Identification, isolation and cultivation of pluripotent endothelial progenitor cells with high angiogenic potential from human aortic valve tissue

Objectives: Endothelial progenitor cells (EPCs) isolated from calcified aortic valve leaflets show an impressive angiogenic potential in 3D cultures. They contribute to revascularisation of ischemic tissue and may work as target for medical therapy and as source for vascularization of engineered tissues. The purpose of the study was selection, isolation and cultivation of these specific EPCs derived from diseased aortic valves in significant numbers.

Methods: Primary cell cultures and cultures in collagen matrix from 20 patients with aortic valve disease were isolated. Grade of sclerosis was low (n=6), moderate (n=7) or severe (n=7). Cell identification was performed 30 days after cultivation using the immunocytochemic endothelial progenitor cell markers CD34, aSMA, Tie-2 and VEGFR-2. FACS-analysis of cells isolated from collagen gel was used for quantification of CD34 positive cell populations. CD34 marked magnetic dynabeads were then used for further cell separation. Isolated progenitor cells were specifically cultivated up to an amount of 108 cells for further use.

Results: We demonstrated that EPCs initially exhibit an endothelial phenotype with positive signals for CD34, aSMA, Tie-2 and VEGFR-2. The coexpression of Tie-2 and VEGFR-2 as two embryonic vascular progenitor markers with a-SMA suggests an endothelial-mesenchymal origin of the cells involved in valvular degeneration. Separation of CD34 positive cells by CD34 marked magnetic dynabeads led to cultivation of pure EPCs.

Conclusion: Isolation of specific EPCs by antibody marked magnetic separation technology deliver a consistently high yield of pure cells as potential source for tissue vascularization or as target for medical therapy.