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Park J, Feng M, Yang J, Shen H, Qin Z, Guo W, Issadore DA. Agarose Microgel-Based In Situ Cleavable Immuno-Rolling Circle Amplification for Multiplexed Single-Molecule Quantitation on Single Extracellular Vesicles. ACS NANO 2025; 19:17884-17899. [PMID: 40320637 DOI: 10.1021/acsnano.5c04207] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/14/2025]
Abstract
We have developed a platform for the multiplexed and ultrasensitive profiling of individual extracellular vesicles (EVs) directly in plasma, which we call GDEVA─Agarose microGel-based Digital single-molecule-single EV Assay. GDEVA achieves single-molecule sensitivity and moderate multiplexing (demonstrated 3-plex), and can achieve a throughput of ∼104 EVs per minute necessary to resolve EVs directly in human plasma when read out using flow cytometry. Our platform integrates a rolling circle amplification (RCA) immunoassay of EV surface proteins, which are cleaved from single EVs, and amplified within agarose microgels, followed by flow cytometry-based readout or imaging after fluorescence-activated cell sorting (FACS). It overcomes steric hindrance of RCA products, nonspecific binding of RCA templates, and the lack of quantitation of multiple proteins on EVs that have plagued earlier approaches. We evaluated the analytical capabilities of GDEVA through head-to-head comparison with conventional technology and demonstrated a ∼100× improvement in the limit of detection (LOD) of EV subpopulations. We evaluate GDEVA's potential in cancer immunology, by analyzing single EVs in plasma samples from patients with melanoma, where EV heterogeneity plays a critical role in disease progression and response to therapy. We demonstrate profiling of individual EVs for key immune markers PD-L1, CD155, and the melanoma marker TYRP-1, and showed that GDEVA can precisely quantify EVs, offering the resolution to detect rare EV subpopulations in complex clinical specimens.
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Affiliation(s)
- Juhwan Park
- Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
- Department of Bio and Fermentation Convergence Technology, Kookmin University, Seoul 02707, Republic of Korea
| | - Michelle Feng
- Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
| | - Jingbo Yang
- Department of Biology, School of Arts and Science, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
| | - Hanfei Shen
- Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
| | - Zhiyuan Qin
- Department of Biology, School of Arts and Science, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
| | - Wei Guo
- Department of Biology, School of Arts and Science, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
| | - David A Issadore
- Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
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Scuteri A, Donzelli E. Dual role of extracellular vesicles in neurodegenerative diseases. World J Stem Cells 2024; 16:1002-1011. [PMID: 39734484 PMCID: PMC11669982 DOI: 10.4252/wjsc.v16.i12.1002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/30/2024] [Revised: 10/17/2024] [Accepted: 11/22/2024] [Indexed: 12/13/2024] Open
Abstract
Extracellular vesicles (EVs) are cell-to-cell interaction tools that are attracting increasing interest in the literature in two opposing areas. In addition to their role in physiological development, there is growing evidence of their involvement in healing and protective processes. However, EVs also mediate pathological conditions, particularly contributing to the progression of several chronic diseases, such as neurodegenerative diseases. On the other hand, EVs also form the core of a new therapeutic strategy for neuroprotection, which is based on the administration of EVs derived from a wide range of donor cells. In particular, the possibility of obtaining numerous EVs from stem cells of different origins, which is feasible for therapeutic aims, is now under investigation. In this review, we focused on neurodegenerative diseases, in which EVs could have a propagative detrimental effect or could also be exploited to deliver protective factors. This review explores the different hypotheses concerning the dual role of EVs, with the aim of shedding light on the following question: Can vesicles be used to fight vesicle-propagated diseases?
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Affiliation(s)
- Arianna Scuteri
- Experimental Neurology Unit and Milan Center for Neuroscience, School of Medicine and Surgery, University of Milano-Bicocca, Monza 20900, Italy.
