Abstract
The existence of circulating endothelial progenitor cells (EPC) in adult humans is under intensive investigation due to its potential clinical application. In the present study we have attempted to identify the EPC with colony forming capacity and compare their characteristics with those of the monocytic-macrophage lineage.
42 healthy donors were analysed (7 Bone Marrow, 20 steady-state Peripheral Blood (PB), 4 apheresis products from mobilised PB and 9 buffy-coat products). Median age was 38 years and M/F ratio was 19/23. EPC were obtained by culturing MNC in IMDM with VEGF and beta-FGF. At day 7 the colonies were counted and flow cytometry and immunohistochemistry studies were carried out. Sequential studies were performed on days +21, +28 and +35 of culture with vascular growth factors. Monocytic cells were obtained by adhesion to plastic surface or CD14+ cell selection using magnetic microbeads (Miltenyi, Biotech). Then, monocytic cells were cultured using the same conditions as EPC until day +21 or alternatively with VEGF, beta-FGF and IGF. Von Willebrand gene expression was analysed by RT-PCR and the formation of vascular structures was analyzed by Matrigel assays on both cell sources, EPC and monocytic cells. The mean number of EPC colonies at day 7 was significantly higher in BM (813±695) than in steady-state PB (21.2±2.5), while mobilized PB displayed intermediate values (272±274). By contrast, using the same medium as EPC, monocytic cells did not form colonies at day 7, but cord-like structures could be seen in 7 out 9 cases. However, when the cells were cultured by adding IGF to the medium, a greater number of colonies could be observed. By immunohistochemistry colonies were positive for CD45, CD31 and lysozyme but negative for vWf. Flow cytometry analysis showed that colonies were positive for CD4, CD13, CD14, CD31, CD33, CD45, lysozyme and VE-cadherin, weak positive for CD15 and CD105, and did not express CD16, CD34, CD133 or KDR. This phenotypic profile remained unchanged at all time-points analysed. The immunophenotype of cultured monocytes at day +21 was identical to that of pre-cultured monocytes and both were similar to those obtained in EPC, even for the “specific” markers lysozyme and VE-cadherin. PCR analysis showed small amounts of vWF transcripts in EPC as well as in monocytes but the expression increased after culture with VEGF. Finally, when matrigel assays were carried out, we observed that monocytes formed cord- and tubular-like structures to a higher extent than EPC. Our results in both cell subtypes suggest that the so “called” EPC belongs to the monocytic cell lineage and both populations express some “specific” endothelial antigens such as CD31 or VE-cadherin, as well as monocytic markers such as lysozyme.
Detection of circulating endothelial cells and endothelial progenitor cells by flow cytometry
