Abstract 1038: Colony Forming Unit-Endothelial Cells are Derived from CD14+ Monocytes and Need Support by T-Cells
Endothelial progenitor cells (EPC) in peripheral blood are of interest for cellular therapy, but also as diagnostic parameter. However, controversies exist on the identity of the EPC. Culture of CFU-EC is a way to quantify numbers of EPC in peripheral blood. We determined the contribution of several cell types to CFU-EC formation. Hereto, peripheral blood mononuclear cells (PBMC) isolated from healthy donors, were either positively selected or depleted for several cell populations by immunomagnetic cell sorting. Subsequently, CFU-EC were cultured in EndoCult™ medium. Results show that CFU-EC are not derived from mature endothelial cells nor from immature EPC, as similar number of colonies per well were found when the starting population was depleted for CD146+/CD34+/KDR+ cells (18±9 vs. 20±14 in controls; n=4). In contrast, when CD14+ cells were depleted from the starting population, no colonies were formed (0±0 vs. 29±23 in controls; n=7, p<0.02). CD14+ monocytes, labelled with phagocyte-specific PKH2 and combined with untreated CD14− cells, were found to form both the colonies and the spindle shaped cells surrounding the colonies, indicating that the CFU-EC originate from monocytes. Surprisingly, cultures of only CD14+ cells did not result in a similar number of colonies as compared to the control (4±4 vs. 29±23 in controls; n=7, p<0.03). To investigate a possible role for a CD14− population in the CFU-EC culture, CD3+, CD19+ or CD56+ cells were depleted from the starting population. Only depletion of CD3+ cells gave a significant decrease in colony formation (6±3 after depletion vs. 23±8 in controls). While culturing CD3+ cells above CD14+ cells in a 0.4 μm transwell system increased colony formation (19±22 with CD3+ vs. 1±1 without CD3+ cells; n=3, p<0.05), direct co-culture of CD3+ and CD14+ cells improved colony formation even more (34±35; n=3). This indicates that paracrine factors and cell-cell contact of CD3+ cells influences colony formation. In conclusion, CFU-EC are derived from CD14+ cells and need the presence of CD14− cells, i.e. T-cells, partly due to the paracrine factors secreted. This study further clarifies the identity of the EPC as determined by CFU-EC, and may give more insight to the role of several cell populations in vascular repair.