| | - Elisabetta Donzelli
- Experimental Neurology Unit and Milan Center for Neuroscience, School of Medicine and Surgery, University of Milano-Bicocca, Monza 20900, Italy
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Kumari S, Lausted C, Scherler K, Ng AHC, Lu Y, Lee I, Hood L, Wang K. Approaches and Challenges in Characterizing the Molecular Content of Extracellular Vesicles for Biomarker Discovery. Biomolecules 2024; 14:1599. [PMID: 39766306 PMCID: PMC11674167 DOI: 10.3390/biom14121599] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2024] [Revised: 12/04/2024] [Accepted: 12/11/2024] [Indexed: 01/11/2025] Open
Abstract
Extracellular vesicles (EVs) are lipid bilayer nanoparticles released from all known cells and are involved in cell-to-cell communication via their molecular content. EVs have been found in all tissues and body fluids, carrying a variety of biomolecules, including DNA, RNA, proteins, metabolites, and lipids, offering insights into cellular and pathophysiological conditions. Despite the emergence of EVs and their molecular contents as important biological indicators, it remains difficult to explore EV-mediated biological processes due to their small size and heterogeneity and the technical challenges in characterizing their molecular content. EV-associated small RNAs, especially microRNAs, have been extensively studied. However, other less characterized RNAs, including protein-coding mRNAs, long noncoding RNAs, circular RNAs, and tRNAs, have also been found in EVs. Furthermore, the EV-associated proteins can be used to distinguish different types of EVs. The spectrum of EV-associated RNAs, as well as proteins, may be associated with different pathophysiological conditions. Therefore, the ability to comprehensively characterize EVs' molecular content is critical for understanding their biological function and potential applications in disease diagnosis. Here, we set out to provide an overview of EV-associated RNAs and proteins as well as approaches currently being used to characterize them.
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Affiliation(s)
- Suman Kumari
- Institute for Systems Biology, Seattle, WA 98109, USA; (S.K.); (C.L.); (K.S.); (L.H.)
| | - Christopher Lausted
- Institute for Systems Biology, Seattle, WA 98109, USA; (S.K.); (C.L.); (K.S.); (L.H.)
| | - Kelsey Scherler
- Institute for Systems Biology, Seattle, WA 98109, USA; (S.K.); (C.L.); (K.S.); (L.H.)
| | - Alphonsus H. C. Ng
- Department of Molecular Pharmaceutics, University of Utah, Salt Lake City, UT 84112, USA; (A.H.C.N.); (Y.L.)
- Department of Biomedical Engineering, University of Utah, Salt Lake City, UT 84112, USA
| | - Yue Lu
- Department of Molecular Pharmaceutics, University of Utah, Salt Lake City, UT 84112, USA; (A.H.C.N.); (Y.L.)
| | - Inyoul Lee
- Institute for Systems Biology, Seattle, WA 98109, USA; (S.K.); (C.L.); (K.S.); (L.H.)
| | - Leroy Hood
- Institute for Systems Biology, Seattle, WA 98109, USA; (S.K.); (C.L.); (K.S.); (L.H.)
| | - Kai Wang
- Institute for Systems Biology, Seattle, WA 98109, USA; (S.K.); (C.L.); (K.S.); (L.H.)
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4
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Ali Moussa HY, Shin KC, de la Fuente A, Bensmail I, Abdesselem HB, Ponraj J, Mansour S, Al-Shaban FA, Stanton LW, Abdulla SA, Park Y. Proteomics analysis of extracellular vesicles for biomarkers of autism spectrum disorder. Front Mol Biosci 2024; 11:1467398. [PMID: 39606031 PMCID: PMC11599737 DOI: 10.3389/fmolb.2024.1467398] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2024] [Accepted: 10/24/2024] [Indexed: 11/29/2024] Open
Abstract
Background Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by symptoms that include social interaction deficits, language difficulties and restricted, repetitive behavior. Early intervention through medication and behavioral therapy can eliminate some ASD-related symptoms and significantly improve the life-quality of the affected individuals. Currently, the diagnosis of ASD is highly limited. Methods To investigate the feasibility of early diagnosis of ASD, we tested extracellular vesicles (EVs) proteins obtained from ASD cases. First, plasma EVs were isolated from healthy controls (HCs) and ASD individuals and were analyzed using proximity extension assay (PEA) technology to quantify 1,196 protein expression level. Second, machine learning analysis and bioinformatic approaches were applied to explore how a combination of EV proteins could serve as biomarkers for ASD diagnosis. Results No significant differences in the EV morphology and EV size distribution between HCs and ASD were observed, but the EV number was slightly lower in ASD plasma. We identified the top five downregulated proteins in plasma EVs isolated from ASD individuals: WW domain-containing protein 2 (WWP2), Heat shock protein 27 (HSP27), C-type lectin domain family 1 member B (CLEC1B), Cluster of differentiation 40 (CD40), and folate receptor alpha (FRalpha). Machine learning analysis and correlation analysis support the idea that these five EV proteins can be potential biomarkers for ASD. Conclusion We identified the top five downregulated proteins in ASD EVs and examined that a combination of EV proteins could serve as biomarkers for ASD diagnosis.
