Conference Registration, Materials Pick-Up, Morning Tea, Coffee and Networking

The Characterisation and Function of Exosomes Derived from Human T Regulatory Cells
Lesley Smyth, Senior Lecturer in Immunology, University of East London, United Kingdom

Regulatory T-cells (Tregs) maintain immune tolerance to self-antigens and prevent excessive pro-inflammatory actions of T-effectors (Teffs) and antigen presenting cells responses. In a murine setting, these cells inhibit the aforementioned cells in a variety of ways including the release of extracellular vesicles (EVs). Release of these vesicles plays a key role in the suppressive capability of murine Tregs through the expression of the ectoenzyme CD73 and specific miRNA species. We have now extended this observation to human CD4+CD25+127lo T-cells isolated from blood, and cultured in the presence of anti-CD3/CD28-coated beads, rapamyacin and IL-2. These cells were suppressive and display key molecules associated with regulation. Activated human Tregs released 50-100nm-sized EVs expressing CD63 and CD81 as well as CD25 and CD39. Excitingly, Treg Evs were capable of suppressing Teffs responses resulting in less activation and the production of an anti-inflammatory cytokine profile. Their suppressive capabilities were also evident in vivo. Adoptive transfer of Treg EVs prevented T cell infiltration and skin damage in a humanised model of transplantation. Our data highlights that human Tregs produce EVs following activation and that this is a novel mechanism by which human Tregs modify immune Teff responses in vivo.

Procoagulant Extracellular Vesicles in Hemostasis and Thrombosis
Nigel Mackman, Distinguished Professor of Medicine, University of North Carolina at Chapel Hill, United States of America

The coagulation cascade consists of a series of cofactor/protease complexes (tissue factor (TF)/FVIIa; FVIIIa/FIXa; FVa/FXa) that assemble on membrane surfaces and result in the formation of fibrin. TF is a transmembrane receptor for FVII/FVIIa and the TF/FVIIa complex is the major initiator of the clotting cascade. The clotting cascade plays an essential role in hemostasis but also contributes to pathologic thrombosis. Several coagulation proteases (FIX, FX, FVII and prothrombin) contain a Gla domain that is positively-charged. Normal cells have an asymmetric membrane with negatively-charged phospholipids, such as phosphatidylserine (PS), located on the inner leaflet of the membrane. However, after cell damage PS flips to the outer leaflet of the membrane where it facilitates the binding and assembly of Gla-containing coagulation proteases and activation of coagulation at sites of injury. Extracellular vesicles (EVs) are small vesicles derived from activated or apoptotic cells. There are several types of EVs that can enhance coagulation depending on the presence of exposed PS and TF. For instance, PS^-,TF^- EVs have the lowest procoagulant activity (PCA) whereas PS⁺,TF⁺ EVs have the highest PCA. The majority of EVs in blood are derived from platelets and were originally described as platelet dust. These PS⁺ and PS^-EVs can enhance ongoing coagulation but cannot trigger coagulation. In contrast, EVs derived from activated monocytes and many types of tumors express PS and TF and can activate coagulation. PS⁺,TF⁺ EVs have been detected in the blood of patients with experimental endotoxemia, liver injury, cirrhosis, sickle cell disease, influenza A infection and cancer. These EVs likely contribute to thrombosis in these patients. Several prospective studies found that increased levels of TF⁺ EV precede venous thrombosis in pancreatic cancer patients. These studies suggest that TF⁺EVs could be used as a biomarker to identify patients at increased risk for venous thrombosis.

Coffee and Tea Break and Networking in the Exhibit Hall

Cellular Stress Sensor mTORC1 Switches Exosome Biogenesis and Secretion Pathways to Influence the Tumour Microenvironment
Deborah Goberdhan, Associate Professor of Cell Signalling, University of Oxford, United Kingdom

I will discuss our data describing a new mechanism of exosome biogenesis, conserved between humans and flies.  We show that reduced signalling from the cellular mTORC1 micro-environmental sensor, in glutamine-depleted conditions, causes changes in membrane trafficking and an upregulation of this alternate mechanism of exosome production.  This leads to the secretion of stress-induced exosomes, which induce adaptive responses in recipient cancer cells.

