Extracellular Vesicles Isolation Research Articles

IPSC-derived lung and lung cancer organoid model to evaluate cisplatin encapsulated autologous iPSC-derived mesenchymal stromal cell-isolated extracellular vesicles

BackgroundLung cancer remains a leading cause of cancer-related mortality globally. Although recent therapeutic advancements have provided targeted treatment approaches, the development of resistance and systemic toxicity remain primary concerns. Extracellular vesicles (EVs), especially those derived from mesenchymal stromal cells (MSC), have gained attention as promising drug delivery systems, offering biocompatibility and minimal immune responses. Recognizing the limitations of conventional 2D cell culture systems in mimicking the tumor microenvironment, this study aims to describe a proof-of-principle approach for using patient-specific organoid models for both lung cancer and normal lung tissue and the feasibility of employing autologous EVs derived from induced pluripotent stem cell (iPSC)-MSC in personalized medicine approaches.MethodsFirst, we reprogrammed healthy fibroblasts into iPSC. Next, we differentiated patient-derived iPSC into branching lung organoids (BLO) and generated patient-matched lung cancer organoids (LCO) from patient-derived tumor tissue. We show a streamlined process of MSC differentiation from iPSC and EV isolation from iPSC-MSC, encapsulated with 0.07 µg/mL of cytotoxic agent cisplatin and applied to both organoid models. Cytotoxicity of cisplatin and cisplatin-loaded EVs was recorded with LDH and CCK8 tests.ResultsFibroblast-derived iPSC showed a normal karyotype, pluripotency staining, and trilineage differentiation. iPSC-derived BLO showed expression of lung markers, like TMPRSS2 and MUC5A while patient-matched LCO showed expression of Napsin and CK5. Next, we compared the effects of iPSC-MSC derived EVs loaded with cisplatin against empty EVs and cisplatin alone in lung cancer organoid and healthy lung organoid models. As expected, we found a cytotoxic effect when LCO were treated with 20 µg/mL cisplatin. Treatment of LCO and BLO with empty EVs resulted in a cytotoxic effect after 24 h. However, EVs loaded with 0.07 µg/mL cisplatin failed to induce any cytotoxic effect in both organoid models.ConclusionWe report on a proof-of-principle pipeline towards using autologous or allogeneic iPSC-MSC EVs as drug delivery tests for lung cancer in future. However, due to the time and labor-intensive processes, we conclude that this pipeline might not be feasible for personalized approaches at the moment.

Extracellular vesicles as mediators of stress response in embryo-maternal communication

Introduction: The pivotal role of extracellular vesicles (EVs) in facilitating effective communication between the embryo and maternal cells during the preimplantation stage of pregnancy has been extensively explored. Nonetheless, inquiries persist regarding the alterations in EV cargo from endometrial cells under stress conditions and its potential to elicit specific stress responses in trophoblast cells. Thus, the aim of this study was to elucidate the involvement of EV miRNA miRNAs in transmitting stress signals from maternal cells to trophoblasts.Methods: The receptive endometrial epithelium analogue RL95-2 cells were subjected to stress induction with 200 µM CoCl2 for 24 h before EV isolation. JAr trophoblast spheroids, which serve as embryos, were subjected to treatment with stressed or unstressed EVs derived from RL95-2 cells for 24 h. Transcriptomic alterations in the treated JAr spheroids as well as in the untreated group, as a negative control, were investigated by mRNA sequencing. Furthermore, the changes in EV miRNAs were assessed by sequencing EV samples.Results: A comprehensive analysis comparing the miRNA profiles between stressed and unstressed EVs revealed significant changes in 25 miRNAs. Furthermore, transcriptomic analysis of JAr spheroids treated with stressed RL95-2EVs versus unstressed EVs or the untreated group demonstrated 6 and 27 differentially expressed genes, respectively. Pathway enrichment analysis showed that stressed EVs induce alterations in gene expression in trophoblast cells, which is partially mediated by EV microRNAs.Discussion: Our results suggest that EVs can transfer stress signals from endometrial cells to the embryo. These discoveries shed new light on the mechanism underlying implantation failures under stress conditions. Unraveling the role of EVs in transmitting stress signals, can extend our knowledge to pave the way for targeted interventions to manage stress-related implantation failures.