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Affiliation(s)
- Houda Yasmine Ali Moussa
- Neurological Disorders Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, Doha, Qatar
| | - Kyung Chul Shin
- Neurological Disorders Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, Doha, Qatar
| | - Alberto de la Fuente
- Neurological Disorders Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, Doha, Qatar
| | - Ilham Bensmail
- Proteomics Core Facility, Hamad Bin Khalifa University (HBKU), Qatar Foundation, Doha, Qatar
| | - Houari B. Abdesselem
- Proteomics Core Facility, Hamad Bin Khalifa University (HBKU), Qatar Foundation, Doha, Qatar
| | | | - Said Mansour
- HBKU Core Labs, Hamad Bin Khalifa University (HBKU), Doha, Qatar
| | - Fouad A. Al-Shaban
- Neurological Disorders Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, Doha, Qatar
- College of Health and Life Sciences (CHLS), Hamad Bin Khalifa University (HBKU), Qatar Foundation, Doha, Qatar
| | - Lawrence W. Stanton
- Neurological Disorders Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, Doha, Qatar
- College of Health and Life Sciences (CHLS), Hamad Bin Khalifa University (HBKU), Qatar Foundation, Doha, Qatar
| | - Sara A. Abdulla
- Neurological Disorders Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, Doha, Qatar
| | - Yongsoo Park
- Neurological Disorders Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, Doha, Qatar
- College of Health and Life Sciences (CHLS), Hamad Bin Khalifa University (HBKU), Qatar Foundation, Doha, Qatar
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Park J, Feng M, Yang J, Shen H, Qin Z, Guo W, Issadore DA. High-throughput, multiplexed quantification, and sorting of single EVs at single-molecule level. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.10.31.621423. [PMID: 39553943 PMCID: PMC11565983 DOI: 10.1101/2024.10.31.621423] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2024]
Abstract
We have developed a platform for the high-throughput, multiplexed, and ultra-sensitive profiling of individual extracellular vesicles (EVs) directly in plasma, which we call BDEVS - Agarose B ead-based D igital Single Molecule-Single EV S orting. Unlike conventional approaches, BDEVS achieves single molecule sensitivity and moderate multiplexing (demonstrated 3-plex) without sacrificing the throughput (processing ten thousand of EVs per minute) necessary to resolve EVs directly in human plasma. Our platform integrates rolling circle amplification (RCA) of EV surface proteins, which are cleaved from single EVs, and amplified within agarose droplets, followed by flow cytometry-based readout and sorting, overcoming steric hindrance, non-specific binding, and the lack of quantitation of multiple proteins on EVs that have plagued earlier approaches. We evaluated the analytical capabilities of BDEVS through head-to-head comparison with gold-standard technologies, and demonstrated a ∼100x improvement in the limit of detection of EV subpopulations. We demonstrate the high throughput (∼100k beads / minute) profiling of individual EVs for key immune markers PD-L1, CD155, and the melanoma tumor marker TYRP-1, and showed that BDEVS can precisely quantify and sort EVs, offering unprecedented resolution for analyzing tumor-immune interactions and detecting rare EV subpopulations in complex clinical specimens. We demonstrate BDEVS's potential as a transformative tool for EV-based diagnostics and therapeutic monitoring in the context of cancer immunology by analyzing plasma samples from patients with melanoma, where EV heterogeneity plays a critical role in disease progression and response to therapy.
