title: Human bone marrow- and adipose-mesenchymal stem cells secrete exosomes enriched in distinctive miRNA and tRNA species.
abstract: BACKGROUND
  Administration of mesenchymal stem cells (MSCs) represents a promising treatment option for patients suffering from immunological and degenerative disorders. Accumulating evidence indicates that the healing effects of MSCs are mainly related to unique paracrine properties, opening opportunities for secretome-based therapies. Apart from soluble factors, MSCs release functional small RNAs via extracellular vesicles (EVs) that seem to convey essential features of MSCs. Here we set out to characterize the full small RNAome of MSC-produced exosomes.
  
  
  METHODS
  We set up a protocol for isolating exosomes released by early passage adipose- (ASC) and bone marrow-MSCs (BMSC) and characterized them via electron microscopy, protein analysis and small RNA-sequencing. We developed a bioinformatics pipeline to define the exosome-enclosed RNA species and performed the first complete small RNA characterization of BMSCs and ASCs and their corresponding exosomes in biological replicates.
  
  
  RESULTS
  Our analysis revealed that primary ASCs and BMSCs have highly similar small RNA expression profiles dominated by miRNAs and snoRNAs (together 64-71 %), of which 150-200 miRNAs are present at physiological levels. In contrast, the miRNA pool in MSC exosomes is only 2-5 % of the total small RNAome and is dominated by a minor subset of miRNAs. Nevertheless, the miRNAs in exosomes do not merely reflect the cellular content and a defined set of miRNAs are overrepresented in exosomes compared to the cell of origin. Moreover, multiple highly expressed miRNAs are precluded from exosomal sorting, consistent with the notion that these miRNAs are involved in functional repression of RNA targets. While ASC and BMSC exosomes are similar in RNA class distribution and composition, we observed striking differences in the sorting of evolutionary conserved tRNA species that seems associated with the differentiation status of MSCs, as defined by Sox2, POU5F1A/B and Nanog expression.
  
  
  CONCLUSIONS
  We demonstrate that primary MSCs release small RNAs via exosomes, which are increasingly implicated in intercellular communications. tRNAs species, and in particular tRNA halves, are preferentially released and their specific sorting into exosomes is related to MSC tissue origin and stemness. These findings may help to understand how MSCs impact neighboring or distant cells with possible consequences for their therapeutic usage.
authors.affiliation: Laboratory for Orthopedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna, 40136, Italy. s.baglio@vumc.nl.|Department of Pathology, Cancer Center Amsterdam, VU University Medical Center, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands. s.baglio@vumc.nl.
authors.forename: Serena Rubina
authors.initials: SR
authors.lastname: Baglio
authors.affiliation: Department of Biological Stress Response, Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands. k.rooijers@nki.nl.
authors.forename: Koos
authors.initials: K
authors.lastname: Rooijers
authors.affiliation: Department of Pathology, Cancer Center Amsterdam, VU University Medical Center, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands. d.lalic@vumc.nl.
authors.forename: Danijela
authors.initials: D
authors.lastname: Koppers-Lalic
authors.affiliation: Department of Pathology, Cancer Center Amsterdam, VU University Medical Center, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands. frederikverweij@gmail.com.
authors.forename: Frederik J
authors.initials: FJ
authors.lastname: Verweij
authors.affiliation: Department of Pathology, Cancer Center Amsterdam, VU University Medical Center, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands. mariaperezlanzon@gmail.com.
authors.forename: M
authors.initials: M
authors.lastname: Pérez Lanzón
authors.affiliation: CNR-National Research Council of Italy, IGM, Bologna, 40136, Italy. nzini@area.bo.cnr.it.|SC Laboratory of Musculoskeletal Cell Biology, Istituto Ortopedico Rizzoli, Bologna, 40136, Italy. nzini@area.bo.cnr.it.
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Zini
authors.affiliation: Department of Pathology, Cancer Center Amsterdam, VU University Medical Center, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands. bennonaaijkens@gmail.com.
authors.forename: Benno
authors.initials: B
authors.lastname: Naaijkens
authors.affiliation: Laboratory for Orthopedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna, 40136, Italy. francesca.perut@ior.it.
authors.forename: Francesca
authors.initials: F
authors.lastname: Perut
authors.affiliation: Department of Pathology, Cancer Center Amsterdam, VU University Medical Center, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands. jwm.niessen@vumc.nl.
authors.forename: Hans W M
authors.initials: HW
authors.lastname: Niessen
authors.affiliation: Laboratory for Orthopedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna, 40136, Italy. nicola.baldini@ior.it.
authors.forename: Nicola
authors.initials: N
authors.lastname: Baldini
authors.affiliation: Department of Pathology, Cancer Center Amsterdam, VU University Medical Center, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands. d.pegtel@vumc.nl.
authors.forename: D Michiel
authors.initials: DM
authors.lastname: Pegtel
mesh_terms: D020413:3' Untranslated Regions; D000273:Adipose Tissue / Q000166:cytology*; D001483:Base Sequence; D001854:Bone Marrow Cells / Q000166:cytology*; D002454:Cell Differentiation; D002478:Cells, Cultured; D055354:Exosomes / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D035683:MicroRNAs / Q000378:metabolism*; D008854:Microscopy, Electron; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification; D012343:RNA, Transfer / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D059467:Transcriptome
chemical_list: D020413:3' Untranslated Regions; D035683:MicroRNAs; D012313:RNA; D012343:RNA, Transfer