Size-Exclusion Chromatography: A Path to Higher Yield and Reproducibility Compared to Sucrose Cushion Ultracentrifugation for Extracellular Vesicle Isolation in Multiple Myeloma

Small extracellular vesicles (EVs) play a pivotal role in intercellular communication across various physiological and pathological contexts. Despite their growing significance as disease biomarkers and therapeutic targets in biomedical research, the lack of reliable isolation techniques remains challenging. This study characterizes vesicles that were isolated from conditioned culture media (CCM) sourced from three myeloma cell lines (MM.1S, ANBL-6, and ALMC-1), and from the plasma of healthy donors and multiple myeloma patients. We compared the efficacy, reproducibility, and specificity of isolating small EVs using sucrose cushion ultracentrifugation (sUC) vs. ultrafiltration combined with size-exclusion chromatography (UF-SEC). Our results demonstrate that UF-SEC emerges as a more practical, efficient, and consistent method for EV isolation, outperforming sUC in the yield of EV recovery and exhibiting lower variability. Additionally, the comparison of EV characteristics among the three myeloma cell lines revealed distinct biomarker profiles. Finally, our results suggest that HBS associated with Tween 20 improves EV recovery and preservation over PBS. Standardization of small EV isolation methods is imperative, and our comparative evaluation represents a significant step toward achieving this goal.

Scalable isolation of surface-engineered extracellular vesicles and separation of free proteins via tangential flow filtration and size exclusion chromatography (TFF-SEC)

BackgroundExtracellular vesicles (EVs) represent small lipid bilayer structures pivotal in mediating intercellular communication via biomolecular transfer. Their inherent characteristics, including packaging, non-immunogenicity, and biofluid stability, position EVs as promising drug delivery vectors. However, developing clinical quality EVs requires multifaceted technological advancement.MethodsIn this study, a method is introduced for engineering extracellular vesicles (eEVs) from cultured cells and their subsequent isolation using lab-scale tangential flow filtration (TFF). This is the first study to evaluate DNA loading efficacy into EVs isolated by TFF, marking a significant milestone in the field of targeted drug delivery. Initially, cells are transfected with EV-display constructs to facilitate the secretion of eEVs bearing the desired coding molecules. Following brief centrifugation, the cell culture media undergoes filtration using hollow fiber filters. TFF, by applying a constant flow, effectively segregates molecules based on designated molecular weight cut-off (MWCO), enriching particles between 50 and 650 nm.ResultsCompared to conventional methods like ultracentrifugation, TFF demonstrates higher efficiency in removing undesired molecules/aggregates while exerting less stress on EVs. Characterization of eEVs through various assays confirms TFF’s superiority in isolating pure EV populations. Additionally, the necessity of size-exclusion chromatography (SEC) after tangential flow filtration (TFF) becomes evident for effectively removing unbound protein contaminants.ConclusionIn conclusion, TFF-SEC emerges as a scalable and superior approach for eEV isolation, promising significant advancements in clinical applications.

Establishing the capacity to monitor proteins relevant to the study of drug exposure and response using liver-derived extracellular vesicles.

Drug exposure and response is determined by pharmacokinetic (PK) and pharmacodynamic (PD) profiles. Interindividual differences in abundance of drug metabolizing enzymes (DMEs) and drug target proteins underpin PK and PD variability and impact treatment efficacy and tolerability. Extracellular vesicles (EVs) carry protein cargo inherited from originating cells and may be useful for defining differences in key proteins related to hepatic drug metabolism and the treatment of metabolic-associated fatty liver disease (MAFLD). We sought to quantify these proteins in liver-derived EVs and establish the profile relative to paired tissue. EVs were recovered from human liver tissue samples (LT-EV, n = 11). Targeted liquid chromatography with tandem mass spectrometry (LC-MS/MS) assays were employed for absolute quantification of proteins in EV isolates and matched liver tissue. DMEs and MAFLD drug targets were readily detected and quantified in LT-EVs. Twelve of 15 DMEs exhibited moderate to strong correlation (Spearman ⍴ = 0.618-0.973) between tissue and EVs. Correlation in protein abundance was influenced by the extent of extra-hepatic expression of the target. This study provides evidence that key proteins related to PK and PD profiles can be measured in liver-derived EVs and abundance of liver-enriched DMEs are robustly correlated between paired tissue and EVs. The robust detection of protein markers related to drug PD profile in MAFLD opens the possibility to track within-subject changes in MAFLD and lays the foundation for future development of a liver-derived EV liquid biopsy to assess markers of drug exposure and response in vivo.

