Clinical research using extracellular vesicles: insights from the International Society for Extracellular Vesicles 2018 Annual Meeting

The abstracts presented at the 2018 International Society for Extracellular Vesicles Annual Meeting offer unique insight into the newest discoveries related to the biology and applied use of extracellular vesicles (EVs). As an extension of a recent “Clinical-Wrap Up” discussion at the International Society for Extracellular Vesicles 2018 Annual Meeting, a systematic review of each abstract was performed to determine which abstracts could be considered clinical research. Once the clinical research abstracts were identified, systematic data extraction included: the major focus of each clinical research abstract; the countries in which the work was done; and the sample size, if provided in the abstract. Each abstract was reviewed by two independent authors, with a third author resolving discrepancies in cases of disagreement. 174 out of 656 (27%) unique abstracts were determined to be clinical research. Oncology was a principal research focus (51 of the 174 clinical research abstracts, 29%). Many other clinical research abstracts presented at the International Society for Extracellular Vesicles 2018 Annual Meeting focused on the use of human samples for development of methods for potential application in the clinic. Beyond oncology and methods development, a wide range of topics was represented, including cardiovascular disease, neurodegenerative disease, genetics, and many others. Current research involving EVs highlights the common, but false dichotomy of science into curiosity-driven basic science or application-driven clinical research, when in fact both quest for understanding and intent to apply the findings appeared to drive much of the work at the International Society for Extracellular Vesicles 2018 Annual Meeting. Using Pasteur’s Quadrant as a framework, we discuss where the field of EV research is heading and how we may gain insight into the biological function of EVs in tandem with how they may benefit individual health

Contribution of extracellular vesicles from Adult-derived human liver stem cells to the correction of Urea Cycle Disorders

Introduction Adult-derived human liver stem cells (ADHLSCs) are currently in clinical development for the treatment of Urea Cycle Disorders (UCD). Clinical and preclinical data seem to indicate a higher clinical effect than what could be expected from the number of cells that have engrafted, suggesting that other mechanisms may be at play. We have previously demonstrated that ADHLSCs produce Extracellular Vesicles, EVs (MP, microparticles; and EXO, exosomes), which have been shown to mediate intracellular communication in other systems by delivering proteins, lipids and/or genetic information (coding and non-coding RNAs) to recipient cells. Therefore, the aim of this study was to determine the precise role of EVs in ADHLSC-mediated correction of UCD. Methods ADHLSCs were cultured for 2 days in DMEM supplemented with 10% EXO-free FBS and 1% P/S. The conditioned medium was collected, and MP and EXO fractions were harvested by serial ultracentrifugation. Transmission electron microscopy (TEM), western blotting and nanoparticle tracking analysis were used to evaluate the presence, purity and abundance of MP and EXO. RNA from EVs was stained with SytoRNA, which only fluoresces upon integration into RNA, to investigate RNA transfer from EVs to rat hepatocytes. Droplet digital PCR (ddPCR) was performed on RNA extracted from the MP and EXO as well as rat hepatocytes previously incubated with EVs to investigate the presence of human mRNAs of interest. Results We confirmed that ADHLSCs produce both MP and EXO. Characterization of the mRNA by ddPCR showed expression of ASL, ASS, and CPS1 in EVs, mainly in MPs. SytoRNA staining of the EV RNA allowed us to show transfer of EV RNA to over 60% of rat hepatocytes in vitro. Finally, we demonstrated transfer of human mRNAs of interest from EVs to rat hepatocytes using ddPCR. Summary/Conclusion In summary, our study shows that ADHLSC-derived EVS contain mRNA encoding for some of the deficient enzymes in UCD and are capable of transfering their mRNA content to recipient cells. mRNA transfer via EVs may therefore be one of the modes of action of ADHLSCs in UCD

Recommendations for reproducibility of cerebrospinal fluid extracellular vesicle studies.

Publication status: PublishedFunder: OHSU Center for Women's Health Circle of GivingCerebrospinal fluid (CSF) is a clear, transparent fluid derived from blood plasma that protects the brain and spinal cord against mechanical shock, provides buoyancy, clears metabolic waste and transports extracellular components to remote sites in the brain. Given its contact with the brain and the spinal cord, CSF is the most informative biofluid for studies of the central nervous system (CNS). In addition to other components, CSF contains extracellular vesicles (EVs) that carry bioactive cargoes (e.g., lipids, nucleic acids, proteins), and that can have biological functions within and beyond the CNS. Thus, CSF EVs likely serve as both mediators of and contributors to communication in the CNS. Accordingly, their potential as biomarkers for CNS diseases has stimulated much excitement for and attention to CSF EV research. However, studies on CSF EVs present unique challenges relative to EV studies in other biofluids, including the invasive nature of CSF collection, limited CSF volumes and the low numbers of EVs in CSF as compared to plasma. Here, the objectives of the International Society for Extracellular Vesicles CSF Task Force are to promote the reproducibility of CSF EV studies by providing current reporting and best practices, and recommendations and reporting guidelines, for CSF EV studies. To accomplish this, we created and distributed a world-wide survey to ISEV members to assess methods considered 'best practices' for CSF EVs, then performed a detailed literature review for CSF EV publications that was used to curate methods and resources. Based on responses to the survey and curated information from publications, the CSF Task Force herein provides recommendations and reporting guidelines to promote the reproducibility of CSF EV studies in seven domains: (i) CSF Collection, Processing, and Storage; (ii) CSF EV Separation/Concentration; (iii) CSF EV Size and Number Measurements; (iv) CSF EV Protein Studies; (v) CSF EV RNA Studies; (vi) CSF EV Omics Studies and (vii) CSF EV Functional Studies