Enrichment of Astrocyte-Derived Extracellular Vesicles from Human Plasma
Summary
This protocol describes the enrichment of astrocyte-derived extracellular vesicles (ADEVs) from human plasma. It is based on the separation of EVs by polymer precipitation, followed by ACSA-1 based immunocapture of ADEVs. Analysis of ADEVs may offer clues to study changes in inflammatory pathways of living patients, non-invasively by liquid biopsy.
Abstract
Extracellular vesicles (EVs) are biological nanoparticles secreted by all cells for cellular communication and waste elimination. They participate in a vast range of functions by acting on and transferring their cargos to other cells in physiological and pathological conditions. Given their presence in biofluids, EVs represent an excellent resource for studying disease processes and can be considered a liquid biopsy for biomarker discovery. An attractive aspect of EV analysis is that they can be selected based on markers of their cell of origin, thus reflecting the environment of a specific tissue in their cargo. However, one of the major handicaps related to EV isolation methods is the lack of methodological consensuses and standardized protocols. Astrocytes are glial cells with essential roles in the brain. In neurodegenerative diseases, astrocyte reactivity may lead to altered EV cargo and aberrant cellular communication, facilitating/enhancing disease progression. Thus, analysis of astrocyte EVs may lead to the discovery of biomarkers and potential disease targets. This protocol describes a 2-step method of enrichment of astrocyte-derived EVs (ADEVs) from human plasma. First, EVs are enriched from defibrinated plasma via polymer-based precipitation. This is followed by enrichment of ADEVs through ACSA-1-based immunocapture with magnetic micro-beads, where resuspended EVs are loaded onto a column placed in a magnetic field. Magnetically labeled ACSA-1+ EVs are retained within the column, while other EVs flow through. Once the column is removed from the magnet, ADEVs are eluted and are ready for storage and analysis. To validate the enrichment of astrocyte markers, glial fibrillary acidic protein (GFAP), or other specific astrocytic markers of intracellular origin, can be measured in the eluate and compared with the flow-through. This protocol proposes an easy, time-efficient method to enrich ADEVs from plasma that can be used as a platform to examine astrocyte-relevant markers.
Introduction
Extracellular vesicles (EVs) are a heterogeneous group of membranous nanoparticles secreted by all types of cells, carrying proteins, lipids, and nucleic acids1. Microvesicles (100-1000 nm), exosomes (30-100 nm), and apoptotic bodies (1000-5000 nm) constitute the main EV types, as distinguished by their site of origin2,3. EVs regulate important physiological processes, such as antigen presentation and immune responses4, receptor recycling, metabolite elimination5, and cellular communication6. The regulation of these processes may occur by direct binding between proteins enriched in the EV cell membrane and targets in recipient cells, and/or through the internalization and release of their cargo in the cytoplasm of the recipient cell7. While EVs perform essential cellular functions, they have gained increasing interest from a pathological perspective in the fields of cancer and neurology. Indeed, several studies have shown EVs can help promote tumor cell migration8,9 or seed toxic protein aggregates in neurodegenerative diseases, such as Alzheimer's disease10,11.
EVs can be selected and enriched from biofluids based on cell surface markers related to their cell of origin, thus reflecting the environment of a specific tissue in their cargo12,13,14,15,16,17,18,19,20. In addition, given their presence in blood, cerebrospinal fluid (CSF), saliva, urine, and breast milk, EVs represent an excellent, non-invasive tool for diagnosis, and can be considered a liquid biopsy for biomarker discovery. This is of special interest in neurology, given the difficulties of studying brain analytes in accessible fluids other than CSF.
