Abstract There is evidence that angiogenic cells exist in the blood that are able to home to sites of ischemia and induce angiogenesis. Angiogenic cell populations identified in human blood include endothelial progenitor cells (EPC) and circulating angiogenic monocytes (CAM). Under basal conditions, however, the number of angiogenic cells in the circulation is small, potentially limiting their delivery to sites of ischemia and subsequent stimulation of angiogenesis. To circumvent this limitation, animal studies have shown that angiogenic cells can be mobilized into blood by certain cytokines; whether a similar mobilization of angiogenic cells can be achieved in humans is not clear. Herein, we report the results of a clinical trial that examined the ability of G-CSF and AMD3100 to mobilize EPC and CAM into the blood. Donors were treated with a single injection of AMD3100 (5mg/kg) after which pheresis was performed. One week later, donors were treated with G-CSF (250microg/kg/d x 5 days) and pheresed. Blood was collected at baseline (prior to the initiation of the mobilizing regimen) and after treatment with AMD3100 or G-CSF (at the time of peak HSC mobilization). In addition, pheresis product was collected after mobilization by AMD3100 or G-CSF. Mononuclear cells (MNC) were isolated from the blood products and cultured under angiogenic conditions. EPC were identified by the formation of discrete colonies of endothelial cells on days 14-28. A novel method to quantify CAM was developed to avoid the pitfalls of counting adherent cells in culture. Briefly, adherent cells were recovered from the culture, counted, and analyzed by flow cytometry. CAM were identified as CD45+ CD14+ CD31+ cells. The angiogenic potential of the CAM was assessed by their transplantation into NOD/SCID/b2M−/− mice following surgical induction of hindlimb ischemia. Indeed, reperfusion was significantly enhanced in mice that received CAM compared to control mice [ratio±SEM) of ischemic to non-ischemic limb perfusion: 0.47±0.05 (saline treated); 0.78±0.09 (CAM transplant); p<.001]. To date, a total of 9 patients have been analyzed. AMD3100 resulted in a 9.2-fold increase in CAM in the blood [CAM/ml peripheral blood±SEM: 3.5x105±0.09 (AMD3100) vs. 0.4x105±0.01 (baseline); p<.05]. Likewise, treatment with G-CSF resulted in a 12.3-fold increase in CAM (4.5x105±0.21; p<.05). Both AMD3100 and G-CSF also mobilized EPC into the blood [EPC/mL peripheral blood±SEM: 0.53±0.22 (AMD3100); 0.98±0.24 (G-CSF); vs. 0.05±0.01 (baseline); p<.05 for both AMD3100 and G-CSF compared with baseline]. Interestingly, data suggests that the G-CSF-mobilized CAM may have an enhanced capacity to stimulate angiogenesis in the hindlimb ischemia model [the ratio of ischemic to non-ischemic limb perfusion±SEM: 0.47±0.05 (saline treated); 0.64+0.11 (G-CSF) p<.001; 0.58±0.06 (AMD3100); p=NS]. Importantly, CAM and EPC were readily recovered from the pheresis product after both AMD3100 and G-CSF treatment. Collectively these data show that treatment with AMD3100 or G-CSF mobilizes a significant number of CAM and EPC into the peripheral circulation. These mobilized angiogenic cells have the capacity to stimulate angiogenesis in vivo. Finally, CAM and EPC can be efficiently harvested by leukopheresis, providing a method to obtain large numbers of circulating angiogenic cells for subsequent clinical use.±