Common endothelial progenitor cell assays identify discrete endothelial progenitor cell populations

Abstract

Background: Multiple measures of endothelial progenitor cells (EPCs) have been described, but there has been limited study of the comparability of these assays. We sought to determine the reproducibility of and correlation between alternative EPC assay methodologies.

Methods: We simultaneously assessed EPC numbers in 140 patients undergoing cardiac catheterization using the 2 most commonly used culture techniques: endothelial cell outgrowth and colony-forming unit (CFU). In the final 77 patients, EPCs were also identified on the basis of cell surface marker expression (CD133, CD34, and vascular endothelial growth factor receptor-2 [VEGFR-2]) and aldehyde dehydrogenase (ALDH) activity.

Results: Endothelial progenitor cell enumeration based on fluorescence activated cell sorting was more precise than culture assays. There was limited correlation between EPC numbers determined using the 2 common culture-based assays; however, endothelial CFUs correlated with VEGFR-2 and CD34/VEGFR-2-expressing cells. Endothelial progenitor cells defined by expression of CD133, CD34, CD133/CD34, and ALDH activity correlated with each other, but not with VEGFR-2(+) cells.

Conclusions: Endothelial progenitor cells can be broadly classified into 2 classes: VEGFR-2-expressing cells, which give rise to endothelial CFUs, and CD133/CD34 or ALDH(br) cells. These observations underscore the need for better assay standardization and a more precise definition of EPCs in cell therapy research.

Fig. 1. EPC Identification Based on ALDH Activity Using Aldefluor

ALDH activity is shown on the X-axis, and side scatter on the Y-axis in the absence and presence (left panel) of DEAB.

Fig. 2. Correlation between Independent EPC Enumeration Assays

Analysis of the precision of EPC enumeration on the basis of endothelial cell outgrowth [top left panel], endothelial CFUs [top right panel], CD133 and CD34 expression [bottom left panel], and ALDH activity [bottom right panel]. Duplicate samples were obtained at the same time point from patients and analyzed in duplicate. Pearson correlation coefficients and p values are shown.

Fig. 3. Temporal stability of EPCs measured by ALDH or FACS

Blood samples obtained from volunteers 24 hours apart were analyzed for EPC content on the basis of a culture based assay (endothelial CFUs) and FACS based assay (CD133/CD34 expression). n = 11. A Pearson correlation value and p-value for the correlation are shown.

Fig. 4. Distribution of EPCs Identified by each technique

Distribution of EPCs as identified on the basis of endothelial cell outgrowth [A], endothelial CFUs [B], CD133 and CD34 expression [C], CD34 and VEGFR-2 expression [D], and ALDH activity [E]. EPC numbers are not normally distributed based on D’Agostino & Pearson omnibus normality test (p<0.0001 for each).

Fig. 5. Correlation of Endothelial CFU Numbers with other EPC assays

Correlation of EPCs identified on the basis of the endothelial CFUs (x-axis) vs. EPC numbers as determined using endothelial outgrowth assay (Panel A), EPCs identified on the basis of VEGFR-2 expression (Panel B), CD34 and VEGFR-2 expression (panel C), CD133 expression (panel D), CD34 expression (Panel E), or CD133 and CD34 expression (panel F). A Spearman correlation coefficient is shown for those correlations which reached statistical significance.

Fig. 6. Correlation of EPCs Identified Based on Cell Surface Protein Expression

Correlation of EPCs identified on the basis of CD34 expression with those expressing CD133 (panel A), and correlation of CD133⁺CD34⁺ cells with CD34⁺ VEGFR-2⁺ cells (panel B). Correlation of cells expressing VEGFR-2 with cells expressing CD133 and CD34 (panels C and D). A Spearman correlation coefficient is shown for those correlations which reached statistical significance.

Fig. 7. Correlation of ALDH^br EPCs with other EPC types

Correlation of EPCs identified on the basis of ALDH activity (x-axis) vs. EPC numbers as determined on the basis of endothelial CFUs (Panel A), CD133 expression (Panel B), CD34 expression (Panel C) and CD133⁺CD34⁺ expression (Panel D) A Spearman correlation coefficient is shown for those correlations which reached statistical significance.