Astrocytes have gained rising interest, as they are at the intersection of neuro-vascular communication21. Under physiological conditions, they are responsible for the preservation of the blood-brain barrier, the recycling of neurotransmitters, the supply of nutrients and growth factors to neurons and other glial cells22,23,24 as well as neuro-immune defense, given their metabolic plasticity from pro-inflammatory to anti-inflammatory states and vice versa25,26,27. An important mechanism by which astrocytes accomplish their regulatory functions is by communication through EVs28,29. Reactive astrocytosis is a key hallmark of several neurodegenerative diseases, such as Alzheimer's disease,30 multiple system atrophy (MSA), progressive supranuclear palsy (PSP)31, and amyotrophic lateral sclerosis (ALS)32. Astrocyte reactivity may lead to altered EV cargo, release of inflammatory mediators, and aberrant cellular communication, thus facilitating the spread of pathology and leading to neurodegeneration10,11. Therefore, studying astrocyte derived EVs (ADEVs) and changes in their cargo is an attractive resource to examine neurodegenerative processes in a non-invasive manner.
Currently, several methodologies exist for the isolation of EVs, each with its corresponding advantages and disadvantages33. It is essential to consider which method is more suitable for a specific use, depending on the final application of interest. In the neurology field, and more specifically, in astrocyte studies, polymer-based precipitation followed by immunocapture has been the predominantly used method12,18,19,20,34. However, even when applying the same approach, there remains heterogeneity between studies in the different steps applied for EV isolation. Therefore, there is a need for a clear, step-by-step standardized methodology to facilitate astrocyte EV studies and study reproducibility. Polymer-based precipitation facilitates biomarker screening given that it is a fast, simple procedure that does not require complex equipment, leading to a high yield of EVs without affecting their biological activity35.
The present protocol describes a detailed, simple, two-step method for the enrichment of ADEVs from human plasma. It is based on a polymer-based precipitation of the total EV fraction, followed by an immunocapture of astrocyte EVs. Given the important functions of astrocytes, analysis of ADEVs may shed light for the discovery of biomarkers and brain inflammatory pathways that can be studied in a non-invasive manner.
Protocol
Representative Results
Discussion
EVs have gained strong interest in biomedical research due to their diagnostic and therapeutic potential. Currently, one of the major handicaps related to EV isolation methods is the lack of methodological consensus and standardized protocols. This study provides a detailed protocol for the enrichment of astrocyte EVs from human plasma via polymer-based precipitation and GLAST immunocapture.
Different methodologies exist for the isolation of EVs from body fluids, each with their own a…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge the help of Soraya Torres, Shaimaa El Bounasri El Bennadi, and Oriol Sanchez Lopez for sample handling and preparation. We would also like to acknowledge the collaboration of José Amable Bernabé, from the ICTS "NANBIOSIS", unit 6 (Unit of CIBER in Bioingineering, Biomaterials & Nanomedicine) of the Barcelona Materials Science Institute, Marti de Cabo Jaume from the Electron Microscopy Unit at Universitat Autonoma de Barcelona, Dr. Marta Soler Castany and Lia Ros Blanco from the Flow Cytometry Platform at Sant Pau Biomedical Research Institute (IIB-Sant Pau), as well as Dr. Joan Carles Escolà-Gil from the Pathophysiology of lipid-related diseases group at IIB-Sant Pau for help with the NTA, cryo-EM, Luminex, and ApoB determinations, respectively.
The authors acknowledge financial support from the Jérôme Lejeune Foundation (Project #1941 and #1913 to MFI and MCI), Instituto de Salud Carlos III (PI20/01473 to JF, PI20/01330 to AL, PI18/00435 to DA, and INT19/00016 to DA), the National Institute of Health (1R01AG056850-01A1, R21AG056974, and R01AG061566 to JF), the Alzheimer's Association and Global Brain Health Institute (GBHI_ALZ-18-543740 to MCI), the The Association for Frontotemporal Degeneration (Clinical Research Postdoctoral Fellowship, AFTD 2019–2021) to ODI, and the Societat Catalana de Neurologia (Premi Beca Fundació SCN 2020 to MCI). This work was also supported by the CIBERNED program (Program 1, Alzheimer Disease to AL and SIGNAL study. SS is a recipient of a Postdoctoral grant “Juan de la Cierva-Incorporación” (IJC2019-038962-I) by the Agencia Estatal de Investigación, Ministerio de Ciencia e Innovación (Gobierno de España).