Role of Exosomes in the Transport of Amyloid Precursor Protein Fragments Between Neurons
Rémy Sadoul, Professor, Université Grenoble Alpes, France

Amyloid beta peptide (A?), the main component of senile plaques of Alzheimer’s disease brains, is produced by cleavage of the C-terminal transmembrane fragments (CTFs) of Amyloid Precursor Protein (APP). An unanswered question is how pathological amyoidogenic peptides spread throughout the brain during the course of the disease. We have shown that cortical neurons secrete exosomes which specifically bind to, and are endocytosed by other neurons. Cortical exosomes, carry endogenous APP and are strikingly enriched in CTFs. Using N2a cells expressing human APP and the tetraspanin CD63, we also found that the two proteins are sorted to different subsets of exosomes. We also found that APP carrying exosomes specifically bind to neurons in contrast to CD63-carrying exosomes, which bind to both neurons and glial cells. Thus, neuroblastoma cells secrete distinct populations of exosomes carrying different cargoes and targeting specific cell types. APP-carrying exosomes can be endocytosed by receiving cells allowing the processing of APP acquired by exosomes to give rise to the APP intracellular domain (AICD). Thus, our results show for the first time that neuronal exosomes may indeed act as vehicles for the intercellular transport of APP and its catabolites.

Networking Lunch in the Exhibit Hall -- Meet the Exhibitors and View Posters

Technology Spotlight:
High-Throughout Characterization of Single Exosomes and Extracellular Vesicles
George Daaboul, Chief Scientific Officer, nanoView Biosciences, Inc.

To accurately characterize exosomes it is necessary to validate prepared samples, which is most commonly performed using nanoparticle tracking analysis (NTA) combined with proteomic analysis for exosomal proteins. As an alternative to this, Flow Cytometry (FC) has recently been used to combine the sizing and proteomic information into a single measurement. While FC is a mature tool for large cellular-sized analyses, there are a number of limitations that stem from the small-size of exosomes, specifically in terms of the number of available surface epitopes and low-signal relative to background levels, especially in complex samples. To improve the characterization of exosomes, nanoView Diagnostics has developed a label-free visible-light microarray imaging technique that allows multiplexed enumeration and sizing of individual nanovesicles captured on the sensor in a one-step assay direct-from-sample and can work with samples volumes as small as 5 µl. The exosome characterization technology collapses a number of steps into a 1-step high-throughput technique that can improve standardization of exosome preparations and facilitate translation of exosome based liquid biopsies and therapeutic developments.

Technology Spotlight:
Development of a Sensitive and Robust NGS Protocol for microRNA Sequencing in Exosome and Extracellular Vesicle Samples
Laura Klitten, Global Product Manager, Exiqon – a QIAGEN company

The contents of exosomes are subject of investigative efforts worldwide, from basic research to biomarker discovery, with the goal of using insights for diagnostic and therapeutic purposes. The profile of microRNAs contained in exosomes can elucidate cellular functions and responses in diseased and healthy states. Plasma-derived exosomes are commonly isolated by differential centrifugation, which has proven to be difficult and is susceptible to variability in experience and skill of the person performing the isolation. To assess and optimize methodologies for the isolation and characterization of microRNAs in liquid biopsies, plasma samples from three immunized animals were prepared for next generation sequencing using three different methods: Exosome preparation by differential centrifugation, exosome preparation with the miRCURY™ isolation technology, and no exosome prep -RNA was isolated directly from the plasma sample. Library preparation and subsequent sequencing was successful for all preparation methods, with mapping percentages above 75% for all samples. Overall, provided that protocols optimized for liquid biopsies are used, there is good agreement between the microRNA expression patterns detected from whole plasma and isolated exosomes. This consistency across preparation methods attests to the high-quality and robust results that Exiqon Services achieves through experience and implementation of carefully designed and standardized protocols.

