title: Endothelial progenitor cells, defined by the simultaneous surface expression of VEGFR2 and CD133, are not detectable in healthy peripheral and cord blood.
abstract: Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted subpopulations of peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells, involved in the endothelial homeostasis maintenance. Both CEC and EPC are thought to represent potential biomarkers in several clinical conditions involving endothelial turnover/remodeling. Although different flow cytometry methods for CEC and EPC characterization have been published so far, none of them have reached consistent conclusions, therefore consensus guidelines with respect to CEC and EPC identification and quantification need to be established. Here, we have carried out an in depth investigation of CEC and EPC phenotypes in healthy PB, CB and BM samples, by optimizing a reliable polychromatic flow cytometry (PFC) panel. Results showed that the brightness of CD34 expression on healthy PB and CB circulating cells represents a key benchmark for the identification of CEC (CD45neg/CD34bright/CD146pos) respect to the hematopoietic stem cell (HSC) compartment (CD45dim/CD34pos/CD146neg). This approach, combined with a dual-platform counting technique, allowed a sharp CEC enumeration in healthy PB (n = 38), and resulting in consistent CEC counts with previously reported data (median = 11.7 cells/ml). In parallel, by using rigorous PFC conditions, CD34pos/CD45dim/CD133pos/VEGFR2pos EPC were not found in any healthy PB or CB sample, since VEGFR2 expression was never detectable on the surface of CD34pos/CD45dim/CD133pos cells. Notably, the putative EPC phenotype was observed in all analyzed BM samples (n = 12), and the expression of CD146 and VEGFR2, on BM cells, was not restricted to the CD34bright compartment, but also appeared on the HSC surface. Altogether, our findings suggest that the previously reported EPC antigen profile, defined by the simultaneous expression of VEGFR2 and CD133 on the surface of CD45dim/CD34pos cells, should be carefully re-evaluated and further studies should be conducted to redefine EPC features in order to translate CEC and EPC characterization into clinical practice.
authors: Department of Medicine and Aging Science, School of Medicine and Health Science, University "G. d'Annunzio" of Chieti-Pescara, Chieti, 66013, Italy.|Center for Ageing Sciences (Ce.S.I.), "Università G. d'Annunzio" Foundation, Chieti, 66013, Italy.
authors: Paola
authors: P
authors: Lanuti
authors: BD Biosciences Italia, Milano, 20090, Italy.
authors: Gianluca
authors: G
authors: Rotta
authors: Department of Transfusion Medicine, Laboratory for Stem Cells Manipulation and Cryopreservation, AO Spedali Civili di Brescia, Brescia, 25123, Italy.
authors: Camillo
authors: C
authors: Almici
authors: Department of Hematology, Stem Cell Transplantation, Transfusion Medicine and Cellular Therapy, Campus Bio-Medico University Hospital, Rome, 00128, Italy.
authors: Giuseppe
authors: G
authors: Avvisati
authors: Experimental Pharmacology Unit, Istituto Nazionale Tumori Fondazione G. Pascale-IRCCS, Naples, 80131, Italy.
authors: Alfredo
authors: A
authors: Budillon
authors: Department of Laboratory Medicine, Clinical Pathology Laboratory, "S. Maria Degli Angeli" Hospital, Pordenone, 33170, Italy.
authors: Paolo
authors: P
authors: Doretto
authors: Department of Health Science, Interregional Research Center for Food Safety and Health (IRC-FSH), University "Magna Graecia" of Catanzaro, Catanzaro, 88100, Italy.|Department of Experimental and Clinical Medicine, BioNEM Lab, University "Magna Graecia" of Catanzaro, Catanzaro 88100, Italy.
authors: Natalia
authors: N
authors: Malara
authors: Department of Transfusion Medicine, Laboratory for Stem Cells Manipulation and Cryopreservation, AO Spedali Civili di Brescia, Brescia, 25123, Italy.
authors: Mirella
authors: M
authors: Marini
authors: Department of Transfusion Medicine, Laboratory for Stem Cells Manipulation and Cryopreservation, AO Spedali Civili di Brescia, Brescia, 25123, Italy.
