[Transplantation of cord blood endothelial progenitor cells ameliorates limb ischemia].

OBJECTIVE: To investigate the feasibility of transplanting cord blood CD133+ cells derived endothelial progenitor cells (EPC) in therapeutic vasculogenesis.

METHODS: CD133+ cells from the cord blood of 52 neonates were cultured in fibronectin-coated flask in M199 medium supplemented with 10% fetal bovine serum, 50 ng/ml vascular endothelial growth factor (VEGF), 20 ng/ml interleukin-3 (IL-3) and 50 ng/ml stem cell factor (SCF). The cell markers of spindle-shaped adherent cells were determined with flow cytometry. The left femoral artery, great saphenous artery, iliac circumflex artery, and vein, and muscular branch of 22 Balb/c nude mice were cut to cause limb ischemia. One day after the unilateral ischemic limb surgery half million adherent cells were transplanted into 12 nude mice via tail vein (EPC group) and M199 was injected into the tail veins of 10 nude mice (M199 group). Fluorescence Dil, laser Doppler perfusion imaging (LDPI) and immunohistochemistry were Laser Doppler perfusion imaging (LDPI) was used to trace the transplanted cells and monitor the blood perfusion and capillary density of ischemic limbs. The ratio between the blood perfusion of the operated limb and of the non-operated opposite limb was recorded. Two to four mice in each group were killed 4, 7, 14, and 21 days after the operation and the gastrocnemius muscles of bilateral hind limbs were taken to count the number of capillaries. The VEGF mRNA levels of the ischemic and nonischemic limbs were examined with semi-quantitative RT-PCR. Seven days after the operation, fluorescein isothiocyanate (FITC)-binded ulex europaeus agglutinin-1 (UEA-1) was injected via tail vein to 3 EPC group mice. Thirty minutes later, the mice were killed. The heart, lung, liver, spleen and limb muscles were taken and examined with fluorescence microscopy. EPC were added into the upper chamber of Coster Transwell and chemotactic fluids of M199 with or without VEGE were added into the lower chamber. Four hours later the number of EPC in the lower chamber was counted so as to examine the chemotactic effect of VEGE.

RESULTS: Numerous cell clusters, spindle-shaped adherent cells and cord-like structures, developed from the culture of cord blood CD133+ cells. These adherent cells expressed vascular endothelial growth factor receptor 2 (VEGFR-2), VE-cadherin, CD31, von Willebrand factor (vWF) and combined with ulex europaeus agglutinin-1 (UEA-1). Transplanted EPC survived and were incorporated into the capillary networks in the ischemic limbs of nude mice. The ratio between the blood perfusion of the ischemic limb and non-ischemic limbs was 19.1% +/- 3.1%. Two weeks after the transplantation, the ratio between the blood perfusion of the ischemic limb and non-ischemic limbs of the EPC group was 77.3% +/- 5.6%, significantly higher than that of the M199 group (40.6% +/- 3.4%, P<0.001). CD31 histochemical staining showed that the density of capillaries in the gastrocnemius muscles of ischemic hind limb was significantly higher 7, 14, and 21 days after operation in the EPC group than in the M199 group (P<0.05) RT-PCR showed obvious VEGF bands in the ischemic hind limb muscles, but not in the non-ischemic muscles. The number of EPC immigrating into the lower chamber of the Coster Transwell was 817 +/- 32.5, significantly higher than that of the control group (473.5 +/- 61.5, P<0.05).

CONCLUSION: Cord blood CD133+ cells derived EPC is a robust cell source for therapeutic neovascularization. Upregulated expression of VEGF may account for the homing of transplanted EPC to ischemic tissue.