Composition and Regulatory Activities of Extracellular Vesicles Released by Osteoclasts
Shannon Holliday, Associate Professor of Orthodontics and Anatomy & Cell Biology, University of Florida, United States of America

Communication between osteoclasts and osteoblasts is vital to maintain healthy bone. We have found osteoclasts release EVs that regulate osteoblasts. We have performed comprehensive proteomic profiling of these EVs, and are exploring their use in bone regeneration.

Afternoon Coffee and Tea Break and Networking in the Exhibit Hall

Live Tracking of Endogenous Exosomes in vivo
Guillaume Van Niel, Team leader Endosomal Dynamic in Neuropathies, Institute Curie, France

Despite of their promising use as biomarkers and drug vehicles, very little is known about the dynamic of extracellular vesicles (EVs) secretion and their physiology in vivo. Here we proposed to fill this gap by developing a hCD63-based fluorescent reporter that allowed us to analyze their secretion in live cells and to track them from their site of production to their final destination in in vivo models. The use of this construct in cell lines revealed specific features and new stimulatory pathway of exosome secretion. The use of this construct in zebrafish embryos allowed us to observe exosome release in vivo and track a massive pool of endogenous exosomes in the blood flow by combining light- and electron microscopy techniques. After cell specific expression of the construct in vivo, we tracked endogenous exosomes by live imaging in the blood flow to identify their main targets. The mapping of the transit routes and final destination of EVs in zebrafish embryos support their role in nutrient delivery during development. Altogether, our work highlights the use of fluorescent reporter to study the regulation of exosome secretion and the use of the zebrafish embryo as a relevant vertebrate model to track endogenous EVs in vivo.

Mechanisms for the Selective Incorporation of Tissue Factor into Extracellular Vesicles
Camille Ettelaie, Lecturer in Biomedical Science, University of Hull , United Kingdom

Incorporation of tissue factor into cell-derived microvesicles results in the release of procoagulant microvesicles. The incorporation of tissue factor into the microvesicles is regulated through mechanisms that include various elements within the cytoplasmic and transmembrane domain of this protein. The presentation will examine some of these known regulatory elements.

EVs as Modulators of Cellular Response During Chemotherapy
David Carter, Reader in Biomedical Science, Oxford Brookes University, United Kingdom

We have previously shown that stress induces a bystander effect that is mediated by extracellular vesicles (EVs). These EVs can be taken up by bystander cells and induce a stress response that can last several generations. Here we show that bystander cells are more resistant to future stress, and that blocking EV-mediated communication during chemotherapy can sensitise cells to the effects of such drugs.

Profiling EV Subsets and Cargo to Enable Adaptive Tumor- and Immuno-therapies
Jennifer Jones, NIH Stadtman Investigator, Head of Transnational Nanobiology, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, United States of America
Joshua Welsh, Research Fellow, National Cancer Institute (NCI), NCI, United States of America

Exosomes and small viruses fall below the detection limits of conventional flow cytometers, which has limited ways to identify, sort, and study, distinct subsets of EVs and other nanoparticles as single particles. In order to maximize information and material that can be obtained with high speed, high resolution flow cytometers we have developed nanoscale Fluorescence Associated Cytometric Sorting (nanoFACS). We have demonstrated nanoFACS to be capable of detecting tumor-cell derived EVs with specific tumor antigens, such as Prostate Specific Membrane Antigen (PSMA), with fluorescence and scattered light parameters, as well as being capable of sorting two distinct HIV strains (85-125 nm) to >95% purity. The development of our nanoFACS method provides a unique way to analyze and sort functional EV- and viral-subsets, while preserving vesicular structure, surface protein specificity, and RNA cargo activity.

