Adult endothelial progenitor cells from human peripheral blood maintain monocyte/macrophage function throughout in vitro culture

Mononuclear cells (NINCs) isolated from peripheral blood by density gradient centrifugation were plated on human fibronectin-coated culture plates and cultured in EGM-2 medium. Attached spindle-shaped cells, reported as endothelial progenitor cells (EPCs) by some investigators, had elongated from adherent round cells, but had not proliferated from a small number of cells as supposed previously. The growth curve of the primary EPCs showed that the cells had little proliferative capacity. Flow cytometry analysis showed that the cells could express some of the endothelial lineage markers, while they could also express CD14, which is considered a marker of monocyte/macrophage lineages throughout culture. In endothelial function assays, the cells demonstrated a lower level of expression of eNOS than mature endothelial cells in the reverse transcription-polymerase chain reaction and did not show an ability to develop tube-like structures in angiogenesis assay in vitro. In this study, we identified the monocytold function of EPCs by the combined Dil-labeled acetylated low-density lipoprotein (Dil-Ac-LDL) and Indian ink uptake tests. All the cells were double positive for Dil-Ac-LDL and Indian ink uptake at days 4, 14 and 28 of culture, which means the EPCs maintained. monocytold function throughout the culture. Therefore, although adult EPCs from peripheral MNCs have some endothelial lineage properties, they maintain typical monocytic function and have little proliferative capacity.