Standardization of sample collection, isolation and analysis methods in extracellular vesicle research

The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)”, 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments

Synthesis and Characterization of Giant Plasma Membrane Vesicles and its Effect on Human Mesenchymal Stem Cells

Differentiation of mesenchymal stem cells to osteogenic lineage is a crucial step of in vivo bone remodelling and bone regeneration. In bone tissue engineering, numerous effort has been made for directed osteogenic differentiation of mesenchymal stem cells. A critical analysis of the literature revealed that in vivo, osteoblast itself induce the differentiation of mesenchymal stem cells towards osteogenic lineage. Keeping that perspective in mind here we hypothesized that giant plasma membrane vesicles (GPMV) derived from osteoblast can be as a ‘packaged cues’ for osteogenic differentiation of mesenchymal stem cells. Here we have reported the synthesis of giant plasma membrane vesicles (GPMVs) from the osteoblast MG-63 using a combination of paraformaldehyde (PFA) / dithiotheritol (DTT). To analyse the effect of different cellular constituents on the physico-chemical properties of the GPMV, osteoblast cells were treated with an actin blocker (cytochalacin-D) and a cholesterol sequester (methyl-β-cyclo dextrin) prior to GPMV generation. Size, shape and structural complexicty of the GPMV was characterized by image based analysis (phase contrast microscopy, fluorescence microscopy and electron microscopy) and by flow cytometry. The lipid extracted from the GPMVs was analysed by biochemical assay and NMR spectroscopy. Further, we fused the GPMV with the hMSCs and the fusion efficiency was analysed by confocal microscopy and flow cytometry. More than 15% of Fusion efficiency was observed. Proliferation of hMSC after fusion was analysed by MTT assay, PI based live/dead assay and confocal microscopy. The osteogenic differentiation was investigated by checking the activity of alkaline phosphatase, extent of biomineralization and collagen synthesis. The RT-PCR based analysis of the expression of osteogenic markers revealed that hMSCs cultured in the presence of GPMVs shows increased expression of early and late osteogenic markers

Identification of storage conditions stabilizing extracellular vesicles pre

Extracellular vesicles (EVs) play a key role in many physiological and pathophysiological processes and hold great potential for therapeutic and diagnostic use. Despite significant advances within the last decade, the key issue of EV storage stability remains unresolved and under investigated. Here, we aimed to identify storage conditions stabilizing EVs and comprehensively compared the impact of various storage buffer formulations at different temperatures on EVs derived from different cellular sources for up to 2 years. EV features including concentration, diameter, surface protein profile and nucleic acid contents were assessed by complementary methods, and engineered EVs containing fluorophores or functionalized surface proteins were utilized to compare cellular uptake and ligand binding. We show that storing EVs in PBS over time leads to drastically reduced recovery particularly for pure EV samples at all temperatures tested, starting already within days. We further report that using PBS as diluent was found to result in severely reduced EV recovery rates already within minutes. Several of the tested new buffer conditions largely prevented the observed effects, the lead candidate being PBS supplemented with human albumin and trehalose (PBS-HAT). We report that PBS-HAT buffer facilitates clearly improved short-term and long-term EV preservation for samples stored at -80°C, stability throughout several freeze-thaw cycles, and drastically improved EV recovery when using a diluent for EV samples for downstream applications

Charakterisierung extrazellulärer Vesikel und Analyse ihrer Bedeutung als Regulatoren der Immunantwort

Extrazelluläre Vesikel (EV) werden von nahezu allen Säugerzellen freigesetzt. Dabei sind verschiedene EV-Populationen in der Literatur bereits beschrieben worden. Mikrovesikel stehen für große Vesikel (GEV), die von der Plasmamembran freigesetzt werden, wohingegen Exosomen kleine Vesikel (KEV) sind, die von einem intrazellulärenKompartiment freigesetzt werden. In der Pathogenese des Systemischen Lupus Erythematodes (SLE) liegt eine dysregulierte Apoptose vor, die sich in einer erhöhten Apoptoserate kombiniert mit einer verringerten Clearence darstellt. Dabei akkumulieren apoptotische Zellreste und Autoantigene, wobei noch ungeklärt ist, weswegen die Autoimmunität ausbricht. Ein Kennzeichen der Apoptose ist die Freisetzung von Vesikeln, wobei Zell-Aktivierung ebenso zur Freisetzung von Vesikeln führt. Diese gegensätzlichen Freisetzungsstimuli lassen vermuten, dass verschiedene Stimuli die Freisetzung diverser EV-Populationen verursachen. Diese Arbeit widmete sich der Charakterisierung von EV, die von T-Zellen freigesetzt wurden, unter Einbeziehung des Freisetzungsstimulus (Zell-Aktivierung vs. Apoptose-Induktion). Außerdem wurde untersucht welchen Einfluss EV auf dendritische Zellen (DZ) ausüben sowie der Frage, ob und welche Rolle EV in der Pathogenese des SLE einnehmen. Die Analyse zeigt, dass EV, isoliert von humanen T-Lymphozyten, zwei voneinander trennbare EV-Populationen (KEV = 6) eine Rolle. Zusammengefasst konnte in der hier vorliegenden Arbeit gezeigt werden, (1) dass sich zwei EV-Populationen (GEV, KEV) in Größe und Proteinexpressionsmuster unterscheiden, (2) dass die differentielle Proteinexpressionen in EV abhängig vom Freisetzungsstimulus sind, (3) dass freigesetzte EV die DZ-Reifung und das von den DZ sezernierte Zytokinprofil beeinflussen können (deutliche Inhibition der MHC- Klasse II-Expression), (4) dass beim Vergleich der Proben von SLE-Patienten vs. gesunder Individuen eine deutlich reduzierte Freisetzung von GEV und KEV bei SLE-Patienten auftritt. Außerdem zeigen Proteinanalysen von SLE-Patienten verglichen mit denen gesunder Individuen Unterschiede in der Proteinbeladung der verschiedenen EV-Subpopulationen