Close of Day 1 of the Conference

Morning Coffee, Tea and Networking

Bioproduction and Clinical Translation of Extracellular Vesicles for Treatment of Inflammatory Diseases
Marcin Jurga, R&D Director, The Cell Factory bvba, Netherlands

Recent progress in development of experimental cell-based therapies demonstrated a promising efficacy of somatic cells in stimulation and modulation of tissue’s regenerative capabilities. Commercialization of cell therapeutics remains challenging mainly due to complex and expensive production process. It has been demonstrated that extracellular vesicles (EVs) could be an attractive alternative for many allogenic cell-based therapeutics. EVs are nanometer-size particles secreted by different types of cells in vivo and in vitro. EVs are presenting a similar therapeutic properties to the parent cells with several additional benefits: (i) manufacturing is more effective and cheaper, (ii) higher stability, (iii) lower immunogenicity, (iv) no risk of uncontrolled ectopic differentiation and proliferation, (v) better penetration into injury site e.g. cross BBB.

Coffee and Tea Break and Networking in the Exhibit Hall

Technology Spotlight:
A Standardized, Automated Approach for Exosome Isolation and Characterization
Lutz Ehrhardt, Marketing Manager Centrifugation Europe, Beckman Coulter

This presentation is describing an standardized method for the isolation and characterization of exosomes.

Networking Lunch in the Exhibit Hall -- Meet the Exhibitors and View Posters

Exosome-Mediated Functional Delivery of Integrins Promotes Cancer Progression
Lucia Languino, Professor of Cancer Biology, Thomas Jefferson University, United States of America

Our investigations focus on exosome-mediated transfer of integrin surface receptors among subsets of cancer cells, and among cancer cells and the surrounding microenvironment.

Our recent studies demonstrate that exosomes from cancer cells contribute to horizontal propagation of integrin-associated invasive phenotypes in a paracrine fashion.

The presentation will cover the role of exosomal integrins in cancer progression, and will discuss their potential use as clinical biomarkers for non-invasive diagnosis and/or as therapeutic targets in cancer.

Overcoming Barriers to Therapeutic Exosome Production using Highly Scalable and Pure Embryonic Progenitor Cell Lines
David Larocca, Vice President, Research and Development, ReCyte Therapeutics, United States of America

Stem cell derived exosomes have shown promise in animal models as an alternative to stem cells for a wide range of regenerative medicine applications including ischemia, myocardial infarct, stroke, atherosclerosis, and wound healing.  However, production of therapeutic exosomes for clinical use will require scalable, stable and relatively pure production cell lines.  Commonly used adult stem cells such as MSCs typically suffer from poor proliferative capacity, donor variability, and population heterogeneity. These limitations may present a formidable barrier to translation of early preclinical studies to the clinic.  To address the limitations of stem cell purity and scalability, we derived hundreds of clonally pure and highly scalable human embryonic progenitor cell lines from pluripotent stem cells. We are currently investigating their potential as a scalable source of therapeutic exosomes.  Using angiogenic exosomes as a case-study, we identified 6 scalable embryonic progenitor cell lines with angiogenic activity in a HUVEC tube forming assay including endothelial, pericyte and smooth muscle cell lines.  The embryonic endothelial cell line, 30-MV2-6, expanded to over 75 population doublings (pd) compared to 16pd for adult bone-marrow MSCs.  Moreover, 30-MV2-6 exosomes had >50-fold higher levels of the angiogenic miR-126 and 6-fold higher angiogenic potency than MSC exosomes. The 30-MV2-6 exosome production was stable to at least 50pd and the potential to scale on a hollow fiber bioreactor was demonstrated. We have identified a variety of distinct cell types including endothelial, smooth muscle, cartilage, bone, fat and pericyte cell lines in our library of over 250 progenitor cell lines. Our data indicates the potential of this library to provide a richly diverse source of exosome production lines that can be mined for variety of therapeutic exosomes.

