Abstract

Isolation and Characterisation of Extracellular Vesicles: Current Perspectives

Richard Simpson, Professor, La Trobe University

EV annotation is a vexed question given that EVs have been variously classified based on cellular origin, biological function and/or mechanism of biogenesis. These concerns are widely discussed in the international EV community and stress the need for standardized nomenclature. In broad terms two predominant EV subtypes exist: 100-1000 nm diameter microvesicles (shed microvesicles, sMVs; membrane blebs) and 30-100 nm diameter exosomes. sMVs are generated by directly outward budding from the plasma membrane while exosome biogenesis involves the early/late endosomal pathway and the inward budding of multivesicular body (MVB) luminal membranes to form intraluminal vesicles (ILVs); MVBs then traffic to and fuse with the plasma membrane whereupon they release their ILV contents into extracellular space (exosomes). Given that most EV functional studies are undertaken using ill-defined (heterogeneous) EV preparations it is very difficult to ascribe function to a specific EV subtype – this may have possible ramifications in EV therapeutics design, especially in the event of possible EV-subtype-associated clinical side effects. These issues have engendered great interest in establishing how many naturally-occurring EV subtypes there are, and improving EV isolation methodologies. In this lecture I will provide an overview of EV subtype classification, including methods used for their isolation and characterization. Our recent findings show that human colon cancer cell line-derived EVs have specific protein and RNA (mRNA, splice-variant mRNA, miRNA, and lncRNA) profiles that allow their discrimination. These data, along with our recent demonstration that the protein content of exosomes is re-programmed during epithelial-mesenchymal transition (EMT) will be discussed.