Are endothelial outgrowth cells a potential source for future re-vascularization therapy?

• Early and late passage EOCs grown from MNCs and CD34+ cells are indistinguishable.
• The replicative potential and morphology of EOC changes over time in culture.
• EOCs undergo senescence during long-term expansion.
• Reduced tube formations and increased SA-β-gal activity in senescent EOC cultures.

Endothelial progenitor cells (EPCs) represent a heterogeneous cell population that is believed to be involved in vasculogenesis after ischemic diseases. EPCs could have a potential for future therapies with the purpose of enhancing endothelial repair. However, due to the low amount of these cells in circulation they have to be expanded in vitro before administration into recipients. The purpose of this study was to analyse and evaluate possible changes in morphology and functionality as a result of in vitro ageing of a subtype of EPCs called endothelial outgrowth cells (EOCs), since such changes might compromise the cells' ability to participate in vasculogenesis.EOCs were isolated and grown from human umbilical cord blood using two methodologies with varying degree of cell purification. The changes between the two culture setups and the changes occurring in EOCs over time were traced by flow cytometry and assays for growth, tube formation, and beta-galactosidase production.The cells showed to be indistinguishable from each other during the first weeks of culture. The cells showed a changed morphology with bigger and more granular cells and the growth rate decreased with time. The cells also showed an increased Beta-galactosidase expression and decreased tube formation ability in late passage EOCs.Our data indicates that EOCs undergo senescence during long-term expansion and therefore time for cell harvest has to be validated in order to achieve functional cells still maintaining a therapeutic potential. A possible application in large animal or humans could be local injection of EOCs into affected areas and thereby reducing the need for long-term expansion of the cells.