title: MSC surface markers (CD44, CD73, and CD90) can identify human MSC-derived extracellular vesicles by conventional flow cytometry.
abstract: BACKGROUND
  Human mesenchymal stromal cells (hMSC) are multipotent cells with both regenerative and immunomodulatory activities making them an attractive tool for cellular therapy. In the last few years it has been shown that the beneficial effects of hMSC may be due to paracrine effects and, at least in part, mediated by extracellular vesicles (EV). EV have emerged as important mediators of cell-to-cell communication. Flow cytometry (FCM) is a routine technology used in most clinical laboratories and could be used as a methodology for hMSC-EV characterization. Although several reports have characterized EV by FCM, a specific panel and protocol for hMSC-derived EV is lacking. The main objective of our study was the characterization of hMSC-EV using a standard flow cytometer.
  
  
  METHODS
  Human MSC from bone marrow of healthy donors, mesenchymal cell lines (HS-5 and hTERT) and a leukemic cell line (K562 cells) were used to obtain EV for FCM characterization. EV released from the different cell lines were isolated by ultracentrifugation and were characterized, using a multi-parametric analysis, in a conventional flow cytometer. EV characterization by transmission electron microscopy (TEM), western blot (WB) and Nano-particle tracking analysis (NTA) was also performed.
  
  
  RESULTS
  EV membranes are constituted by the combination of specific cell surface molecules depending on their cell of origin, together with specific proteins like tetraspanins (e.g. CD63). We have characterized by FCM the EV released from BM-hMSC, that were defined as particles less than 0.9 μm, positive for the hMSC markers (CD90, CD44 and CD73) and negative for CD34 and CD45 (hematopoietic markers). In addition, hMSC-derived EV were also positive for CD63 and CD81, the two characteristic markers of EV. To validate our characterization strategy, EV from mesenchymal cell lines (hTERT/HS-5) were also studied, using the leukemia cell line (K562) as a negative control. EV released from mesenchymal cell lines displayed the same immunophenotypic profile as the EV from primary BM-hMSC, while the EV derived from K562 cells did not show hMSC markers. We further validated the panel using EV from hMSC transduced with GFP. Finally, EV derived from the different sources (hMSC, hTERT/HS-5 and K562) were also characterized by WB, TEM and NTA, demonstrating the expression by WB of the exosomal markers CD63 and CD81, as well as CD73 in those from MSC origin. EV morphology and size/concentration was confirmed by TEM and NTA, respectively.
  
