Combination of granulocyte colony-stimulating factor and CXCR4 antagonist AMD3100 for effective harvest of endothelial progenitor cells from peripheral blood and in vitro formation of primitive endothelial networks

Endothelial progenitor cells (EPC) derived from the circulation may be used to enhance neovascularization. Since the combination of granulocyte colony-stimulating factor (GCSF) and CXCR4 antagonist AMD3100 efficiently mobilizes hematopoietic stem cells into peripheral circulation, it may increase the pool of endogenously circulating EPC. We tested this hypothesis by administering GCSF and AMD3100 to adult rabbits and rats, isolating mononuclear cells from peripheral blood by Ficoll density gradient centrifugation, and characterizing the blood-derived EPC based on morphology, immunophenotyping, gene expression and other functional analyses. These EPC showed clonal growth similar to that of human umbilical vein endothelial cells when cultured in complete EGM-2 medium on collagen I-precoated culture plates. The EPC exhibited a typical cobblestone-like morphology and were relatively homogeneous by the third passage. The cells expressed the typical endothelial marker CD31 based on flow cytometry and fluorescence microscopy, formed capillary-like structures when cultured in Matrigel, internalized DiI-acetylated low-density lipoprotein, bound Ulex europaeus agglutinin-1, and expressed CD31 and several other endothelial markers (VEGFR2, VE-cadherin, Tie-2, eNOS, vWF) at significantly higher levels than bone marrow-derived mesenchymal stem cells. These results suggest that the combination of GCSF and AMD3100 can efficiently release stem cells into peripheral circulation and generate EPC that show the desired morphological, immunophenotypic and functional characteristics. This minimally invasive approach may be useful for autologous cell transplantation for postnatal neovasculogenesis and tissue repair.