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Xu S, Zhang Z, Melvin BC, Basu Ray N, Ikezu S, Ikezu T. Comparison of nanoimaging and nanoflow based detection of extracellular vesicles at a single particle resolution. JOURNAL OF EXTRACELLULAR BIOLOGY 2024; 3:e70016. [PMID: 39416671 PMCID: PMC11481688 DOI: 10.1002/jex2.70016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 04/18/2024] [Revised: 09/08/2024] [Accepted: 10/02/2024] [Indexed: 10/19/2024]
Abstract
The characterization of single extracellular vesicle (EV) has been an emerging tool for the early detection of various diseases despite there being challenges regarding how to interpret data with different protocols or instruments. In this work, standard EV particles were characterized for single CD9+, single CD81+ or double CD9+/CD81+ tetraspanin molecule positivity with two single EV analytic technologies in order to optimize their EV sample preparation after antibody labelling and analysis methods: NanoImager for direct stochastic optical reconstruction microscopy (dSTORM)-based EV imaging and characterization, and Flow NanoAnalyzer for flow-based EV quantification and characterization. False positives from antibody aggregates were found during dSTORM-based NanoImager imaging. Analysis of particle radius with lognormal fittings of probability density histogram enabled the removal of antibody aggregates and corrected EV quantification. Furthermore, different machine learning models were trained to differentiate antibody aggregates from EV particles and correct EV quantification with increased double CD9+/CD81+ population. With Flow NanoAnalyzer, EV samples were prepared with different dilution or fractionation methods, which increased the detection rate of CD9+/CD81+ EV population. Comparing the EV phenotype percentages measured by two instruments, differences in double positive and single positive particles existed after percentage correction, which might be due to the different detection limit of each instrument. Our study reveals that the characterization of individual EVs for tetraspanin positivity varies between two platforms-the NanoImager and the Flow NanoAnalyzer-depending on the EV sample preparation methods used after antibody labelling. Additionally, we applied machine learning models to correct for false positive particles identified in imaging-based results by fitting size distribution data.
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Affiliation(s)
- Shihan Xu
- Department of NeuroscienceMayo Clinic FloridaJacksonvilleFloridaUSA
| | - Zhengrong Zhang
- Department of NeuroscienceMayo Clinic FloridaJacksonvilleFloridaUSA
| | | | | | - Seiko Ikezu
- Department of NeuroscienceMayo Clinic FloridaJacksonvilleFloridaUSA
| | - Tsuneya Ikezu
- Department of NeuroscienceMayo Clinic FloridaJacksonvilleFloridaUSA
- Regenerative Science Graduate ProgramMayo Clinic College of Medicine and ScienceJacksonvilleFloridaUSA
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Dayarathna T, Roseborough AD, Gomes J, Khazaee R, Silveira CRA, Borron K, Yu S, Coleman K, Jesso S, Finger E, MacDonald P, Borrie M, Wells J, Bartha R, Zou G, Whitehead SN, Leong HS, Pasternak SH. Nanoscale flow cytometry-based quantification of blood-based extracellular vesicle biomarkers distinguishes MCI and Alzheimer's disease. Alzheimers Dement 2024; 20:6094-6106. [PMID: 38958575 PMCID: PMC11497682 DOI: 10.1002/alz.14087] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2024] [Revised: 05/09/2024] [Accepted: 05/30/2024] [Indexed: 07/04/2024]
Abstract
INTRODUCTION Accurate testing for Alzheimer's disease (AD) represents a crucial step for therapeutic advancement. Currently, tests are expensive and require invasive sampling or radiation exposure. METHODS We developed a nanoscale flow cytometry (nFC)-based assay of extracellular vesicles (EVs) to screen biomarkers in plasma from mild cognitive impairment (MCI), AD, or controls. RESULTS Circulating amyloid beta (Aβ), tau, phosphorylated tau (p-tau)181, p-tau231, p-tau217, p-tauS235, ubiquitin, and lysosomal-associated membrane protein 1-positive EVs distinguished AD samples. p-tau181, p-tau217, p-tauS235, and ubiquitin-positive EVs distinguished MCI samples. The most sensitive marker for AD distinction was p-tau231, with an area under the receiver operating characteristic curve (AUC) of 0.96 (sensitivity 0.95/specificity 1.0) improving to an AUC of 0.989 when combined with p-tauS235. DISCUSSION This nFC-based assay accurately distinguishes MCI and AD plasma without EV isolation, offering a rapid approach requiring minute sample volumes. Incorporating nFC-based measurements in larger populations and comparison to "gold standard" biomarkers is an exciting next step for developing AD diagnostic tools. HIGHLIGHTS Extracellular vesicles represent promising biomarkers of Alzheimer's disease (AD) that can be measured in the peripheral circulation. This study demonstrates the utility of nanoscale flow cytometry for the measurement of circulating extracellular vesicles (EVs) in AD blood samples. Multiple markers including amyloid beta, tau, phosphorylated tau (p-tau)181, p-tau231, p-tau217, and p-tauS235 accurately distinguished AD samples from healthy controls. Future studies should expand blood and cerebrospinal fluid-based EV biomarker development using nanoflow cytometry approaches.