authors: Arabella
authors: A
authors: Neva
authors: Department of Medicine and Aging Science, School of Medicine and Health Science, University "G. d'Annunzio" of Chieti-Pescara, Chieti, 66013, Italy.|Center for Ageing Sciences (Ce.S.I.), "Università G. d'Annunzio" Foundation, Chieti, 66013, Italy.
authors: Pasquale
authors: P
authors: Simeone
authors: Experimental Pharmacology Unit, Istituto Nazionale Tumori Fondazione G. Pascale-IRCCS, Naples, 80131, Italy.
authors: Elena
authors: E
authors: Di Gennaro
authors: Experimental Pharmacology Unit, Istituto Nazionale Tumori Fondazione G. Pascale-IRCCS, Naples, 80131, Italy.
authors: Alessandra
authors: A
authors: Leone
authors: Department of Laboratory Medicine, Clinical Pathology Laboratory, "S. Maria Degli Angeli" Hospital, Pordenone, 33170, Italy.
authors: Alessandra
authors: A
authors: Falda
authors: Department of Laboratory Medicine, Clinical Pathology Laboratory, "S. Maria Degli Angeli" Hospital, Pordenone, 33170, Italy.
authors: Renato
authors: R
authors: Tozzoli
authors: Department of Hematology, Stem Cell Transplantation, Transfusion Medicine and Cellular Therapy, Campus Bio-Medico University Hospital, Rome, 00128, Italy.
authors: Chiara
authors: C
authors: Gregorj
authors: Department of Hematology, Stem Cell Transplantation, Transfusion Medicine and Cellular Therapy, Campus Bio-Medico University Hospital, Rome, 00128, Italy.
authors: Melania
authors: M
authors: Di Cerbo
authors: Department of Health Science, Interregional Research Center for Food Safety and Health (IRC-FSH), University "Magna Graecia" of Catanzaro, Catanzaro, 88100, Italy.
authors: Valentina
authors: V
authors: Trunzo
authors: Department of Health Science, Interregional Research Center for Food Safety and Health (IRC-FSH), University "Magna Graecia" of Catanzaro, Catanzaro, 88100, Italy.
authors: Vincenzo
authors: V
authors: Mollace
authors: Department of Medicine and Aging Science, School of Medicine and Health Science, University "G. d'Annunzio" of Chieti-Pescara, Chieti, 66013, Italy.|Center for Ageing Sciences (Ce.S.I.), "Università G. d'Annunzio" Foundation, Chieti, 66013, Italy.
authors: Marco
authors: M
authors: Marchisio
authors: Department of Medicine and Aging Science, School of Medicine and Health Science, University "G. d'Annunzio" of Chieti-Pescara, Chieti, 66013, Italy.|Center for Ageing Sciences (Ce.S.I.), "Università G. d'Annunzio" Foundation, Chieti, 66013, Italy.
authors: Sebastiano
authors: S
authors: Miscia
mesh_terms: D000071916:AC133 Antigen / Q000235:genetics* / Q000276:immunology; D000293:Adolescent; D000328:Adult; D000368:Aged; D018952:Antigens, CD34 / Q000235:genetics / Q000276:immunology; D019985:Benchmarking; D051929:CD146 Antigen / Q000235:genetics / Q000276:immunology; D002452:Cell Count; D066026:Endothelial Progenitor Cells / Q000166:cytology* / Q000276:immunology; D005260:Female; D005312:Fetal Blood / Q000166:cytology* / Q000276:immunology; D005434:Flow Cytometry / Q000592:standards*; D005456:Fluorescent Dyes / Q000737:chemistry; D015870:Gene Expression; D064368:Healthy Volunteers; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000276:immunology; D006801:Humans; D016130:Immunophenotyping / Q000592:standards*; D017493:Leukocyte Common Antigens / Q000235:genetics / Q000276:immunology; D008297:Male; D008875:Middle Aged; D017410:Practice Guidelines as Topic; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics* / Q000276:immunology
chemical_list: D000071916:AC133 Antigen; D018952:Antigens, CD34; D051929:CD146 Antigen; D005456:Fluorescent Dyes; C497902:MCAM protein, human; C000606023:PROM1 protein, human; C000588866:KDR protein, human; D040301:Vascular Endothelial Growth Factor Receptor-2; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human
keywords: CD133; circulating endothelial cells; endothelial progenitor cells; polychromatic flow cytometry; vascular endothelial growth factor receptor 2