title: Therapeutic MSC exosomes are derived from lipid raft microdomains in the plasma membrane.
abstract: BACKGROUND
  Mesenchymal stem cell (MSC) was previously shown to secrete lipid vesicles that when purified by high performance liquid chromatography as a population of homogenously sized particles with a hydrodynamic radius of 55-65 nm reduce infarct size in a mouse model of myocardial ischemia/reperfusion injury. As these vesicles exhibit many biophysical and biochemical properties of exosomes, they were identified as exosomes. Here we investigated if these lipid vesicles were indeed exosomes that have an endosomal biogenesis.
  
  
  METHODS
  In most cells, endocytosis is thought to occur at specialized microdomains known as lipid rafts. To demonstrate an endosomal origin for MSC exosomes, MSCs were pulsed with ligands e.g. transferrin (Tfs) and Cholera Toxin B (CTB) that bind receptors in lipid rafts. The endocytosed ligands were then chased to determine if they were incorporated into the exosomes.
  
  
  RESULTS
  A fraction of exogenous Tfs was found to recycle into MSC exosomes. When MSCs were pulsed with labelled Tfs in the presence of chlorpromazine, an inhibitor of clathrin-mediated endocytosis, Tf incorporation in CD81-immunoprecipitate was reduced during the chase. CTB which binds GM1 gangliosides that are enriched in lipid rafts extracted exosome-associated proteins, CD81, CD9, Alix and Tsg101 from MSC-conditioned medium. Exogenous CTBs were pulse-chased into secreted vesicles. Extraction of Tf- or CTB-binding vesicles in an exosome preparation mutually depleted each other. Inhibition of sphingomyelinases reduced CTB-binding vesicles.
  
  
  CONCLUSIONS
  Together, our data demonstrated that MSC exosomes are derived from endocytosed lipid rafts and that their protein cargo includes exosome-associated proteins CD81, CD9, Alix and Tsg101.
authors.affiliation: Institute of Medical Biology, ASTAR, Singapore.
authors.forename: Soon Sim
authors.initials: SS
authors.lastname: Tan
authors.affiliation: Institute of Medical Biology, ASTAR, Singapore.
authors.forename: Yijun
authors.initials: Y
authors.lastname: Yin
authors.affiliation: Institute of Medical Biology, ASTAR, Singapore.
authors.forename: Tricia
authors.initials: T
authors.lastname: Lee
authors.affiliation: Institute of Medical Biology, ASTAR, Singapore.
authors.forename: Ruenn Chai
authors.initials: RC
authors.lastname: Lai
authors.affiliation: Institute of Medical Biology, ASTAR, Singapore.
authors.forename: Ronne Wee Yeh
authors.initials: RW
authors.lastname: Yeo
authors.affiliation: Institute of Medical Biology, ASTAR, Singapore.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Bioprocessing Technology Institute, ASTAR, Singapore.
authors.forename: Andre
authors.initials: A
authors.lastname: Choo
authors.affiliation: Institute of Medical Biology, ASTAR, Singapore ; Department of Surgery, YLL School of Medicine, National University of Singapore, Singapore.
authors.forename: Sai Kiang
authors.initials: SK
authors.lastname: Lim
keywords: cholera toxin B; clathrin mediated endocytosis; exosomes; lipid rafts; mesenchymal stem cells; transferrin