Therapeutic Application of Extracellular Vesicles Derived from Mesenchymal Stem Cells in Domestic Animals

Recently, the therapeutic potential of extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) has been extensively studied in both human and veterinary medicine. EVs are nano-sized particles containing biological components commonly found in other biological materials. For that reason, EV isolation and characterization are critical to draw precise conclusions during their investigation. Research on EVs within veterinary medicine is still considered in its early phases, yet numerous papers were published in recent years. The conventional adult tissues for deriving MSCs include adipose tissue and bone marrow. Nonetheless, alternative sources such as synovial fluid, endometrium, gingiva, and milk have also been intermittently used. Fetal adnexa are amniotic membrane/fluid, umbilical cord and Wharton’s jelly. Cells derived from fetal adnexa exhibit an intermediate state between embryonic and adult cells, demonstrating higher proliferative and differentiative potential and longer telomeres compared to cells from adult tissues. Summarized here are the principal and recent preclinical and clinical studies performed in domestic animals such as horse, cattle, dog and cat. To minimize the use of antibiotics and address the serious issue of antibiotic resistance as a public health concern, they will undoubtedly also be utilized in the future to treat infections in domestic animals. A number of concerns, including large-scale production with standardization of EV separation and characterization techniques, must be resolved for clinical application.

Isolation, characterization, and immunomodulatory effects of extracellular vesicles isolated from fish pathogenic Aeromonas hydrophila

Bacterial extracellular vesicles (BEVs) are natural nanocarriers that have shown great potential for biomedical applications such as biomarkers, cancer therapy, immunomodulators, vaccines, wound healing, tissue engineering, and drug carriers. In the present study, BEVs were isolated from the gram-negative bacterium, Aeromonas hydrophila using the ultracentrifugation method and denoted as AhEVs. Using transmission electron microscopy imaging, we confirmed the ultrastructure and spherical shape morphology of AhEVs. Nanoparticle-tracking analysis results showed a mean particle size of 105.5 ± 2.0 nm for AhEVs. Moreover, the particle concentration of AhEVs was 2.34 ± 0.12 × 1011 particles/mL of bacterial supernatant. AhEV-treated fathead minnow (FHM) cells did not show cytotoxicity effects up to 50 μg/mL with no significant decrease in cells. Moreover, no mortality was observed in larval zebrafish up to 50 μg/mL which indicates that the AhEVs are biocompatible at this concentration. Furthermore, fluorescent-labeled AhEVs were internalized into FHM cells. Results of qRT-PCR analysis in FHM cells revealed that cellular pro-inflammatory cytokines such as nuclear factor (NF)-κB, interferon (Ifn), Irf7, interleukin (Il) 8, and Il11 were upregulated while downregulating the expression of anti-inflammatory Il10 in a concentration-dependent manner. AhEV-treated adult zebrafish (5 μg/fish) induced toll-like receptor (tlr) 2 and tlr4; tumor necrosis factor-alpha (tnfα); heat shock protein (hsp) 70; and il10, il6, and il1β in kidney. Protein expression of NF-κB p65 and Tnfα presented amplified levels in the spleen of AhEVs-treated zebrafish. Based on the collective findings, we conclude that AhEVs exhibited morphological and physicochemical characteristics to known EVs of gram (−)ve bacteria. At biocompatible concentrations, the immunomodulatory activity of AhEVs was demonstrated by inducing different immune response genes in FHM cells and zebrafish. Hence, we suggest that AhEVs could be a novel vaccine candidate in fish medicine due to their ability to elicit strong immune responses.

