Evaluation of mobilized peripheral blood CD34(+) cells from patients with severe coronary artery disease as a source of endothelial progenitor cells

Abstract

Background aims: The distinction between hematopoietic stem cells (HSC) and endothelial progenitor cells (EPC) is poorly defined. Co-expression of CD34 antigen with vascular endothelial growth factor (VEGF) receptor (VEGFR2) is currently used to define EPC ( 1 ).

Methods: We evaluated the phenotypic and genomic characteristics of peripheral blood-derived CD34(+) cells in 22 granulocyte-colony-stimulating factor (G-CSF)-mobilized patients with severe coronary artery disease and assessed the influence of cell selection and storage on CD34(+) cell characteristics.

Results: The median CD34(+) cell contents in the products before and after enrichment with the Isolex 300i Magnetic Cell Selection System were 0.2% and 82.5%, respectively. Cell-cycle analysis showed that 80% of CD34(+) cells were in G0 stage; 70% of the isolated CD34(+) cells co-expressed CD133, a marker for more immature progenitors. However, less than 5% of the isolated CD34(+) cells co-expressed the notch receptor Jagged-1 (CD339) and only 2% of the isolated CD34(+) population were positive for VEGFR2 (CD309). Molecular assessment of the isolated CD34(+) cells demonstrated extremely low expression of VEGFR2 and endothelial nitric oxide synthase (eNOS) and high expression of VEGF-A. Overnight storage at 4 degrees C did not significantly affect CD34(+) cell counts and viability. Storage in liquid nitrogen for 7 weeks did not affect the percentage of CD34(+) cells but was associated with a 26% drop in cell viability.

Conclusions: We have demonstrated that the majority of isolated CD34(+) cells consist of immature and quiescent cells that lack prototypic markers of EPC. High VEGF-A gene expression might be one of the mechanisms for CD34(+) cell-induced angiogenesis.

Figure 1

Assessment of co-expression of CD34 antigen with CD133 and VEFR2 antigens by immunocytochemistry. The figure shows cytospins of selected CD34⁺ cells labeled with (A) anti-CD34–FITC antibodies, with more than 90% of the cells expressing CD34 antigen; (B) anti-CD34–FITC and anti-CD133–PE antibodies, with the majority of the CD34⁺ cells co-expressing CD133 antigen; and (C) anti-CD34–FITC and anti-CD309–PE (VEGFR2) antibodies, with less than 5% of the CD34⁺ cells expressing CD309 (arrow). Magnification for all images ×60.

Figure 2

Box-plots showing flow cytometric evaluation of selected CD34⁺ cells that co-express CD133, CD309 (VEGFR2) and CD339 (Jagged-1). The median percentage purity of the CD34-selected cells was 82.5%; 70% of the cells co-expressed CD133 antigen. Less than 5% of the CD34⁺ cells co-expressed CD309 and CD339 (n=22).

Figure 3

Quantitative real-time RT-PCR of selected CD34⁺ cells. Box-plots show gene expression change relative to 18S rRNA expression of CD133, CD309 (VEGFR2) and CD339 (Jagged-1) normalized to CD34 expression. CD34-expressing cells showed significant high co-expression of CD133 compared with Jagged-1 (n=6).

Figure 4

Flow cytometric analysis: figure shows dot-plot of PY (Pyronin Y) on the y-axis and Ho (Hoechst) on the x-axis.

Figure 5

Cell-cycle analyzes of G-CSF-mobilized CD34-selected cells using an Isolex 3000. The median percentage of CD34⁺ cells in the G0 stage of the cell cycle was 78.5%.

Figure 6

Molecular characterization of G-CSF-mobilized CD34⁺ cells for VEGFR2, VEGF-A and eNOS. (A) PCR amplification products prepared from MNC (G, P) and sorted CD34⁺ cells (S). G, sample with good mobilized CD34⁺ cell yield; P, sample with poor mobilized CD34⁺ cell yield; H, HUVEC cells, positive control. (B) VEGFR2, VEGF-A and eNOS gene expression relative to 18S rRNA by sorted CD34⁺ cells from six G-CSF-mobilized patients and HUVEC cells as a control. Both figures show that mobilized MNC and sorted CD34⁺ cells significantly expressed VEGF-A but not VEGFR2 and eNOS.

Figure 7

Impact of CD34⁺ cell selection and storage on CD34⁺ cell count and viability. Statistically significant drop in cell viability (P < 0.05). Impact of CD34⁺ cell selection using an Isolex 300i Magnetic Cell Selection System on the MNC profile.