H4236

Human CD34⁺, CD34⁻, or both CD34⁺ and CD34⁻ cells isolated from HUCB were cultured in methylcellulose-containing medium, H4236 (StemCell Technologies, Vancouver, Canada), supplemented with 20 ng/mL stem cell-derived factor (Kirin, Tokyo, Japan), 50 ng/mL vascular endothelial growth factor (VEGF; R&D Systems, Minneapolis, MN, USA), 20 ng/mL interleukin (IL)-3 (Kirin), 50 ng/mL basic fibroblast growth factor (bFGF; Wako, Osaka, Japan), 50 ng/mL epidermal growth factor (EGF; Wako), 50 ng/mL insulin-like growth factor (IGF)-1 (Wako), 2 U/mL heparin (Ajinomoto, Tokyo, Japan), and 10% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA) on a 35-mm dish (Thermo SCIENTIFIC, Rockford, IL) for 8 d. The cell density of each sample was 5×10² cells per dish or was adjusted depending on the assay. The EPCs were identified as small EPC-CFUs or large EPC-CFUs by visual inspection using a light microscope (OLYMPUS, Tokyo, Japan) under 40x magnification. Small EPC-CFUs were composed of round adhesive cells, and large EPC-CFUs were composed of spindle-shaped cells. Nonattached cells were isolated as small EPCs by washing with PBS (WELGENE, Daegu, Korea), while attached cells were harvested as large EPCs by treatment with 5 mM EDTA (Sigma-Aldrich, St. Louis, MO) in PBS (5 mmol/L) for 5 min at 37°C.

DIF-3 was added to K562 cell lines or primary cells (103 CD34+ or CD34- cells/ml) growing in semisolid methylcellulose medium. Metho-Cult H4100 or H4236 were used for cell lines and primary CD34+ cells respectively (StemCell Technologies Inc., Vancouver, Canada). Colonies were detected after 10 days of culture by adding 1 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent and were scored by Image J quantification software (U.S. National Institutes of Health, Bethesda, MD, USA).