Prospects and challenges in using neuronal extracellular vesicles in biomarker research.

Extracellular vesicles (EVs) hold promise as a source of disease biomarkers. The diverse molecular cargo of EVs can potentially indicate the status of their tissue of origin, even against the complex background of whole plasma. The main tools currently available for assessing biomarkers of brain health include brain imaging and analysis of the cerebrospinal fluid of patients. Given the costs and difficulties associated with these methods, isolation of EVs of neuronal origin (NEVs) from the blood is an attractive approach to identify brain-specific biomarkers. This perspective describes current key challenges in EV- and NEV-based biomarker research. These include the relative low abundance of EVs, the lack of validated isolation methods, and the difficult search for an adequate target for immunocapturing NEVs. We discuss that these challenges must be addressed before NEVs can fulfill their potential for biomarker research. HIGHLIGHTS: NEVs are promising sources of biomarkers for brain disorders. Immunocapturing NEVs from complex biofluids presents several challenges. The choice of surface target for capture will determine NEV yield. Contamination by non-EV sources is relevant for biomarkers at low concentrations.

Self-Assembled DNA Microflowers for Platelet-Derived Extracellular Vesicle Isolation and Infected Wound Healing.

Platelet-derived extracellular vesicles (PEVs) showing great potential in wound healing have attracted increasing attention recently. Nondestructive isolation and effective utilization strategies are highly conducive for PEVs developing into recognized therapeutic entities. Here, we present an efficient strategy for PEV isolation and bacterial infected wound healing based on self-assembled DNA microflowers. First, DNA microflowers are prepared using rolling circle amplification. Then, the hydrophobic interaction between cholesteryl modified on DNA microflowers and the phospholipid bilayer membrane of PEVs leads to the formation of a network structure with improved mechanical strength and the separation of PEVs from biological samples. Finally, controlled release of PEVs is achieved through bacterial-induced hydrogel degradation. In vitro experiments demonstrate the obtained DNA hydrogel with good cytocompatibility and therapeutic potential. Taken together, the DNA microflower-based hydrogels with bioadhesive, self-healing, tunable mechanical properties and bacteria-responsive behavior offer substantial potential for EV isolation and wound healing.

Rapid isolation method for extracellular vesicles based on Fe3O4@ZrO2.

Extracellular vesicles (EVs) are pivotal in intercellular communication, disease mechanisms. Despite numerous methods for EVs isolation, challenges persist in yield, purity, reproducibility, cost, time, and automation. We introduce a EVs isolation technique using Fe3O4@ZrO2 beads, leveraging ZrO2-phosphate interaction. The results indicated that EVs were efficiently separated from large volumes of samples in 30minutes without preconcentration. Our method demonstrated capture efficiency (74%-78%) compared to ultracentrifugation, purity (97%), and reproducibility (0.3%-0.5%), with excellent linearity (R2 > 0.99). EVs from urine samples showed altered expression of miRNAs. The logistic regression model achieved an AUC of 0.961, sensitivity of 0.92, and specificity of 0.94. With potential for automation, this magnetic bead-based method holds promise for clinical applications, offering an efficient and reliable tool for EVs research and clinical studies.

Bioprocessing of human platelet concentrates to generate lysates and extracellular vesicles for therapeutic applications

This work describes protocols for preparing specific forms of human platelet lysates from pooled platelet concentrates (PCs) and the isolation of platelet-derived extracellular vesicles (p-EVs). Clinical-grade PCs can be sourced from blood establishments immediately following expiration for transfusion use. Here, we describe methods to process PCs into specific lysates from which p-EVs can be isolated. Each lysate type is prepared using platelet activation and processing methods which produce distinct products that may be useful in different applications. For example, serum-converted platelet lysate (SCPL)-EVs were recently shown to have powerful therapeutic properties following myocardial infarction in mice. EVs can be isolated from all products using size exclusion chromatography, producing pure and consistent p-EVs from multiple batches. Together, these methods allow isolation of p-EVs with excellent potential for clinical and preclinical applications.•Platelet concentrates (PCs) obtained from local blood establishments are reliable and sustainable sources to generate biomaterials.•We outline five distinct methods of platelet lysate generation and one method for extracellular vesicle isolation.•Each platelet lysate form has different biological properties which may be suitable for certain applications.

