miRNA analysis of hMSC-derived exosomes compared across isolation techniques

Exosomes are a class of nano-sized extracellular vesicles, typically 40-150 nm in diameter, that are released from endocytic multivesicular bodies. Exosomal contents are unique to the cell from which they are secreted, and the resulting combination of functional proteins, enzymes, and genes suggests exosomes with a variety of functions. In recent years, exosomes have been found to play fundamental roles in cellular communication and are secreted by nearly all cell types, including tumor and stem cells. Recently, exosomes have been majorly investigated for potential diagnostic use. For example, tumor cells secrete a higher number of exosomes than normal tissue cells, such that exosome count alone could be used as a diagnostic tool for tumorigenesis. Exosomes are also attractive in regards to cell-free therapy, due to their endogenous cell-specific targeted delivery of genes, protein, and small molecules. In particular, this approach could majorly improve therapies related to mesenchymal stem cells (MSCs). Recent research indicates that the MSC secretome (i.e. the full panel of biomolecules and particles released by the cell) can achieve the same therapeutic results as MSCs themselves. Thus MSC exosomes and other extracellular vesicles are poised to highly impact stromal therapy methods and must be further understood. Despite the explosion of substantial discoveries related to exosome structure and function over the past few years, the basics of isolation, handling, characterization, and even the very definition of exosomes have yet to be standardized. Despite the prevalence of three major methods of exosome isolation: (i) differential ultracentrifugation which relies on a series of centrifugation steps, (ii) density gradient ultracentrifugation, isolating exosomes based on their density, and (iii) commercial precipitation reagents whose composition and mechanism is proprietary, very few studies perform in-depth side by side comparisons of the resulting exosome biomolecular profile. The project described here focuses on the optimization of exosome isolation and characterization, followed by a novel large-scale case study of hMSC-derived exosomes and their genomic profile by next-generation microRNA microarray analysis. In particular we discovered that miRNA expression levels actually vary by exosome collection methodology. The alteration in miRNA expression levels indicate a subpopulation of exosomes based on different exosome collection methodology. This discovery is of central importance for research employing hMSC-exosomes for therapy and extracellular vesicles in general.