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Affiliation(s)
- Thamara Dayarathna
- Institute for Genomic MedicineAbigail Wexner Research Institute at Nationwide Children's HospitalColumbusOhioUSA
| | - Austyn D. Roseborough
- Vulnerable Brain Lab, Department of Anatomy and Cell Biology, Schulich School of Medicine and DentistryWestern UniversityLondonOntarioCanada
| | - Janice Gomes
- Robarts Research Institute, Schulich School of Medicine and DentistryWestern UniversityLondonOntarioCanada
| | - Reza Khazaee
- Department of BiologyWestern UniversityLondonOntarioCanada
- Biotron Integrated Microscopy FacilityWestern UniversityLondonOntarioCanada
| | - Carolina R. A. Silveira
- Cognitive Neurology and Alzheimer's Disease Research CentreParkwood Institute, St. Joseph's Health Care CentreLondonOntarioCanada
| | - Kathy Borron
- Cognitive Neurology and Alzheimer's Disease Research CentreParkwood Institute, St. Joseph's Health Care CentreLondonOntarioCanada
| | - Soojung Yu
- Cognitive Neurology and Alzheimer's Disease Research CentreParkwood Institute, St. Joseph's Health Care CentreLondonOntarioCanada
| | - Kristy Coleman
- Cognitive Neurology and Alzheimer's Disease Research CentreParkwood Institute, St. Joseph's Health Care CentreLondonOntarioCanada
| | - Sarah Jesso
- Cognitive Neurology and Alzheimer's Disease Research CentreParkwood Institute, St. Joseph's Health Care CentreLondonOntarioCanada
| | - Elizabeth Finger
- Robarts Research Institute, Schulich School of Medicine and DentistryWestern UniversityLondonOntarioCanada
- Cognitive Neurology and Alzheimer's Disease Research CentreParkwood Institute, St. Joseph's Health Care CentreLondonOntarioCanada
- Department of Clinical Neurological Sciences, Schulich School of Medicine and DentistryWestern UniversityLondonOntarioCanada
| | - Penny MacDonald
- Department of Clinical Neurological Sciences, Schulich School of Medicine and DentistryWestern UniversityLondonOntarioCanada
| | - Michael Borrie
- Department of Geriatric Medicine, Schulich School of Medicine and DentistryWestern UniversityLondonOntarioCanada
| | - Jennie Wells
- Department of Geriatric Medicine, Schulich School of Medicine and DentistryWestern UniversityLondonOntarioCanada
| | - Robert Bartha
- Robarts Research Institute, Schulich School of Medicine and DentistryWestern UniversityLondonOntarioCanada
| | - Guangyong Zou
- Robarts Research Institute, Schulich School of Medicine and DentistryWestern UniversityLondonOntarioCanada
- Department of Epidemiology and Biostatistics, Schulich School of Medicine and DentistryWestern UniversityLondonOntarioCanada
| | - Shawn N. Whitehead
- Vulnerable Brain Lab, Department of Anatomy and Cell Biology, Schulich School of Medicine and DentistryWestern UniversityLondonOntarioCanada
| | - Hon S. Leong
- Sunnybrook Research InstituteUniversity of TorontoTorontoOntarioCanada
| | - Stephen H. Pasternak
- Robarts Research Institute, Schulich School of Medicine and DentistryWestern UniversityLondonOntarioCanada
- Cognitive Neurology and Alzheimer's Disease Research CentreParkwood Institute, St. Joseph's Health Care CentreLondonOntarioCanada
- Department of Clinical Neurological Sciences, Schulich School of Medicine and DentistryWestern UniversityLondonOntarioCanada
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Brooks B, D'Egidio F, Borlongan MC, Borlongan MC, Lee JY. Stem cell grafts enhance endogenous extracellular vesicle expression in the stroke brain. Brain Res Bull 2024; 214:110999. [PMID: 38851436 DOI: 10.1016/j.brainresbull.2024.110999] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2024] [Revised: 06/04/2024] [Accepted: 06/05/2024] [Indexed: 06/10/2024]
Abstract
Endogenous brain repair occurs following an ischemic stroke but is transient, thus unable to fully mount a neuroprotective response against the evolving secondary cell death. Finding a treatment strategy that may render robust and long-lasting therapeutic effects stands as a clinically relevant therapy for stroke. Extracellular vesicles appear to be upregulated after stroke, which may represent a candidate target for neuroprotection. In this study, we probed whether transplanted stem cells could enhance the expression of extracellular vesicles to afford stable tissue remodeling in the ischemic stroke brain. Aged rats were initially exposed to the established ischemic stroke model of middle cerebral artery occlusion then received intravenous delivery of either bone marrow-derived mesenchymal stem cell transplantation or vehicle. A year later, the animals were assayed for brain damage, inflammation, and extracellular vesicle expression. Our findings revealed that while core infarction was not reduced, the stroke animals transplanted with stem cells displayed a significant reduction in peri-infarct cell loss that coincided with downregulated Iba1-labeled inflammatory cells and upregulated CD63-positive extracellular vesicles that appeared to be co-localized with GFAP-positive astrocytes. Interestingly, grafted stem cells were not detected at one year post-transplantation period, suggesting that the extracellular vesicles likely originated within the host brain. That long-lasting functional benefits persisted in the absence of surviving transplanted stem cells, but with upregulation of endogenous extracellular vesicles, advances the concept that transplantation of stem cells acutely after stroke propels host extracellular vesicles to the ischemic brain, altogether promoting chronic brain remodeling.