Tumor-Derived Extracellular Vesicles as Liquid Biopsy for Diagnosis and Prognosis of Solid Tumors: Their Clinical Utility and Reliability as Tumor Biomarkers.

Early cancer detection and accurate monitoring are crucial to ensure increased patient survival. Recent research has focused on developing non-invasive biomarkers to diagnose cancer early and monitor disease progression at low cost and risk. Extracellular vesicles (EVs), nanosized particles secreted into extracellular spaces by most cell types, are gaining immense popularity as novel biomarker candidates for liquid cancer biopsy, as they can transport bioactive cargo to distant sites and facilitate intercellular communications. A literature search was conducted to discuss the current approaches for EV isolation and the advances in using EV-associated proteins, miRNA, mRNA, DNA, and lipids as liquid biopsies. We discussed the advantages and challenges of using these vesicles in clinical applications. Moreover, recent advancements in machine learning as a novel tool for tumor marker discovery are also highlighted.

Nanoscale flow cytometry-based quantification of blood-based extracellular vesicle biomarkers distinguishes MCI and Alzheimer's disease.

Accurate testing for Alzheimer's disease (AD) represents a crucial step for therapeutic advancement. Currently, tests are expensive and require invasive sampling or radiation exposure. We developed a nanoscale flow cytometry (nFC)-based assay of extracellular vesicles (EVs) to screen biomarkers in plasma from mild cognitive impairment (MCI), AD, or controls. Circulating amyloid beta (Aβ), tau, phosphorylated tau (p-tau)181, p-tau231, p-tau217, p-tauS235, ubiquitin, and lysosomal-associated membrane protein 1-positive EVs distinguished AD samples. p-tau181, p-tau217, p-tauS235, and ubiquitin-positive EVs distinguished MCI samples. The most sensitive marker for AD distinction was p-tau231, with an area under the receiver operating characteristic curve (AUC) of 0.96 (sensitivity 0.95/specificity 1.0) improving to an AUC of 0.989 when combined with p-tauS235. This nFC-based assay accurately distinguishes MCI and AD plasma without EV isolation, offering a rapid approach requiring minute sample volumes. Incorporating nFC-based measurements in larger populations and comparison to "gold standard" biomarkers is an exciting next step for developing AD diagnostic tools. Extracellular vesicles represent promising biomarkers of Alzheimer's disease (AD) that can be measured in the peripheral circulation. This study demonstrates the utility of nanoscale flow cytometry for the measurement of circulating extracellular vesicles (EVs) in AD blood samples. Multiple markers including amyloid beta, tau, phosphorylated tau (p-tau)181, p-tau231, p-tau217, and p-tauS235 accurately distinguished AD samples from healthy controls. Future studies should expand blood and cerebrospinal fluid-based EV biomarker development using nanoflow cytometry approaches.

P-290 Isolation and characterization of extracellular vesicles from human endometrial organoids

