Characterizing Extracellular Vesicles and Their Diverse RNA Contents

Abstract

Cells release nanometer-scale, lipid bilayer-enclosed biomolecular packages (extracellular vesicles; EVs) into their surrounding environment. EVs are hypothesized to be intercellular communication agents that regulate physiological states by transporting biomolecules between near and distant cells. The research community has consistently advocated for the importance of RNA contents in EVs by demonstrating that: (1) EV-related RNA contents can be detected in a liquid biopsy, (2) disease states significantly alter EV-related RNA contents, and (3) sensitive and specific liquid biopsies can be implemented in precision medicine settings by measuring EV-derived RNA contents. Furthermore, EVs have medical potential beyond diagnostics. Both natural and engineered EVs are being investigated for therapeutic applications such as regenerative medicine and as drug delivery agents. This review focuses specifically on EV characterization, analysis of their RNA content, and their functional implications. The NIH extracellular RNA communication (ERC) program has catapulted human EV research from an RNA profiling standpoint by standardizing the pipeline for working with EV transcriptomics data, and creating a centralized database for the scientific community. There are currently thousands of RNA-sequencing profiles hosted on the Extracellular RNA Atlas alone (Murillo et al., 2019 ), encompassing a variety of human biofluid types and health conditions. While a number of significant discoveries have been made through these studies individually, integrative analyses of these data have thus far been limited. A primary focus of the ERC program over the next five years is to bring higher resolution tools to the EV research community so that investigators can isolate and analyze EV sub-populations, and ultimately single EVs sourced from discrete cell types, tissues, and complex biofluids. Higher resolution techniques will be essential for evaluating the roles of circulating EVs at a level which impacts clinical decision making. We expect that advances in microfluidic technologies will drive near-term innovation and discoveries about the diverse RNA contents of EVs. Long-term translation of EV-based RNA profiling into a mainstay medical diagnostic tool will depend upon identifying robust patterns of circulating genetic material that correlate with a change in health status.

Longitudinal saliva omics responses to immune perturbation: a case study

“…We have also profiled the RNA of salivary EVs. There has been expansive interest in the diverse RNA content of EVs 63 , including by the Extracellular RNA Communication (ERC) program 64 , for applications in liquid biopsies, as markers in disease states and for cell-free precision medicine diagnostics. EVs are being evaluated as mediators of intercellular communication through molecular transport, offer stable containment of RNA, and can easily be collected for potential diagnostics 63 .…”

“…There has been expansive interest in the diverse RNA content of EVs 63 , including by the Extracellular RNA Communication (ERC) program 64 , for applications in liquid biopsies, as markers in disease states and for cell-free precision medicine diagnostics. EVs are being evaluated as mediators of intercellular communication through molecular transport, offer stable containment of RNA, and can easily be collected for potential diagnostics 63 . EVs have been detected and may move across biofluids, with RNAs from bacteria, fungi, and other species having been reported in human plasma and saliva [65][66][67][68] .…”

Expression Pattern of Seminal Plasma Extracellular Vesicle Small RNAs in Boar Semen

“…Western blot analysis showed that the boar SP-EVs expressed the EVs markers CD63 and CD9 and were free of cell contamination ( Figure 1C ). However, recent study has indicated that ultracentrifugation alone may not be sufficient to isolate clean EVs; an additional purification process such as a density gradient ultracentrifugation made with iodixanol might be applied to remove the aggregates ( 36 ). Although we tried to isolate boar SP-EVs using membrane-based affinity binding and precipitation, methods that are commonly used to isolate EVs from bioliquids, the yield was too small for TEM analysis (data are not shown).…”

Extracellular microvesicles/exosomes: discovery, disbelief, acceptance, and the future?

“…Moreover, additional evidence has accumulated that ExMVs play a role in the spread of certain viruses and prions [ 2 , 27 , 28 ]. Currently under investigation is the possibility that ExMV molecular signature and content will be important tools in diagnostics as part of so-called liquid biopsies [ 20 , 29 ].…”