Direct Comparison of Umbilical Cord Blood versus Bone Marrow–Derived Endothelial Precursor Cells in Mediating Neovascularization in Response to Vascular Ischemia

Characteristics and yield of UCB- and BM-derived EPCs. Mononuclear cells (MNCs) from fresh UCB or BM were isolated and cultured under endothelial conditions for 7 days [13]. Adherent cell yields at day 7 from UCB cultures were on average 2.4% ± 0.3% of the initial MNC input, compared with 18.6% ± 2.2% obtained from BM MNCs (A). Fluorescent microscopy of adherent cells was used to assess the uptake of acetylated low-density lipoprotein (acLDL; red) and nuclear staining with Hoechst 33258 (blue) after 7 days of culture (B). Representative images shown here were taken at ×40 with a Zeiss (Jena, Germany) LSM510 confocal microscope. For flow analysis, cultured cells were dissociated (2 mmol/L ethylenediaminetetraacetic acid), and the fluorescence of acetylated low-density lipoprotein and UEA-lectin was quantified (C).

Flow cytometric analysis of EPC cells derived from UCB versus BM. UCB and BM MNCs were cultured for 7 days, and adherent cells were trypsinized, washed, and stained for stromal (Stro-1, CXCR4, CD105, and CD73), endothelial (CD31, CD146, and VE-cadherin [VE-Cad]), stem cell (CD34 and CD133), and monocyte (CD14) surface markers and analyzed by flow cytometry.

Perfusion ratios after injection of EPCs into NOD/SCID mice with induced hind-limb ischemia. NOD/SCID mice underwent femoral artery ligation and excision followed by intracardiac injection of EPCs. Control animals were injected with saline or complete EBM2 media. Adherent UCB or BM EPCs on the day of injection (day 7 of culture) were removed with trypsin, and 1 × 10⁶ cells per mouse were injected. Doppler measurements were taken immediately after ligation and then on days 7, 14, and 28. Perfusion ratios were determined between the ischemic and nonischemic leg of each study animal.

Comparison of capillary density in treated and nontreated mice. At 28 days after isolation and ligation of the right femoral artery, tissue was harvested from the lower calf muscle of study and control mice. Tissue was fresh-frozen and stained for alkaline phosphatase by the indoxyl-tetrazolium method. Two blinded investigators counted 20 fields per sample, and capillary density was expressed as capillaries per square millimeter.

Histologic assessment of ischemic hind limbs with CD31. Tissue from the lower calf muscles of both hind limbs was harvested at day 28 for histologic evaluation. The samples were fixed in formalin. Sections (6-μm thickness) were mounted on saline-coated glass slides and stained with anti-human CD31 antibody to identify EPCs derived from human cells. Control mice (A) were negative for CD31 staining. Specimens from mice that were injected with UCB-derived EPCs (B) or BM-derived EPCs (C) showed positive staining for CD31 expression by human cells within the microvasculature.