EFFECTS OF MICROVESICLES DERIVED FROM BONE MARROW MESENCHYMAL STEM CELLS IN EXPERIMENTAL MODELS OF ALZHEIMER¿S DISEASE

Alzheimer's disease (AD) is the most common form of neurodegenerative illness leading to dementia characterized by the accumulation of abnormally folded \u3b2-Amyloid (A\u3b2) and tau proteins, forming amyloid plaques and neurofibrillary tangles, respectively. Recent evidences have highlighted that inflammation plays a critical role in AD, even though it remains unclear whether it represents a cause or a consequence of the pathology. Deposition of A\u3b2 peptides and tangles are able to stimulate a chronic inflammatory reaction, involving microglial activation and production of inflammatory cytokines likely contributing to neuronal dysfunction and cell death per se. Regarding immunomodulatory strategy development, during the last years, it has been shown that Mesenchymal Stem Cells (MSCs) play a strongly immunomodulatory role, protecting the injured tissue and guiding anti-inflammatory processes by the secretion of cytokines and microvesicles (MVs), involved in their paracrine effects. Furthermore many clinical studies are now performing therapies with MSCs and some phase I and phase II clinical trials in the oncology field are also studying MSC derived MVs (Dai et al, 2008 and Chaput and Th\ue9ry, 2011). Since the possibility that inflammation is not a mere consequence but a primary contributing factor in AD is becoming concrete, and given upregulation of inflammatory molecules (pro-inflammatory cytokines and chemokines) and activated glial cells surrounding the senile plaques in AD patients brains and AD transgenic animal models are now recognized as typical features of AD, the aim of this project is to assess the anti-inflammatory effects of MVs released by Bone Marrow Mesenchymal Stem Cells (BM-MSCs) in an AD context. Both in vitro and in vivo experimental approaches are used to investigate the MV immunomodulatory effect and their ability to affect A\u3b2 deposition and degradation. Pro- inflammatory (TNF\u3b1 and IL6) and anti-inflammatory cytokines (IL10) release was investigated by in vitro experiments performed on both microglial N9 cell line and on primary cortical cells exposed to human-A\u3b21-42 (h- A\u3b21-42 ) and MSC derived MVs. For a more complete analysis of the cell inflammatory state, the microglia phenotype was assessed in order to determine whether a change from M1 to M2 cell phenotype was detectable. The in vivo approach consisted of MV intracranial injections in a well-established transgenic AD chimeric murine model (APPswe/PS1dE9) that recapitulates many of the aspects of the human disease; to investigate whether MVs effects could be effective on the clearance and production of A\u3b2, possibly ameliorating the neurodegenerative context, we analyzed A\u3b2 load, plaque area and density in three different brain areas: cerebral cortex, hippocampus and cerebellum. The results obtained in vitro indicate that i) the administration of MVs promotes in vitro the secretion of anti-inflammatory cytokines, such as IL10; ii) MVs promote the switch of microglial cell to M2 phenotype, characterized by the typical amoeboid morphology, and increase the expression of CD206, a marker associated to the anti-inflammatory abilities; iii) MV administration significantly inhibits the release of the pro- inflammatory cytokines TNF\u3b1 and IL 6 in vitro and accordingly, MHC II expression, which is associated with a pro-inflammatory phenotype, is down regulated in the presence of MSC-MVs. On the other hand, in vivo results show that a single MV administration causes a significant reduction of A\u3b2 load into amyloid plaques and a decreased plaque area, into all three brain regions analyzed in 6 month old mice. In 3 month old treated mice, MV administration only affected A\u3b2 plaque density, that resulted smaller respect to untreated mice. In conclusion, MVs released from BM-derived MSCs can exert, in vivo, a protective effect reducing the accumulation in plaques of A\u3b2, possibly promoting the degradation and elimination of A\u3b242 and participating in the regulation of neuroinflammation. Future experiments will be directed to assess the safety of MV administration and to define whether a significant improvement of cognitive impairment is detectable in AD mice treated with BM-derived MSCs . \u200