Endothelial colony forming units: are they a reliable marker of endothelial progenitor cell numbers?

BACKGROUND. Flow cytometry and cell culture, the two main laboratory techniques employed for counting endothelial progenitor cells (EPCs), have serious limitations. Mononuclear cells cultured in media favouring endothelial growth allow cells to replicate and differentiate/mature. EPCs under these circumstances tend to form groups of cells called endothelial colony forming units (EC-CFUs). EC-CFUs are widely accepted as a surrogate as an estimate of EPC number and function in cell culture. However, some important limitations may restrict the assumption that EC-CFUs reflect EPC numbers accurately. OUR FINDINGS. Our own experience of EPC culture in atrial fibrillation has demonstrated that: 1) the size of EC-CFUs and proportion of single cells fluctuate significantly, even on the same culture plate; 2) the ability of EPCs to migrate towards one another to form EC-CFUs varies; and 3) the rate of EPC differentiation and proliferation may significantly affect the number of EC-CFUs, despite similarities in EPC counts on separate plates. In contrast, the count of differentiated cultured EPCs by flow cytometry with specific mature endothelial markers (e.g. CD146, vascular endothelial (VE) cadherin) is a potentially more objective alternative. SUMMARY. Endothelial CFU counts represent the cumulative characteristics of EPC quantity and their functional characteristics, and cannot be reliably used for the estimation of EPC numbers in peripheral blood or the bone marrow. Until stronger definition(s) of bone marrow or peripheral blood population(s) of EPCs are developed, flow cytometry may be the more optimal technique for EPC quantification.