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Affiliation(s)
- Beverly Brooks
- Department of Neurosurgery and Brain Repair, Center of Excellence for Aging and Brain Repair, University of South Florida Morsani College of Medicine, 12901 Bruce B. Downs Blvd., Tampa, FL 33612, United States
| | - Francesco D'Egidio
- Department of Neurosurgery and Brain Repair, Center of Excellence for Aging and Brain Repair, University of South Florida Morsani College of Medicine, 12901 Bruce B. Downs Blvd., Tampa, FL 33612, United States
| | - Maximillian C Borlongan
- Department of Neurosurgery and Brain Repair, Center of Excellence for Aging and Brain Repair, University of South Florida Morsani College of Medicine, 12901 Bruce B. Downs Blvd., Tampa, FL 33612, United States
| | - Mia C Borlongan
- Department of Neurosurgery and Brain Repair, Center of Excellence for Aging and Brain Repair, University of South Florida Morsani College of Medicine, 12901 Bruce B. Downs Blvd., Tampa, FL 33612, United States
| | - Jea-Young Lee
- Department of Neurosurgery and Brain Repair, Center of Excellence for Aging and Brain Repair, University of South Florida Morsani College of Medicine, 12901 Bruce B. Downs Blvd., Tampa, FL 33612, United States.
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Wu CC, Tsantilas KA, Park J, Plubell D, Sanders JA, Naicker P, Govender I, Buthelezi S, Stoychev S, Jordaan J, Merrihew G, Huang E, Parker ED, Riffle M, Hoofnagle AN, Noble WS, Poston KL, Montine TJ, MacCoss MJ. Mag-Net: Rapid enrichment of membrane-bound particles enables high coverage quantitative analysis of the plasma proteome. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2023.06.10.544439. [PMID: 38617345 PMCID: PMC11014469 DOI: 10.1101/2023.06.10.544439] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/16/2024]
Abstract
Membrane-bound particles in plasma are composed of exosomes, microvesicles, and apoptotic bodies and represent ~1-2% of the total protein composition. Proteomic interrogation of this subset of plasma proteins augments the representation of tissue-specific proteins, representing a "liquid biopsy," while enabling the detection of proteins that would otherwise be beyond the dynamic range of liquid chromatography-tandem mass spectrometry of unfractionated plasma. We have developed an enrichment strategy (Mag-Net) using hyper-porous strong-anion exchange magnetic microparticles to sieve membrane-bound particles from plasma. The Mag-Net method is robust, reproducible, inexpensive, and requires <100 μL plasma input. Coupled to a quantitative data-independent mass spectrometry analytical strategy, we demonstrate that we can collect results for >37,000 peptides from >4,000 plasma proteins with high precision. Using this analytical pipeline on a small cohort of patients with neurodegenerative disease and healthy age-matched controls, we discovered 204 proteins that differentiate (q-value < 0.05) patients with Alzheimer's disease dementia (ADD) from those without ADD. Our method also discovered 310 proteins that were different between Parkinson's disease and those with either ADD or healthy cognitively normal individuals. Using machine learning we were able to distinguish between ADD and not ADD with a mean ROC AUC = 0.98 ± 0.06.