Abstract Study question Do human endometrial organoids produce extracellular vesicles (EVs)? Summary answer Endometrial organoids release EVs (ORG-EVs) that we successfully and comprehensively characterize according to guidelines. What is known already EVs play a crucial role in cellular communication, garnering increasing attention in physiological and pathological processes. Although the enormous potential of EVs has been extensively demonstrated across a large variety of research fields, the promise held by EVs derived from human endometrial organoids is still largely unexplored. Indeed, nowadays there is limited research available on the presence, role, and function of EVs derived from organoids of female reproductive tissue samples. This underscores the importance of adopting standardized methods for isolating, characterizing, and reporting findings, which is crucial for advancing in this promising and innovative, yet challenging, research field. Study design, size, duration This study involved establishing organoid cultures from endometrial biopsies of healthy women (n = 10) of reproductive age, who underwent ovarian surgery between January and October 2023. ORG-EVs were isolated using differential ultracentrifugation. Characterization of ORG-EVs, including assessment of their biophysical and biochemical properties, adhered to guidelines established by the International Society for Extracellular Vesicles (ISEV). Participants/materials, setting, methods Endometrial specimens from 10 women were employed to establish organoid cultures. ORG-EVs were isolated from spent medium through differential ultracentrifugation. Morphology, size distribution and concentration analysis were performed using Transmission Electron Microscopy (TEM) and Nanoparticle Tracking Analysis (NTA). Western blotting was used to confirm the presence of EVs markers. Main results and the role of chance TEM analysis confirmed the presence of isolated ORG-EVs, elucidating their morphology and structural integrity. NTA revealed that ORG-EVs had an average size of 160.4 ± 6.2 nm (mean ± SD) and a mean concentration of 5.2E+11 ± 2.1E+10 particles/ml (mean ± SD). Western blot analysis further confirmed the vesicular nature of the assessed particles, demonstrating positivity for standard EV markers such as tetraspanins (CD63 and CD9), and soluble proteins (ALIX and TSG101), while excluding the endoplasmic reticulum-resident protein calnexin (negative control). Additionally, ORG-EVs exhibited a positive signal for mucin1 (MUC1), a glycoprotein present on the surface of endometrial epithelial cells, thereby confirming their cellular origin. Limitations, reasons for caution This is a pilot study, characterized by a small sample size requiring larger cohorts to validate results and, at the same time, to undertake deeper exploration of ORG-EVs’ molecular cargo. Emphasizing the critical need for standardized isolation and characterization methods is paramount. Wider implications of the findings Endometrial organoids enhance EVs production, providing a powerful tool for reproductive aspects studies, and improving regenerative medicine and artificial reproductive technology. These initial findings propel further exploration, unveiling of ORG-EVs’ molecular cargo aiming to elucidate their potential role in human female reproductive tissues and mechanism related to embryo implantation process. Trial registration number not applicable

Extracellular vesicle isolation and counting system (EVics) based on simultaneous tandem tangential flow filtration and large field-of-view light scattering.

Although the isolation and counting of small extracellular vesicles (sEVs) are essential steps in sEV research, an integrated method with scalability and efficiency has not been developed. Here, we present a scalable and ready-to-use extracellular vesicle (EV) isolation and counting system (EVics) that simultaneously allows isolation and counting in one system. This novel system consists of (i) EVi, a simultaneous tandem tangential flow filtration (TFF)-based EV isolation component by applying two different pore-size TFF filters, and (ii) EVc, an EV counting component using light scattering that captures a large field-of-view (FOV). EVi efficiently isolated 50-200nm-size sEVs from 15µL to 2L samples, outperforming the current state-of-the-art devices in purity and speed. EVc with a large FOV efficiently counted isolated sEVs. EVics enabled early observations of sEV secretion in various cell lines and reduced the cost of evaluating the inhibitory effect of sEV inhibitors by 20-fold. Using EVics, sEVs concentrations and sEV PD-L1 were monitored in a 23-day cancer mouse model, and 160 clinical samples were prepared and successfully applied to diagnosis. These results demonstrate that EVics could become an innovative system for novel findings in basic and applied studies in sEV research.

