Circulating Endothelial Progenitor Cells (EPCs): Variability between Normal Individuals as Measured by a New Standardized, Functional 5-Day In Vitro Colony Assay.

Endothelial cells derived from pre-existing blood vessels are believed to be responsible for minor repairs to the peripheral vasculature; however, a more primitive circulating EPC is thought to contribute to the maintenance and repair of heart tissue and may be useful in treating more significant vascular injuries as well as myocardial infarcts. While numerous attempts to identify the antigen expression of endothelial precursors have been made, to date no conclusive in vitro functional assays have correlated with the proposed phenotypes. Although the exact phenotype of the precursor cell remains elusive, a recently described colony assay for EPCs showed an inverse correlation between the number of circulating colony-forming cells (CFCs) and the risk of cardiovascular disease (Hill et al., NEJM 348(7):2003). This suggests that the precursor cells that read out in this colony assay may play a role in cardiac tissue maintenance and repair. For this assay to be useful in research and clinical studies, the variability in the frequency of circulating EPCs in the general population needs to be determined. We evaluated the EPC number in peripheral blood (PB) of volunteer male (n = 18) and female (n = 13) donors from the general population (age range 23–54, median age 34 years). PB white blood cell counts (WBC) were determined and mononuclear cell fractions were obtained by Ficoll® gradient density centrifugation. Following cell washing, 5 x 10⁶ cells per well were plated in EndoCult™ medium in a 6-well fibronectin coated (FN) plate and incubated for 48 hours. Non-adherent cells were then harvested and replicate cultures were initiated with 1 x 10⁶ cells per well in a 24-well FN plate. EPC derived colonies, consisting of a central core of round cells and more elongated cells at the periphery, were enumerated following an additional 3 days in culture. The frequency of EPCs in PB varied between individuals with a range of 0 to 99 and a mean ± SD of 25 ± 23 per ml of blood. The frequency of EPCs did not correlate with either WBC (r = 0.199) or age (r = −0.083) for either gender. In a subset of individuals studied (n = 5), fluctuations in the frequency of EPCs were determined at multiple time points over an 8-month period. Two of 5 donors consistently had less than 10 EPCs per ml of blood, whereas the other 3 donors never had less than 20 EPCs per ml of blood. These results show that, while there is a relatively large variation in the frequency of EPCs between individuals in the general population, an individual’s colony content is quite consistent over time. In conclusion, the lack of temporal variability in the frequency of circulating EPCs within an individual supports the use of this standardized 5-day EPC assay for the measurement of differences in endothelial progenitor content between individual patients. As these endothelial progenitors may have a clinical association with cardiovascular health, this standardized functional assay could be useful for further comparative clinical trials across multiple centers.