A rigorous method to enrich for exosomes from brain tissue

Abstract

Extracellular vesicles, including exosomes, are released by all cells, including those of the nervous system. Capable of delivering lipid, protein and nucleic acids to both nearby and distal cells, exosomes have been hypothesized to play a role in progression of many diseases of the nervous system. To date, most analyses on the role of these vesicles in the healthy and diseased state have relied on studying vesicles from in vitro sources, such as conditioned cell culture media, or body fluids. Here we have taken a critical approach to the enrichment and characterization of exosomes from human frontal cortex. This method maintains the integrity of the vesicles and their cargo, and comprehensive proteomic and genomic characterization confirms the legitimacy of the resulting extracellular vesicles as endosome-derived exosomes. This method will enable neuroscientists to acquire more detailed information about exosomes in the brain and explore the role(s) this form of intercellular communication and unique source of lipid, protein and RNA has in healthy brain function and pathogenic conditions. Furthermore, this method may have important utility in the isolation of exosomes from other tissues.

Exosomes as secondary inductive signals involved in kidney organogenesis

“…We used DAVID to identify statistically significant overrepresented gene ontology (GO) terms (Figure 2(b)) in the exosomal cargo proteins. They were enriched for cellular components typically associated with exosomal samples: extracellular vesicle, extracellular region, membrane and vesicle [30]. Furthermore, cellular components associated with protein binding, focal adhesion, extracellular matrix, cadherin binding involved in cell–cell adhesion, cell–cell adherence junction and structural molecule activity were enriched, many of which are involved in regulating cell shape and maintaining tissue integrity.…”

“…All p -values less than 10 –5 after Bonferroni correction were considered to be significant. Settings were chosen as previously published [30]. Pathway overview visualization was performed using the Reactome Pathway Analysis Tool (www.reactome.org).…”

Proceedings of the 2017 ISEV symposium on “HIV, NeuroHIV, drug abuse, & EVs”

“…Dr. Pulliam’s group successfully isolated neuron-derived exosomes from plasma of HIV-infected subjects using precipitation and immunoadsorption with an antibody to a neuronal specific cell surface marker (Sun et al 2017). Additionally, Dr. Hill’s group reported a novel method for the successful enrichment of exosomes from human brain tissue (Vella et al 2017). The consensus of the panel was that it is necessary to develop a method for the accurate selection of subpopulations of EVs since the cargo in specific subpopulations of EVs could reflect disease status and could also be used as a diagnostic indicator to determine therapeutic efficacy.…”

Extracellular Vesicles in Oncology: Progress and Pitfalls in the Methods of Isolation and Analysis

“…The enrichment of EVs markers (such as CD63 or CD81) does not exclude the presence of contaminants and for this reason, it should be used only to analyze purified preparations of EVs. Moreover, a specific EVs housekeeping protein does not exist and total protein stainings, such as Ponceau S, are adopted for visualization of total protein and ensure equal loading …”