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Affiliation(s)
- Christine C. Wu
- Department of Genome Sciences, University of Washington, Seattle, WA, USA
| | | | - Jea Park
- Department of Genome Sciences, University of Washington, Seattle, WA, USA
| | - Deanna Plubell
- Department of Genome Sciences, University of Washington, Seattle, WA, USA
| | - Justin A. Sanders
- Department of Computer Science, University of Washington, Seattle, WA, USA
| | | | | | | | | | | | - Gennifer Merrihew
- Department of Computer Science, University of Washington, Seattle, WA, USA
| | - Eric Huang
- Department of Computer Science, University of Washington, Seattle, WA, USA
| | - Edward D. Parker
- Vision Core Lab, Department of Ophthalmology, University of Washington, Seattle, WA, USA
| | - Michael Riffle
- Department of Biochemistry, University of Washington, Seattle, WA, USA
| | - Andrew N. Hoofnagle
- Department of Lab Medicine and Pathology, University of Washington, Seattle, WA, USA
| | - William S. Noble
- Department of Genome Sciences, University of Washington, Seattle, WA, USA
- Department of Computer Science, University of Washington, Seattle, WA, USA
| | - Kathleen L. Poston
- Department of Neurology & Neurological Sciences, Stanford University, Palo Alto CA, USA
| | | | - Michael J. MacCoss
- Department of Genome Sciences, University of Washington, Seattle, WA, USA
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10
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Kumar A, Nader MA, Deep G. Emergence of Extracellular Vesicles as "Liquid Biopsy" for Neurological Disorders: Boom or Bust. Pharmacol Rev 2024; 76:199-227. [PMID: 38351075 PMCID: PMC10877757 DOI: 10.1124/pharmrev.122.000788] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2022] [Revised: 11/11/2023] [Accepted: 11/27/2023] [Indexed: 02/16/2024] Open
Abstract
Extracellular vesicles (EVs) have emerged as an attractive liquid biopsy approach in the diagnosis and prognosis of multiple diseases and disorders. The feasibility of enriching specific subpopulations of EVs from biofluids based on their unique surface markers has opened novel opportunities to gain molecular insight from various tissues and organs, including the brain. Over the past decade, EVs in bodily fluids have been extensively studied for biomarkers associated with various neurological disorders, such as Alzheimer's disease, Parkinson's disease, schizophrenia, bipolar disorder, major depressive disorders, substance use disorders, human immunodeficiency virus-associated neurocognitive disorder, and cancer/treatment-induced neurodegeneration. These studies have focused on the isolation and cargo characterization of either total EVs or brain cells, such as neuron-, astrocyte-, microglia-, oligodendrocyte-, pericyte-, and endothelial-derived EVs from biofluids to achieve early diagnosis and molecular characterization and to predict the treatment and intervention outcomes. The findings of these studies have demonstrated that EVs could serve as a repetitive and less invasive source of valuable molecular information for these neurological disorders, supplementing existing costly neuroimaging techniques and relatively invasive measures, like lumbar puncture. However, the initial excitement surrounding blood-based biomarkers for brain-related diseases has been tempered by challenges, such as lack of central nervous system specificity in EV markers, lengthy protocols, and the absence of standardized procedures for biological sample collection, EV isolation, and characterization. Nevertheless, with rapid advancements in the EV field, supported by improved isolation methods and sensitive assays for cargo characterization, brain cell-derived EVs continue to offer unparallel opportunities with significant translational implications for various neurological disorders. SIGNIFICANCE STATEMENT: Extracellular vesicles present a less invasive liquid biopsy approach in the diagnosis and prognosis of various neurological disorders. Characterizing these vesicles in biofluids holds the potential to yield valuable molecular information, thereby significantly impacting the development of novel biomarkers for various neurological disorders. This paper has reviewed the methodology employed to isolate extracellular vesicles derived from various brain cells in biofluids, their utility in enhancing the molecular understanding of neurodegeneration, and the potential challenges in this research field.
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Affiliation(s)
- Ashish Kumar
- Departments of Cancer Biology (A.K., G.D.), Physiology and Pharmacology (M.A.N.), Radiology (M.A.N.), and Center for Addiction Research (M.A.N., G.D.), Wake Forest University School of Medicine, Winston-Salem, North Carolina; Atrium Health Wake Forest Baptist Comprehensive Cancer Center, Winston-Salem, North Carolina (G.D.); and Sticht Center for Healthy Aging and Alzheimer's Prevention, Wake Forest School of Medicine, Winston-Salem, North Carolina (G.D.)
| | - Michael A Nader
- Departments of Cancer Biology (A.K., G.D.), Physiology and Pharmacology (M.A.N.), Radiology (M.A.N.), and Center for Addiction Research (M.A.N., G.D.), Wake Forest University School of Medicine, Winston-Salem, North Carolina; Atrium Health Wake Forest Baptist Comprehensive Cancer Center, Winston-Salem, North Carolina (G.D.); and Sticht Center for Healthy Aging and Alzheimer's Prevention, Wake Forest School of Medicine, Winston-Salem, North Carolina (G.D.)
