Cd309

Cells from the digested cord tissue and cultured in MSCGM were characterized as UCT-MSC based upon morphology and phenotype determined by flow cytometry as separate biological replicates. Antibodies used for phenotypic characterization included CD31, CD14, CD90, CD44, CD83, CD105, CD45, UEA-1, Stro-1, ICAM-1, CD146, CD11b, CD29, CD80, CD117, CD166, CD34, and CD309 (Becton Dickinson, Franklin Lakes, NJ, United States). UEA-1 was purchased from Sigma-Aldrich (St. Louis, MO, United States) and CD51 was purchased from Immunotec (Beckman-Coulter, Miami, FL, United States).
To obtain cord tissue-derived endothelial progenitor cells (UCT-EPC), cells from the digested cord tissue that had been cultured in EGM-2 were labeled with FITC-conjugated Ulex europaeus agglutinin 1 (UEA-1) (Sigma-Aldrich, St. Louis, MO, United States) and isolated with anti-FITC microbeads (MACS Miltenyi Biotec, San Diego, CA, United States). Cells were then phenotypically defined by flow cytometry as separate biological replicates. Endothelial cell lineage was confirmed by visualizing tube formation with the Cultrex “In Vitro Angiogenesis Assay Tube Formation Kit” (Trevigen, Gaithersburg, MD, United States). UCT-EPC were cultured on gelatin-coated flasks using EGM-2 growth media in a humidified 37°C incubator at 5% CO₂.

Endothelial progenitor cells were isolated and characterized as previously described.18, 19 Peripheral blood mononuclear cells, which were isolated using a Ficoll‐Isopaque Plus (Histopaque‐1077; Sigma) gradient centrifugation method, were seeded in fibronectin‐coated cell culture flasks. Then, the cells were cultured with Endothelial Basal Medium‐2 (EBM‐2; Lonza) in a 37°C, 5% CO₂ incubator. Culture media were replaced after 4 days of incubation. Late EPCs grow into cobble‐stone‐like colonies after 14 days of incubation. The presence of EPCs was validated by both confocal microscopy and flow cytometry as previously described.20, 21 EPCs were identified by double‐positive for DiI‐Ac‐LDL and UEA‐1. Surface makers, including CD31, CD34, CD45, CD133 and CD309 (Becton‐Dickinson), were analysed for the definition of EPCs. The EPCs from 3 to 5 passages were used.

The numbers of EPCs were measured as CD34⁺ KDR⁺ (KDR: vascular endothelial growth factor receptor 2) double positive cells using a Beckman Gallios flow cytometer (Brea, CA, USA). Briefly, 20 µl of whole blood were incubated with CD34-FITC (Beckman Coulter, Brea, CA, USA) and CD309 (Becton Dickinson, Franklin Lakes, New Jersey, USA). Conjugate isotype-matched immunoglobulin (IgG1-FITC, IgG1-PE) with no reactivity against human antigens was used as a negative control. After 30 minutes of incubation in a dark environment, BD cell-fix was added to fixate the samples. Twenty thousand events of leukocytes were collected (based on classical forward scatter/side scatter (size/granularity) characteristics of blood leukocytes) and results are presented as the number of EPC events.