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A comprehensive proteomics profiling identifies NRP1 as a novel identity marker of human bone marrow mesenchymal stromal cell-derived small extracellular vesicles
BACKGROUND: Clinical applications have shown extracellular vesicles (EVs) to be a major paracrine effector in therapeutic responses produced by human mesenchymal stromal/stem cells (hMSCs). As the regenerative capacity of EVs is mainly ascribed to the transfer of proteins and RNA composing its cargo...
Autores principales: | , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6921509/ https://www.ncbi.nlm.nih.gov/pubmed/31852509 http://dx.doi.org/10.1186/s13287-019-1516-2 |
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author | Munshi, Afnan Mehic, Jelica Creskey, Marybeth Gobin, Jonathan Gao, Jun Rigg, Emma Muradia, Gauri Luebbert, Christian C. Westwood, Carole Stalker, Andrew Allan, David S. Johnston, Michael J. W. Cyr, Terry Rosu-Myles, Michael Lavoie, Jessie R. |
author_facet | Munshi, Afnan Mehic, Jelica Creskey, Marybeth Gobin, Jonathan Gao, Jun Rigg, Emma Muradia, Gauri Luebbert, Christian C. Westwood, Carole Stalker, Andrew Allan, David S. Johnston, Michael J. W. Cyr, Terry Rosu-Myles, Michael Lavoie, Jessie R. |
author_sort | Munshi, Afnan |
collection | PubMed |
description | BACKGROUND: Clinical applications have shown extracellular vesicles (EVs) to be a major paracrine effector in therapeutic responses produced by human mesenchymal stromal/stem cells (hMSCs). As the regenerative capacity of EVs is mainly ascribed to the transfer of proteins and RNA composing its cargo, and to the activity attributed by the protein surface markers, we sought to profile the protein composition of small EVs released from hMSCs to identify hMSC-EV biomarkers with potential clinical relevance. METHODS: Small EVs were produced and qualified from five human bone marrow MSC donors at low passage following a 48-h culture in exosome-depleted medium further processed by steps of centrifugation, filtration, and precipitation. Quantitative proteomic analysis comparing the protein profile of the EVs released from hMSCs and their parental cell was conducted using tandem mass tag labeling combined to mass spectrometry (LC-MS/MS) to identify enriched EV protein markers. RESULTS: Nanoparticle tracking analysis showed no differences in the EV concentration and size among the five hMSC donors (1.83 × 10(10) ± 3.23 × 10(9)/mL), with the mode particle size measuring at 109.3 ± 5.7 nm. Transmission electron microscopy confirmed the presence of nanovesicles with bilayer membranes. Flow cytometric analysis identified commonly found exosomal (CD63/CD81) and hMSC (CD105/CD44/CD146) markers from released EVs in addition to surface mediators of migration (CD29 and MCSP). Quantitative proteomic identified 270 proteins significantly enriched by at least twofold in EVs released from hMSCs as compared to parental hMSCs, where neuropilin 1 (NRP1) was identified among 21 membrane-bound proteins regulating the migration and invasion of cells, as well as chemotaxis and vasculogenesis. Validation by western blot of multiple batches of EVs confirmed consistent enrichment of NRP1 in the nanovesicles released from all five hMSC donors. CONCLUSION: The identification and verification of NRP1 as a novel enriched surface marker from multiple batches of EVs derived from multiple hMSC donors may serve as a biomarker for the assessment and measurement of EVs for therapeutic uses. |
format | Online Article Text |