Infection of Alfalfa Cotyledons by an Incompatible but Not a Compatible Species of Colletotrichum Induces Formation of Paramural Bodies and Secretion of EVs

Hemibiotrophic fungi in the genus Colletotrichum employ a biotrophic phase invading host epidermal cells followed by a necrotrophic phase spreading through neighboring mesophyll and epidermal cells. We used serial block face scanning electron microscopy (SBF-SEM) to compare subcellular changes that occur in Medicago sativa (alfalfa) cotyledons during infection by Colletotrichum destructivum (compatible on M. sativa) and C. higginsianum (incompatible on M. sativa). Three-dimensional reconstruction of serial images revealed that alfalfa epidermal cells infected with C. destructivum undergo massive cytological changes during the first 60 hours following inoculation to accommodate extensive intracellular hyphal growth. Conversely, inoculation with the incompatible species C. higginsianum resulted in no successful penetration events and frequent formation of papilla-like structures and cytoplasmic aggregates beneath attempted fungal penetration sites. Further analysis of the incompatible interaction using focused ion beam-scanning electron microcopy (FIB-SEM) revealed formation of large multivesicular body-like structures that appeared spherical and were not visible in compatible interactions. These structures often fused with the host plasma membrane, giving rise to paramural bodies that appeared to be releasing extracellular vesicles (EVs). Isolation of EVs from the apoplastic space of alfalfa leaves at 60h post inoculation showed significantly more vesicles secreted from alfalfa infected with incompatible fungus compared to compatible fungus, which in turn was more than produced by non-infected plants. Thus, the increased frequency of paramural bodies during incompatible interactions correlated with an increase in EV quantity in apoplastic wash fluids. Together, these results suggest that EVs and paramural bodies contribute to immunity during pathogen attack in alfalfa.

Extracellular vesicle isolation methods identify distinct HIV-1 particles released from chronically infected T-cells.

The current study analyzed the intersecting biophysical, biochemical, and functional propertiesof extracellular particles (EPs) with the human immunodeficiency virus type-1 (HIV-1) beyond the currently accepted size range for HIV-1. We isolated five fractions (Frac-A through Frac-E) from HIV-infected cells by sequential differential ultracentrifugation (DUC). All fractions showed a heterogeneous size distribution with median particle sizes greater than 100nm for Frac-A through Frac-D but not for Frac-E, which contained small EPs with an average size well below 50nm. Synchronized and released cultures contained large infectious EPs in Frac-A, with markers of amphisomes and viral components. Additionally, Frac-E uniquely contained EPs positive for CD63, HSP70, and HIV-1 proteins. Despite its small average size, Frac-E contained membrane-protected viral integrase, detectable only after SDS treatment, indicating that it is enclosed in vesicles. Single particle analysis with dSTORM further supported these findings as CD63, HIV-1 integrase, and the viral surface envelope (Env) glycoprotein (gp) colocalized on the same Frac-E particles. Surprisingly, Frac-E EPs were infectious, and infectivity was significantly reduced by immunodepleting Frac-E with anti-CD63, indicating the presence of this protein on the surface of infectious small EPs in Frac-E. To our knowledge, this is the first time that extracellular vesicle (EV) isolation methods have identified infectious small HIV-1 particles (smHIV-1) that are under 50nm. Collectively, our data indicate that the crossroads between EPs and HIV-1 potentially extend beyond the currently accepted biophysical properties of HIV-1, which may have further implications for viral pathogenesis.

A systematic review and meta-analysis of clinical trials assessing safety and efficacy of human extracellular vesicle-based therapy.

Nowadays, it has become clear that extracellular vesicles (EVs) are not a cellular waste disposal vesicle but are an essential part of an intercellular communication system. Besides the use of EVs in biomarker studies and diagnostics, the potential of EV-therapeutics has been seen by many. They provide unique properties for disease therapy, including strong immune-modulatory actions, the possibility of engineering, low immunogenicity, and the capability of crossing biological barriers. Proof-of-concept of EV-therapeutics for various pathologies has been achieved in preclinical studies. However, clinical trials with EVs have only been emerging slowly. Here, we aim to provide a comprehensive overview of the current state-of-the-art concerning clinical studies using EVs in human therapy. By approaching the current knowledge in a systematic manner, we were able to include 21 reports for meta-analysis of safety and evaluation of efficacy outcomes. Overall, we have shown that EV-based therapy is safe with a low incidence of serious adverse events (SAE; 0.7% (95%-CI: 0.1-5.2%), and adverse events (AE; 4.4% (95%-CI: 0.7-22.2%). Subgroup analysis showed no significant difference in SAE when comparing autologous versus allogeneic administration, as well as engineered versus non-engineered EV products. A significantly higher number of AE was seen in autologous versus allogeneic administration. However, the clinical relevance remains questionable. Evaluation of the clinical outcomes of immunostimulatory, immunosuppressive or regenerative EV-therapies indicated improvement in the majority of treated patients. Despite these promising results, data need to be approached with caution due to a high heterogeneity in the EVs manufacturing methods, study design, and reporting of (S)AE. Overall, we conclude that EV-based therapy is safe and presents a promising opportunity in therapy. More efforts are needed in the standardization and harmonization of reporting of EV isolation and characterization data as well as in the reporting of (S)AE to allow inter-study comparison.

