title: Flow cytometric identification and functional characterization of immature and mature circulating endothelial cells.
abstract: OBJECTIVE
  We sought to identify and characterize 2 distinct populations of bona fide circulating endothelial cells, including the endothelial colony-forming cell (ECFC), by polychromatic flow cytometry (PFC), colony assays, immunomagnetic selection, and electron microscopy.
  
  
  RESULTS
  Mononuclear cells from human umbilical cord blood and peripheral blood were analyzed using our recently published PFC protocol. A population of cells containing both ECFCs and mature circulating endothelial cells was determined by varying expressions of CD34, CD31, and CD146 but not AC133 and CD45. After immunomagnetic separation, these cells failed to form hematopoietic colonies, yet clonogenic endothelial colonies with proliferative potential were obtained, thus verifying their identity as ECFCs. The frequency of ECFCs were increased in cord blood and were extremely rare in the peripheral blood of healthy adults. We also detected another mature endothelial cell population in the circulation that was apoptotic. Finally, when comparing this new protocol with a prior method, we determined that the present protocol identifies circulating endothelial cells, whereas the earlier protocol identified extracellular vesicles.
  
  
  CONCLUSIONS
  Two populations of circulating endothelial cells, including the functionally characterized ECFC, are now identifiable in human cord blood and peripheral blood by PFC.
authors.affiliation: Department of Pediatrics, Indiana University School of Medicine, Indianapolis, 46202, USA.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Mund
authors.forename: Myka L
authors.initials: ML
authors.lastname: Estes
authors.forename: Mervin C
authors.initials: MC
authors.lastname: Yoder
authors.forename: David A
authors.initials: DA
authors.lastname: Ingram
authors.forename: Jamie
authors.initials: J
authors.lastname: Case
mesh_terms: D000071916:AC133 Antigen; D000328:Adult; D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34 / Q000032:analysis; D017209:Apoptosis; D015415:Biomarkers / Q000032:analysis; D051929:CD146 Antigen / Q000032:analysis; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D042783:Endothelial Cells / Q000145:classification* / Q000276:immunology / Q000637:transplantation / Q000648:ultrastructure; D005260:Female; D005312:Fetal Blood / Q000166:cytology; D005434:Flow Cytometry; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D018189:Immunomagnetic Separation; D007231:Infant, Newborn; D017493:Leukocyte Common Antigens / Q000032:analysis; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D046529:Microscopy, Electron, Transmission; D010455:Peptides / Q000032:analysis; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000032:analysis; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000145:classification* / Q000276:immunology / Q000648:ultrastructure; D055815:Young Adult
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D018952:Antigens, CD34; D015415:Biomarkers; D051929:CD146 Antigen; D006023:Glycoproteins; C497902:MCAM protein, human; C000606023:PROM1 protein, human; D010455:Peptides; D019408:Platelet Endothelial Cell Adhesion Molecule-1; C000606024:Prom1 protein, mouse; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human