Enrichment of Astrocyte-Derived Extracellular Vesicles from Human Plasma

Published: August 03, 2022

doi:

Natalia Valle-Tamayo², Rocío Pérez-González³, Gemma Chiva-Blanch⁵, Olivia Belbin², Sara Serrano-Requena², Sònia Sirisi², Alba Cervantes González², Oriol Giró⁵, Érika Sánchez-Aced², Oriol Dols-Icardo², Daniel Alcolea², María Carmona-Iragui², Amanda Jimenez⁵, Alberto Lleó², Juan Fortea², M. Florencia Iulita²

¹Sant Pau Memory Unit, Department of Neurology, Hospital de la Santa Creu i Sant Pau, Biomedical Research Institute Sant Pau,Universitat Autònoma de Barcelona, ²Center of Biomedical Investigation Network for Neurodegenerative Diseases (CIBERNED), ³Movement Disorders Unit, Department of Neurology,Hospital de la Santa Creu i Sant Pau, ⁴Department of Endocrinology and Nutrition, Hospital Clínic de Barcelona,August Pi i Sunyer Biomedical Research Institute – IDIBAPS, ⁵Spanish Biomedical Research Network in Physiopathology of Obesity and Nutrition (CIBEROBN)

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

The research described in this protocol has been conducted with human plasma samples from healthy adult donors of both sexes (age range 65.9-81.3 years, 45.5% females), from the Sant Pau Initiative on Neurodegeneration (SPIN) cohort, Barcelona, Spain36. Participants gave informed consent. The study has been conducted following the international ethical guidelines for medical research contained in the declaration of Helsinki and the Spanish law. The Sant Pau Research Ethics Committee (CEIC) reviewe…

Representative Results

The isolation of ADEVs from plasma collected from healthy donors was successfully accomplished. A polymer-based precipitation method was employed to obtain the total EV fraction, followed by an immunocapture with magnetic microbeads to obtain ADEVs. Western blot analysis of the total EV fraction prior to the immuno-capture step indicated the lack of calnexin (cellular contamination marker) and the presence of Alix and the transmembrane protein CD9 in the EV preparations (F…

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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