Materials
| Anti-Alix primary antibody for Western blotting | EMD Millipore | ABC40 | |
| µMACS Separator | Miltenyi Biotec | 130-042-602 | The µMACS Separator is used in combination with µ Columns and MACS MicroBeads. |
| Anti-calnexin primary antibody for Western blotting | Genetex | GTX109669 | |
| Anti-CD9 primary antibody for Western blotting | Cell Signaling | 13174 | |
| Blocker BSA (10%) 200 mL | Thermo Fisher | 37525 | |
| Bransonic 1510E-MT Ultrasonic bath | Branson | ||
| COBAS 6000 autoanalyzer | Roche Diagnostics | Analyzer for immunoturbidimetric determination of ApoB; commercial autoanalyzer | |
| cOmplete Protease Inhibitor Cocktail (EDTA-free) | Roche | 11873580001 | |
| Digital Micrograph 1.8 | micrograph software | ||
| Dulbecco's PBS Mg++, Ca++ free 500 mL | Thermo Fisher | 14190144 | |
| EveryBlot Blocking Buffer | BioRad | 12010020 | |
| Exoquick (exosome precipitation solution 5 mL) + Thrombin | System Bioscience | EXOQ5TM-1 | ExoQuick 20 mL can also be purchased (EXOQ20A-1) |
| Gatan 895 USC 4000 | camera | ||
| GeneGnome XRQ chemiluminiscence imaging system | Syngene | ||
| Human CD81 antigen (CD81) ELISA kit | Cusabio | CSB-EL004960HU | |
| Human Programmed cell death 6-interacting protein (PDCD6IP) ELISA kit | Cusabio | CSB-EL017673HU | |
| Immun-Blot PVDF Membrane | BioRad | 1620177 | |
| JEOL 2011 transmission electron microscope | JEOL LTD | Equipped with a CCD Gatan 895 USC 4000 camera (Gatan 626, Gatan, Pleasanton, USA) | |
| Lavender EDTA BD Vacutainer K2E tubes | Becton dickinson | 367525 | |
| Leica EM GP | Leica Microsystem | commercial plunge freezer | |
| Low binding microtubes 1,5 mL | Deltalab | 4092.3NS | |
| MACS µ Columns with plungers | Miltenyi Biotec | 130-110-905 | µ Columns with plungers are especially designed for isolation of exosomes from body fluids |
| MACS Multistand | Miltenyi Biotec | 130-042-303 | |
| MAGPIX plate reader | Luminex Corporation | 80-073 | Luminex's xMAP multiplexing unit (Luminex xPonent v 4.3 software) |
| MicroBead Kit100 μL Anti-GLAST (ACSA-1)-Biotin, human, mouse, rat – small size; 100 μL Anti-Biotin MicroBeads | Miltenyi Biotec | 130-095- 825 | |
| MILLIPLEX MAP Kit Human cytokine/Chemokine/Growth Factor Panel A magnetic bead panel | EMD Millipore | HCYTA-60K-25 | |
| M-PER Mammalian Protein Extraction Reagent 25 mL | Thermo Fisher | 78503 | For certain applications like Western blot, more aggressive lysis buffers can be used (e.g. RIPA) |
| MultiSkan SkyHigh Microplate Spectrophotometer | Thermofisher | A51119500C | |
| NanoSight NS300 | Malvern Panalytical | NTA; 3.4 version | |
| Pierce Halt Protease and Phosphatase Inhibitor Cocktail | Thermo Fisher | 78441 | |
| Polypropylene syringe (G29) | PeroxFarma | 1mL syringe; 0.33x12mm-G29x1/2" | |
| Secondary anti-rabbit antibody | Thermo Fisher | 10794347 | |
| Simoa GFAP Discovery Kit | Quanterix | 102336 | |
| Simoa, SR-X instrument | Quanterix | SR-X Ultra-Sensitive Biomarker Detection System; commercial biomarker detection technology | |
| Specific Protein Test Apolipoprotein B – APOB (100 det) COBAS C/CI | Roche Diagnostics | 3032574122 | |
| SuperSignal West Femto | Thermo Fisher | 34095 | Ultra-sensitive enhanced chemiluminescent (ECL) HRP substrate |
| Trans-Blot Turbo Transfer System | BioRad | 1704150 |
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