| | - Gagan Deep
- Departments of Cancer Biology (A.K., G.D.), Physiology and Pharmacology (M.A.N.), Radiology (M.A.N.), and Center for Addiction Research (M.A.N., G.D.), Wake Forest University School of Medicine, Winston-Salem, North Carolina; Atrium Health Wake Forest Baptist Comprehensive Cancer Center, Winston-Salem, North Carolina (G.D.); and Sticht Center for Healthy Aging and Alzheimer's Prevention, Wake Forest School of Medicine, Winston-Salem, North Carolina (G.D.)
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Alvarez MM, Salazar FE, Rodriguez T, D’Egidio F, Borlongan CV, Lee JY. Endogenous Extracellular Vesicles Participate in Brain Remodeling after Ischemic Stroke. Int J Mol Sci 2023; 24:16857. [PMID: 38069179 PMCID: PMC10706116 DOI: 10.3390/ijms242316857] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2023] [Revised: 11/21/2023] [Accepted: 11/26/2023] [Indexed: 12/18/2023] Open
Abstract
Brain remodeling after an ischemic stroke represents a promising avenue for exploring the cellular mechanisms of endogenous brain repair. A deeper understanding of these mechanisms is crucial for optimizing the safety and efficacy of neuroprotective treatments for stroke patients. Here, we interrogated the role of extracellular vesicles, particularly exosomes, as potential mediators of endogenous repair within the neurovascular unit (NVU). We hypothesized that these extracellular vesicles may play a role in achieving transient stroke neuroprotection. Using the established ischemic stroke model of middle cerebral artery occlusion in adult rats, we detected a surged in the extracellular vesicle marker CD63 in the peri-infarct area that either juxtaposed or co-localized with GFAP-positive glial cells, MAP2-labeled young neurons, and VEGF-marked angiogenic cells. This novel observation that CD63 exosomes spatially and temporally approximated glial activation, neurogenesis, and angiogenesis suggests that extracellular vesicles, especially exosomes, contribute to the endogenous repair of the NVU, warranting exploration of extracellular vesicle-based stroke therapeutics.
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Affiliation(s)
| | | | | | | | - Cesar V. Borlongan
- Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, Morsani College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd., Tampa, FL 33612, USA; (M.M.A.); (F.E.S.); (T.R.); (F.D.); (J.-Y.L.)
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Zhang J, Wu J, Wang G, He L, Zheng Z, Wu M, Zhang Y. Extracellular Vesicles: Techniques and Biomedical Applications Related to Single Vesicle Analysis. ACS NANO 2023; 17:17668-17698. [PMID: 37695614 DOI: 10.1021/acsnano.3c03172] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/12/2023]
Abstract
Extracellular vesicles (EVs) are extensively dispersed lipid bilayer membrane vesicles involved in the delivery and transportation of molecular payloads to certain cell types to facilitate intercellular interactions. Their significant roles in physiological and pathological processes make EVs outstanding biomarkers for disease diagnosis and treatment monitoring as well as ideal candidates for drug delivery. Nevertheless, differences in the biogenesis processes among EV subpopulations have led to a diversity of biophysical characteristics and molecular cargos. Additionally, the prevalent heterogeneity of EVs has been found to substantially hamper the sensitivity and accuracy of disease diagnosis and therapeutic monitoring, thus impeding the advancement of clinical applications. In recent years, the evolution of single EV (SEV) analysis has enabled an in-depth comprehension of the physical properties, molecular composition, and biological roles of EVs at the individual vesicle level. This review examines the sample acquisition tactics prior to SEV analysis, i.e., EV isolation techniques, and outlines the current state-of-the-art label-free and label-based technologies for SEV identification. Furthermore, the challenges and prospects of biomedical applications based on SEV analysis are systematically discussed.
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Affiliation(s)
- Jie Zhang
- Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, P. R. China
| | - Jiacheng Wu
- Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, P. R. China
| | - Guanzhao Wang
- Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, P. R. China
| | - Luxuan He
- Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, P. R. China
| | - Ziwei Zheng
- Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, P. R. China
| | - Minhao Wu
- Department of Immunology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, P. R. China
| | - Yuanqing Zhang
- Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, P. R. China
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