  
  CONCLUSIONS
  We described a strategy that allows the identification and characterization by flow cytometry of hMSC-derived EV that can be routinely used in most laboratories with a standard flow cytometry facility.
authors.affiliation: Servicio de Hematología, IBSAL-Hospital Universitario de Salamanca, Paseo de San Vicente 58-182, 37007, Salamanca, Spain. teresaclramos@usal.es.|Centro de Investigación del Cáncer, Universidad de Salamanca, Salamanca, Spain. teresaclramos@usal.es.|Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, León, Spain. teresaclramos@usal.es.|Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, Spain. teresaclramos@usal.es.
authors.forename: Teresa
authors.initials: T
authors.lastname: L Ramos
authors.affiliation: Servicio de Hematología, IBSAL-Hospital Universitario de Salamanca, Paseo de San Vicente 58-182, 37007, Salamanca, Spain. abarca@usal.es.|Centro de Investigación del Cáncer, Universidad de Salamanca, Salamanca, Spain. abarca@usal.es.|Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, León, Spain. abarca@usal.es.|Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, Spain. abarca@usal.es.
authors.forename: Luis Ignacio
authors.initials: LI
authors.lastname: Sánchez-Abarca
authors.affiliation: Servicio de Hematología, IBSAL-Hospital Universitario de Salamanca, Paseo de San Vicente 58-182, 37007, Salamanca, Spain. smuntion@usal.es.|Centro de Investigación del Cáncer, Universidad de Salamanca, Salamanca, Spain. smuntion@usal.es.|Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, León, Spain. smuntion@usal.es.|Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, Spain. smuntion@usal.es.
authors.forename: Sandra
authors.initials: S
authors.lastname: Muntión
authors.affiliation: Servicio de Hematología, IBSAL-Hospital Universitario de Salamanca, Paseo de San Vicente 58-182, 37007, Salamanca, Spain. silviapreciadoperez@gmail.com.|Centro de Investigación del Cáncer, Universidad de Salamanca, Salamanca, Spain. silviapreciadoperez@gmail.com.|Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, León, Spain. silviapreciadoperez@gmail.com.|Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, Spain. silviapreciadoperez@gmail.com.
authors.forename: Silvia
authors.initials: S
authors.lastname: Preciado
authors.affiliation: Servicio de Hematología, IBSAL-Hospital Universitario de Salamanca, Paseo de San Vicente 58-182, 37007, Salamanca, Spain. noepuig@gmail.com.|Centro de Investigación del Cáncer, Universidad de Salamanca, Salamanca, Spain. noepuig@gmail.com.|Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, León, Spain. noepuig@gmail.com.|Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, Spain. noepuig@gmail.com.
authors.forename: Noemí
authors.initials: N
authors.lastname: Puig
authors.affiliation: Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, Spain. guiller@usal.es.|Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca, Salamanca, Spain. guiller@usal.es.
authors.forename: Guillermo
authors.initials: G
authors.lastname: López-Ruano
authors.affiliation: Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, Spain. angelhh@usal.es.|Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca, Salamanca, Spain. angelhh@usal.es.
authors.forename: Ángel
authors.initials: Á
authors.lastname: Hernández-Hernández
authors.affiliation: Servicio de Hematología, IBSAL-Hospital Universitario de Salamanca, Paseo de San Vicente 58-182, 37007, Salamanca, Spain. redondoguijo@hotmail.com.|Centro de Investigación del Cáncer, Universidad de Salamanca, Salamanca, Spain. redondoguijo@hotmail.com.|Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, León, Spain. redondoguijo@hotmail.com.|Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, Spain. redondoguijo@hotmail.com.
authors.forename: Alba
authors.initials: A
authors.lastname: Redondo
authors.affiliation: Servicio de Hematología, IBSAL-Hospital Universitario de Salamanca, Paseo de San Vicente 58-182, 37007, Salamanca, Spain. rbk.ortega@hotmail.com.
authors.forename: Rebeca
authors.initials: R
authors.lastname: Ortega
authors.affiliation: Servicio de Hematología, IBSAL-Hospital Universitario de Salamanca, Paseo de San Vicente 58-182, 37007, Salamanca, Spain. rodriguezserrano@yahoo.es.|Centro de Investigación del Cáncer, Universidad de Salamanca, Salamanca, Spain. rodriguezserrano@yahoo.es.|Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, León, Spain. rodriguezserrano@yahoo.es.|Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, Spain. rodriguezserrano@yahoo.es.
authors.forename: Concepción
authors.initials: C
authors.lastname: Rodríguez
authors.affiliation: Servicio de Hematología, IBSAL-Hospital Universitario de Salamanca, Paseo de San Vicente 58-182, 37007, Salamanca, Spain. ferminsg@usal.es.|Centro de Investigación del Cáncer, Universidad de Salamanca, Salamanca, Spain. ferminsg@usal.es.|Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, León, Spain. ferminsg@usal.es.|Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, Spain. ferminsg@usal.es.
authors.forename: Fermín
authors.initials: F
authors.lastname: Sánchez-Guijo
authors.affiliation: Servicio de Hematología, IBSAL-Hospital Universitario de Salamanca, Paseo de San Vicente 58-182, 37007, Salamanca, Spain. concarol@usal.es.|Centro de Investigación del Cáncer, Universidad de Salamanca, Salamanca, Spain. concarol@usal.es.|Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, León, Spain. concarol@usal.es.|Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, Spain. concarol@usal.es.
authors.forename: Consuelo
authors.initials: C
authors.lastname: del Cañizo
mesh_terms: D015720:5'-Nucleotidase / Q000032:analysis*; D000328:Adult; D002460:Cell Line; D002478:Cells, Cultured; D000067128:Extracellular Vesicles / Q000737:chemistry*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D018960:Hyaluronan Receptors / Q000032:analysis*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000737:chemistry / Q000166:cytology*; D008875:Middle Aged; D018800:Thy-1 Antigens / Q000032:analysis*; D055815:Young Adult
chemical_list: D018960:Hyaluronan Receptors; D018800:Thy-1 Antigens; D015720:5'-Nucleotidase