Afternoon Coffee and Tea Break and Networking in the Exhibit Hall

Technology Spotlight:
Imaging Flow Cytometry and Extracellular Vesicles
PJ Chana, UK Imaging and Flow Application Specialist, Merck

A short introduction to the ImagestreamX MKII and its use in phenotyping EVs

Technology Spotlight:
An Integrated Methodology for Fast and Reliable Extracellular Vesicle Purification and Characterization
Dimitri Aubert, Director, Global Sales, iZON Science Ltd.

iZON Science has designed and produced standardised SEC columns that yield clean EVs from biological fluids and cell culture, 99% free of non-vesicular proteins. It has been combined with TRPS technology, which provides detailed, calibrated measurement of EV particle number, size, concentration of each size fraction, and individual particle surface charge.

Production of Functionally Bioactive EVs From an Immortalized Human Neural Stem Cell (hNSC) Line
Randolph Corteling, Head of Research, ReNeuron Ltd., United Kingdom

To ensure the scale required for clinical research and development, producer cell immortalization and clonal isolation is a practical strategy to produce consistent, functionally bioactive exosomes for use as therapeutic agents.

Nano-Extracellular Vesicles: True Perspectives in Therapy for Regenerative Medicine and Oncology
Ciro Tetta, Chief Project Manager, Unicyte AG, Germany

Unicyte AG is an independent subsidiary of Fresenius Medical Care KGaA, the world's largest provider of products and services for individuals with renal diseases. Based on a long-standing cooperation with Prof G.Camussi in Turin (I), Unicyte is bulding its stategy on the nano-EV efficacy in regenerative medicine (renal and vascular injury) and oncology (renal tumors) ectopic tumor models.

Renal Regenerative Potential of Different Extracellular Vesicle Populations Derived from BM-MSCs
Stefania Bruno, Assistant Professor, University of Torino, Italy

I will present in vivo experiments that prove the efficacy of EV treatments in different renal injury models. EVs derived from different cell sources will be presented.

Close of Day 2 of the Conference

Morning Coffee, Tea and Networking

Exosomes in Inflammation and as Cancer Treatment
Susanne Gabrielsson, Professor, Division of Immunology and Allergy, Department of Medicine Solna, Karolinska Institutet, Sweden

Glioblastoma Hijacks Multiple Microglial Functions to Promote Tumor Growth
Marieke Broekman, Neurosurgeon, University Medical Center Utrecht and Massachusetts General Hospital/Harvard Medical School, United States of America

Extracellular vesicles (EVs) have been shown play a role in glioblastoma biology. Contrary to all prior studies, we will present data on EV mediated modification of microglia and macrophages in vivo.

Controlling Reproduction Through Secretions
Yael Heifetz, Principal Investigator, The Hebrew University of Jerusalem, Israel

In preparation for the acquisition of reproductive functionality, the female reproductive tract (RT) undergoes physiological changes. Both male and female contribute important components required for a suitable environment in which a healthy offspring will develop. In Drosophila melanogaster, the major secretory centers in the female RT are the spermathecae and accessory glands. The spermathecae and accessory gland secretory cells provide much of the milieu for the environment where reproductive events occur. Simultaneous disruption of spermatheca and accessory gland development resulted in female sterility. Genetic ablation of the spermathecal secretory cells (SSC) in adulthood impairs sperm storage and egg laying. To better understand how the spermathecae influence female reproductive success, we used Drosophila genetics to study the extracellular vesicles (EVs) involved in intercellular communication within the female RT. We focused on the SSC and examined whether the spermathecal EVs are involved in regulating female fertility. Our results indicate that EVs are essential for achieving high fertility and are used by the SSC as intercellular communication shuttles to modulate processes in the female RT.

Coffee and Tea Break and Networking in the Exhibit Hall

Hyaluronan-Coated EVs in Health and Disease
Kirsi Rilla, Group Leader, Institute of Biomedicine, University of Eastern Finland, Finland

Hyaluronan-coated EVs originate from different plasma membrane protrusions like filopodia of cells with high hyaluronan synthesis activity. This study is focused on the biogenesis of those specific EVs, their interactions and effects on target cells, and putative role in tissue regeneration and cancer progression.

