizing, concentration measurement and phenotyping. The repeatable measurement of vesicle concentration is one of the key-challenges that requires further efforts, in order to obtain comparable results by using different techniques and assure reproducibility. In this study, the performance of measuring the concentration of particles in the size range of 50-300 nm with complementary techniques is thoroughly investigated in a step-by step approach of incremental complexity. The six applied techniques include multi-angle dynamic light scattering (MADLS), asymmetric flow field flow fractionation coupled with multi-angle light scattering (AF4-MALS), centrifugal liquid sedimentation (CLS), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), and high-sensitivity nano flow cytometry (nFCM). To achieve comparability, monomodal samples and complex polystyrene mixtures were used as particles of metrological interest, in order to check the suitability of each technique in the size and concentration range of interest, and to develop reliable post-processing data protocols for the analysis. Subsequent complexity was introduced by testing liposomes as validation of the developed approaches with a known sample of physicochemical properties closer to EVs. Finally, the vesicles in EV containing plasma samples were analysed with all the tested techniques. The results presented here aim to shed some light into the requirements for the complex characterization of biological samples, as this is a critical need for quality assurance by the EV and regulatory community. Such efforts go with the view to contribute to both, set-up reproducible and reliable characterization protocols, and comply with the Minimal Information for Studies of Extracellular Vesicles (MISEV) requirements.

Extracellular vesicle microRNA cargo is correlated with HPV status in oropharyngeal carcinoma.

BACKGROUND: The incidence of human papilloma virus positive (HPV+ ) oropharyngeal squamous cell carcinoma (OPSCC) has increased rapidly in recent decades. These tumours have a favourable outcome compared to HPV-negative (HPV- ) OPSCC. However, HPV+ tumours are more likely to metastasise to distant sites, suggesting a difference in how these tumour subtypes interact with the metastatic niche. Extracellular vesicles (EVs) have emerged as important players in cell-to-cell communication and are a potential source of biomarkers for cancer diagnosis. This study aims to characterise the microRNA cargo of small EVs released by HPV+ and HPV- OPSCC cell lines. METHODS: Extracellular vesicles produced by HPV+ (SCC2 and SCC90) and HPV- (SCC72 an SCC89) OPSCC cells were characterised by tunable resistive pulse sensing (TRPS) and western blotting. RNA was extracted from EVs and analysed by small RNA sequencing. A bioinformatics approach was used to identify EV miRNA signatures associated with HPV status. RESULTS: HPV- OPSCC cells produced more EVs than HPV+ OPSCC cells. EVs were positive for the common EV markers CD63, CD9 and TSG101. Unbiased hierarchical clustering analysis of EV miRNA cargo revealed that samples clustered based on HPV status. 14 miRNA were enriched in HPV+ cell-derived EVs, whereas 19 miRNA were enriched in EVs derived from HPV- cell lines. CONCLUSIONS: Here, we identify EV miRNA signatures indicative of the HPV status of the parent cell. This may provide a platform from which to validate salivary or blood-based biomarkers with utility for early detection and stratifying risk in OPSCC patients.