Exploration of putative endothelial progenitor cells in cells mobilised by granulocyte colony stimulating factor

Identification of circulating endothelial progenitor cells (EPC), which share a common precursor with haematopoietic progenitor cells (HPC), the haemangioblast, has generated considerable interest in isolating, characterising and expanding them for clinical use. There is no definitive phenotype of EPC but there appears to be two main types, the CD14+ monocyte derived early EPC and the CD34+ derived endothelial outgrowth cell (EOC). These populations differ in their proliferative potential and appear quite distinct, though their function in vasculogenesis is debated. Potential sources of such cells include peripheral blood, bone marrow and umbilical cord blood. Peripheral blood stem cell (PBSC) transplantation is the paradigm of adult stem cell therapy. It relies on the use of granulocyte colony stimulating factor (G-CSF) to mobilise HPC from the bone marrow. With EPC and HPC sharing common origins it has been suggested that G-CSF mobilised peripheral blood would be an excellent source of EPC for clinical use. This work centres on the identification of EPC in G-CSF mobilised peripheral blood. G-CSF mobilised and non mobilised peripheral blood samples were obtained at a number of time points from autologous and allogeneic donors referred for PBSC collection using GCSF, given alone or sequentially with chemotherapy. We consistently demonstrated marked reductions in early EPC following the administration of G-CSF, using standard commercially available colony assays (CFU-EPC), which is reversible within a month of G-CSF treatment. We have also been unable to generate EOC from mobilised blood samples. Our goal has been to resolve why, when EPC are contained within the bone marrow, that we cannot find evidence of their mobilisation together with HPC following G-CSF. A series of experiments were performed in order to exclude technical factors as potential influences on CFU-EPC formation in mobilised blood. Flow cytometric analysis showed clear changes in the proportions of leukocyte subpopulations in MNC obtained from whole blood samples following G-CSF. We have explored the influence of cellular factors on CFUEPC formation and present evidence that CD66b+ granulocytes affect CFU-EPC. We have identified phenotypic differences between CD34 positive cells mobilised with G-CSF and CD34 positive cells present in umbilical cord blood, another potential source of CD34 positive cells for clinical use. We believe that these differences contribute to the failure of EOC development in mobilised blood. We have yet to resolve why we are unable to generate CFU-EPC or EOC from mobilised blood but using these results we are moving to explore other areas including G-CSF induced alterations of cell adhesion molecules expressed by CD14+ monocytes.