Towards IVDR-compliance by implementing quality control steps in a quantitative extracellular vesicle-miRNA liquid biopsy assay for response monitoring in patients with classic Hodgkin lymphoma.

Previously, we showed that quantification of lymphoma-associated miRNAs miR-155-5p, -127-3p and let-7a-5p levels in plasma extracellular vesicles (EVs) report treatment response in patients with classic Hodgkin lymphoma (cHL). Prior to clinical implementation, quality control (QC) steps and validation are required to meet international regulatory standards. Most published EV-based diagnostic assays have yet to meet these requirements. In order to advance the assay towards regulatory compliance (e.g., IVDR 2017/746), we incorporated three QC steps in our experimental EV-miRNA quantitative real-time reverse-transcription PCR (q-RT-PCR) assay in an ISO-13485 certified quality-management system (QMS). Liposomes encapsulated with a synthetic (nematode-derived) miRNA spike-in controlled for EV isolation by automated size-exclusion chromatography (SEC). Additional miRNA spike-ins controlled for RNA isolation and cDNA conversion efficiency. After deciding on quality criteria, in total 107 out of 120 samples from 46 patients passed QC. Generalized linear mixed-effect modelling with bootstrapping determined the diagnostic performance of the quality-controlled data at an area under the curve (AUC) of 0.84 (confidence interval [CI]: 0.76-0.92) compared to an AUC of 0.87 (CI: 0.80-0.94) of the experimental assay. After the inclusion of QC steps, the accuracy of the assay was determined to be 78.5% in predicting active disease status in cHL patients during treatment. We demonstrate that a quality-controlled plasma EV-miRNA assay is technically robust, taking EV-miRNA as liquid biopsy assay an important step closer to clinical evaluation.

The impact of ischemic stroke on bone marrow microenvironment and extracellular vesicles: A study on inflammatory and molecular changes

An ischemic stroke (IS) is caused due to the lack of blood flow to cerebral tissue. Most of the studies have focused on how stroke affects the localized tissue, but it has been observed that a stroke can cause secondary complications in distant organs, such as Bone Marrow (BM). Our study focused on the effect of ischemic strokes on the bone marrow microenvironment. Bone marrow (BM) is a vital organ that maintains inflammatory homeostasis and aids in the repair of damaged tissue after injury/IS. We used the middle cerebral artery occlusion (MCAO) model of ischemic stroke on adult mice (6 months) and investigated the changes in the BM environment. BM cells were used for western blot and RT-PCR, and the BM supernatant was used for cytokine analysis and extracellular vesicle (EVs) isolation. We observed a significant increase in the total cell number within the BM and an increase in TNF-alpha and MCP-1, which are known for inducing a pro-inflammatory environment. Western blots analysis on the whole BM cell lysate demonstrated elevated levels of inflammatory factors (IL-6, TNF-alpha, and TLR-4) and senescence markers (p21 p16). EVs isolated from the BM supernatant showed no change in size or concentration; however, we found that the EVs carried increased miRNA-141-3p and miRNA-34a. Proteomic analysis on BM-derived EVs showed an alteration in the protein cargo of IS. We observed an increase in FgB, C3, Fn1, and Tra2b levels. The signaling pathway analysis showed mitochondrial function is most affected within the bone marrow. Our study demonstrated that IS induces changes in the BM environment and EVs secreted in the BM.