TY - JOUR
T1 - Influence of species and processing parameters on recovery and content of brain tissue-derived extracellular vesicles
AU - Huang, Yiyao
AU - Cheng, Lesley
AU - Turchinovich, Andrey
AU - Mahairaki, Vasiliki
AU - Troncoso, Juan C.
AU - Pletniková, Olga
AU - Haughey, Norman J.
AU - Vella, Laura J.
AU - Hill, Andrew F.
AU - Zheng, Lei
AU - Witwer, Kenneth W.
N1 - Funding Information:
This work was supported by grants from the US National Institutes of Health (DA040385, DA047807, AI144997, and MH118164, to KWW) and AG057430 (to VM and KWW); by the Michael J. Fox Foundation (to KWW); by UG3CA241694, supported by the NIH Common Fund, through the Office of Strategic Coordination/Office of the NIH Director; and by the National Center for Research Resources and the Office of Research Infrastructure Programs (ORIP) and the National Institutes of Health, grant number P40 OD013117. This work was also supported by the National Health and Medical Research Council of Australia, GNT1132604 (to AFH). We thank Suzanne Queen, Brandon Bullock, and Emily Mallick from the Johns Hopkins University School of Medicine for generously providing and processing the macaque brain tissues. We thank Connie Talbot, Bob Cole and Tatiana Boronina from the Johns Hopkins University School of Medicine for contributing to proteomics data analysis. We gratefully acknowledge the La Trobe University Comprehensive Proteomics Platform.
Funding Information:
This work was supported by the Michael J. Fox Foundation for Parkinson?s Research; NIH Office of the Director [CA241694]; National Institute of Allergy and Infectious Diseases [AI144997]; National Institute of Mental Health [MH118164]; National Institute on Aging [AG057430]; National Institute on Drug Abuse [DA040385]; National Institute on Drug Abuse [DA047807]; National Health and Medical Research Council of Australia [GNT1132604]. This work was supported by grants from the US National Institutes of Health (DA040385, DA047807, AI144997, and MH118164, to KWW) and AG057430 (to VM and KWW); by the Michael J. Fox Foundation (to KWW); by UG3CA241694, supported by the NIH Common Fund, through the Office of Strategic Coordination/Office of the NIH Director; and by the National Center for Research Resources and the Office of Research Infrastructure Programs (ORIP) and the National Institutes of Health, grant number P40 OD013117. This work was also supported by the National Health and Medical Research Council of Australia, GNT1132604 (to AFH). We thank Suzanne Queen, Brandon Bullock, and Emily Mallick from the Johns Hopkins University School of Medicine for generously providing and processing the macaque brain tissues. We thank Connie Talbot, Bob Cole and Tatiana Boronina from the Johns Hopkins University School of Medicine for contributing to proteomics data analysis. We gratefully acknowledge the La Trobe University Comprehensive Proteomics Platform.
Publisher Copyright:
© 2020, © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.
PY - 2020/1/1
Y1 - 2020/1/1
N2 - Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque, and mouse. Effects of tissue perfusion and a model of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with those of macaque and, especially, mouse, suggesting opportunities for optimization. Where possible, perfusion should be performed in animal models. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Finally, different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease.
AB - Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque, and mouse. Effects of tissue perfusion and a model of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with those of macaque and, especially, mouse, suggesting opportunities for optimization. Where possible, perfusion should be performed in animal models. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Finally, different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease.
KW - Extracellular vesicles
KW - brain
KW - central nervous system
KW - exosomes
KW - neurodegenerative disease
KW - post-mortem interval
KW - proteomics
KW - small RNA sequencing
KW - tissue preparation
UR - http://www.scopus.com/inward/record.url?scp=85087561179&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85087561179&partnerID=8YFLogxK
U2 - 10.1080/20013078.2020.1785746
DO - 10.1080/20013078.2020.1785746
M3 - Article
C2 - 32944174
AN - SCOPUS:85087561179
SN - 2001-3078
VL - 9
JO - Journal of Extracellular Vesicles
JF - Journal of Extracellular Vesicles
IS - 1
M1 - 1785746
ER -