Endothelial progenitor cells (EPCs) are bone marrow-derived immature cells being capable of differentiating into endothelial cells and are involved in endothelial homeostasis as well as physiological and pathological angiogenesis (Asahara et al., 1999).So far, two morphologically distinct EPC subpopulations, named as early EPCs (eEPCs) and endothelial outgrowth cells (EOCs, also termed late-outgrowth EPCs or endothelial colony-forming cells), have been identified in mammals.However, the characteristics of EPCs in avian species remain unknown.The resent study was conducted to isolate EPCs from peripheral blood of broilers.PBMNCs from clinically healthy 4-to 6-week-old Arbor Acres broilers were prepared by density-gradient centrifuging and resuspended in EBM-2 medium supplemented with growth factors as provided in the EGM-2 Bullet Kit, and 10% fetal bovine serum (FBS) , 100 IU/ml penicillin and 100 μg/ml streptomycin.Cells were seeded onto 6-well tissue plates coated with rat tail collagen (5 μg/cm2, Sigma-Aldrich) at 1 × 107 cells per well, and incubated at 39℃ in 5% CO2.Non-adherent cells were removed after 48 h of plating and the medium was refreshed every 2 days thereafter.Cell morphology was monitored daily using a phase contrast microscope.Quantative PCR was performed to detecte the expression of progenitor and endothelial markers.As an alternative approach, cell phenotype was characterized by Dil-ac-LDL uptake and FITC-UEA-I binding.Cell proliferation was determined by a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay.On day 14 of plating, cells were submitted to an in vitro angiogenesis assay on BD MatrigelTM Basement Membrane Matrix.During a 2-week culture,one heterogeneous cell population dominated by spindle-shaped cells (eEPCs) and one homogenous cell population exhibiting cobblestone-like morphology (EOCs) appeared sequentially.Quantitative PCR revealed the expression of progenitor and endothelial cell markers such as CD133, VEGFR-2 and CD31 in both populations.However, another progenitor marker, CD34 gene, was undetectable in either freshly isolated PBMNCs or cultured cells.The endothelial phenotype of the EOCs was further identified by acetylated low-density lipoprotein/lectin double staining, and in vitro tube formation.Collectively, this work demonstrates that chicken EPCs can be isolated and cultured from PBMNCs, and suggests that EPCs obtained from peripheral blood may originate mainly from CD34-subpopulation.