id | pubmed-6921509 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-69215092019-12-30 A comprehensive proteomics profiling identifies NRP1 as a novel identity marker of human bone marrow mesenchymal stromal cell-derived small extracellular vesicles Munshi, Afnan Mehic, Jelica Creskey, Marybeth Gobin, Jonathan Gao, Jun Rigg, Emma Muradia, Gauri Luebbert, Christian C. Westwood, Carole Stalker, Andrew Allan, David S. Johnston, Michael J. W. Cyr, Terry Rosu-Myles, Michael Lavoie, Jessie R. Stem Cell Res Ther Research BACKGROUND: Clinical applications have shown extracellular vesicles (EVs) to be a major paracrine effector in therapeutic responses produced by human mesenchymal stromal/stem cells (hMSCs). As the regenerative capacity of EVs is mainly ascribed to the transfer of proteins and RNA composing its cargo, and to the activity attributed by the protein surface markers, we sought to profile the protein composition of small EVs released from hMSCs to identify hMSC-EV biomarkers with potential clinical relevance. METHODS: Small EVs were produced and qualified from five human bone marrow MSC donors at low passage following a 48-h culture in exosome-depleted medium further processed by steps of centrifugation, filtration, and precipitation. Quantitative proteomic analysis comparing the protein profile of the EVs released from hMSCs and their parental cell was conducted using tandem mass tag labeling combined to mass spectrometry (LC-MS/MS) to identify enriched EV protein markers. RESULTS: Nanoparticle tracking analysis showed no differences in the EV concentration and size among the five hMSC donors (1.83 × 10(10) ± 3.23 × 10(9)/mL), with the mode particle size measuring at 109.3 ± 5.7 nm. Transmission electron microscopy confirmed the presence of nanovesicles with bilayer membranes. Flow cytometric analysis identified commonly found exosomal (CD63/CD81) and hMSC (CD105/CD44/CD146) markers from released EVs in addition to surface mediators of migration (CD29 and MCSP). Quantitative proteomic identified 270 proteins significantly enriched by at least twofold in EVs released from hMSCs as compared to parental hMSCs, where neuropilin 1 (NRP1) was identified among 21 membrane-bound proteins regulating the migration and invasion of cells, as well as chemotaxis and vasculogenesis. Validation by western blot of multiple batches of EVs confirmed consistent enrichment of NRP1 in the nanovesicles released from all five hMSC donors. CONCLUSION: The identification and verification of NRP1 as a novel enriched surface marker from multiple batches of EVs derived from multiple hMSC donors may serve as a biomarker for the assessment and measurement of EVs for therapeutic uses. BioMed Central 2019-12-18 /pmc/articles/PMC6921509/ /pubmed/31852509 http://dx.doi.org/10.1186/s13287-019-1516-2 Text en © The Author(s). 2019 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Munshi, Afnan Mehic, Jelica Creskey, Marybeth Gobin, Jonathan Gao, Jun Rigg, Emma Muradia, Gauri Luebbert, Christian C. Westwood, Carole Stalker, Andrew Allan, David S. Johnston, Michael J. W. Cyr, Terry Rosu-Myles, Michael Lavoie, Jessie R. A comprehensive proteomics profiling identifies NRP1 as a novel identity marker of human bone marrow mesenchymal stromal cell-derived small extracellular vesicles |
title | A comprehensive proteomics profiling identifies NRP1 as a novel identity marker of human bone marrow mesenchymal stromal cell-derived small extracellular vesicles |
title_full | A comprehensive proteomics profiling identifies NRP1 as a novel identity marker of human bone marrow mesenchymal stromal cell-derived small extracellular vesicles |
title_fullStr | A comprehensive proteomics profiling identifies NRP1 as a novel identity marker of human bone marrow mesenchymal stromal cell-derived small extracellular vesicles |
title_full_unstemmed | A comprehensive proteomics profiling identifies NRP1 as a novel identity marker of human bone marrow mesenchymal stromal cell-derived small extracellular vesicles |
title_short | A comprehensive proteomics profiling identifies NRP1 as a novel identity marker of human bone marrow mesenchymal stromal cell-derived small extracellular vesicles |
title_sort | comprehensive proteomics profiling identifies nrp1 as a novel identity marker of human bone marrow mesenchymal stromal cell-derived small extracellular vesicles |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6921509/ https://www.ncbi.nlm.nih.gov/pubmed/31852509 http://dx.doi.org/10.1186/s13287-019-1516-2 |
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