title: MSC secretes at least 3 EV types each with a unique permutation of membrane lipid, protein and RNA.
abstract: Mesenchymal stem cell (MSC), a widely used adult stem cell candidate for regenerative medicine, has been shown to exert some of its therapeutic effects through the secretion of extracellular vesicles (EVs). These homogenously sized EVs of 100-150 ηm exhibited many exosome-like biophysical and biochemical properties and carry both proteins and RNAs. Recently, exosome-associated proteins in this MSC EV preparation were found to segregate primarily to those EVs that bind cholera toxin B chain (CTB), a GM1 ganglioside-specific ligand, and pulse-chase experiments demonstrated that these EVs have endosomal origin and carried many of the exosome-associated markers. Here, we report that only a fraction of the MSC EV proteome was found in CTB-bound EVs. Using Annexin V (AV) and Shiga toxin B subunit (ST) with affinities for phosphatidylserine and globotriaosylceramide, respectively, AV- and a ST-binding EV were identified. CTB-, AV- and ST-binding EVs all carried actin. However, the AV-binding EVs carried low or undetectable levels of the exosome-associated proteins. Only the ST-binding EVs carried RNA and EDA-containing fibronectin. Proteins in AV-binding EVs were also different from those released by apoptotic MSCs. CTB- and AV-binding activities were localized to the plasma membrane and cytoplasm of MSCs, while ST-binding activity was localized to the nucleus. Together, this study demonstrates that cells secrete many types of EVs. Specifically, MSCs secrete at least 3 types. They can be differentially isolated based on their affinities for membrane lipid-binding ligands. As the subcellular sites of the binding activities of these ligands and cargo load are different for each EV type, they are likely to have a different biogenesis pathway and possibly different functions.
authors.affiliation: A*STAR Institute of Medical Biology, Singapore.
authors.forename: Ruenn Chai
authors.initials: RC
authors.lastname: Lai
authors.affiliation: A*STAR Institute of Medical Biology, Singapore.
authors.forename: Soon Sim
authors.initials: SS
authors.lastname: Tan
authors.affiliation: A*STAR Institute of Medical Biology, Singapore.
authors.forename: Ronne Wee Yeh
authors.initials: RW
authors.lastname: Yeo
authors.affiliation: A*STAR Bioprocessing Technology Institute, Singapore.|Department of Biomedical Engineering, Faculty of Engineering, NUS, Singapore.
authors.forename: Andre Boon Hwa
authors.initials: AB
authors.lastname: Choo
authors.affiliation: BioSensor Technologies, AIT-Austrian Institute of Technology GmbH, Vienna, Austria.
authors.forename: Agnes T
authors.initials: AT
authors.lastname: Reiner
authors.affiliation: A*STAR Genome Institute of Singapore, Singapore.
authors.forename: Yan
authors.initials: Y
authors.lastname: Su
authors.affiliation: A*STAR Genome Institute of Singapore, Singapore.
authors.forename: Yang
authors.initials: Y
authors.lastname: Shen
authors.affiliation: A*STAR Genome Institute of Singapore, Singapore.
authors.forename: Zhiyan
authors.initials: Z
authors.lastname: Fu
authors.affiliation: A*STAR Genome Institute of Singapore, Singapore.
authors.forename: Lezhava
authors.initials: L
authors.lastname: Alexander
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Siu Kwan
authors.initials: SK
authors.lastname: Sze
authors.affiliation: A*STAR Institute of Medical Biology, Singapore.|Department of Surgery, YLL School of Medicine, NUS, Singapore; saikiang.lim@imb.a-star.edu.sg.
authors.forename: Sai Kiang
authors.initials: SK
authors.lastname: Lim
keywords: annexin V; cholera toxin B; exosome; mesenchymal stem cell; shiga toxin