Malaria Parasites use DNA-Harbouring Vesicles as a Mechanism to Activate Cytosolic Immune Sensors
Neta Regev-Rudzki, Assistant Professor, Department of Biomolecular Sciences, Weizmann Institute of Science, Israel

Malaria, caused by Plasmodium falciparum (Pf), is a devastating parasitic disease affecting hundreds of millions of people worldwide. The parasite’s transmission cycle between humans and mosquitoes involves a remarkable series of morphological transformations and in each context the parasites face very hostile environment. Here we discovered that at specific stage post-invasion into their host red blood cells (RBCs), the parasites secrete DNA-harbouring vesicles. The vesicles are taken up by human monocytes and the DNA species are released within their cytosol and leading to STING-dependent DNA sensing. The parasitic DNA then induces type I interferon (IFN) via STING pathway, suggesting a decoy mechanism employed by the parasites from a distance, while growing within the RBCs.

Exosome (EV)-mediated Specific Gene Delivery (as mRNA) to Cause Specific Suicide of HER2-positive Breast Cancer by the New Prodrug CNOB
A. C. Matin, Professor, Stanford University, United States of America

I will discuss successful use of extracellular vesicles in specifically delivering the hChrR6 gene (in the form of its mRNA) to HER2+ve cells and tumors, generating their killing by the prodrug, CNOB.

Networking Lunch in the Exhibit Hall -- Meet the Exhibitors and View Posters

Vesicle and RNA Secretion by Helminths: At the Host Interface
Amy Buck, Reader, University of Edinburgh, United Kingdom

The transport of RNA within extracellular vesicles (EVs) has been proposed as a means of cell-to-cell communication within an organism and a mechanism of cross-species communication. We study the functions of EVs and their RNA cargos in helminths, which are parasitic worms that naturally infect plants and animals, including ¼ of the human population. Helminths have co-evolved with their hosts’ immune systems for hundreds of millions of years and establish chronic infections through the secretion of bioactive molecules that modulate host cells. Helminth secretion products contain EVs that we have shown can suppress innate immune responses in vitro and in vivo. Proteomic analyses of the EVs derived from Heligomosomoides polygyrus, a gastrointestinal nematode that naturally infects mice, suggest these derive from the endocytic pathway and also reveal the presence of a newly evolved Argonaute protein in the EVs. Small RNA sequencing analyses demonstrate that miRNAs and Y RNAs are present in the EVs, but the dominant class of vesicular small RNA contains 5’triphosphates, most likely as a result of synthesis by an RNA-dependent RNA polymerase. A subset of these 5’ triphosphate nematode small RNAs are detected in mouse cells and l will discuss our recent work investigating their origins and properties.

Hepatocellular Exosomes and their miRNA Cargo
John Pezacki, Professor, University of Ottawa, Canada

I will discuss studies of microRNA profiling of hepatocytes, viral infection, and the immunometabolic response to infection of the liver. A comparison of intracellular and exosomal microRNAs will be presented as well as properties of exosomes and implications to intercellular signaling.

RNA Binding Proteins and Small RNA Transcriptome of Extracellular Vesicles
Imre Mäger, Postdoctoral Research Scientist and Exosome Team Leader, University of Oxford, United Kingdom

Extracellular vesicles (EVs) mediate specific functions in cell-to-cell communication in health and disease by delivering or displaying biological macromolecules such as proteins and nucleic acids, such as miRNA, to their recipient cells. In order to understand miRNA content in EVs better, in relation to other small RNA species, we conducted next generation sequencing and proteomics experiments of EV samples derived from number of different cell types. Whereas EVs contain significant amount or miRNA, it is not the main EV-associated RNA species even among small RNAs. This is also reflected by the presence of respective RNA binding proteins of EVs. These findings are interesting in the context of EV biology, but also important for EV bioengineering for RNA delivery and for developing EVs for potential biomarkers.

Close of Conference