Cord blood derived endothelial progenitor cells: time dependent in vitro generation capacity.

OBJECTIVES. To test in vitro endothelial progenitor cells (EPCs) generation from fresh cord blood (CBs) cultured at different time after collection. MATERIALS AND METHODS. Mononuclear cells (MNCs) were isolated by density gradient centrifugation using Lympholyte Cell Separation Media (Cedarlane) from CBs collected for banking and discharged either for low TNC count or volume. 20 * 106 MNCs from CBs aged from 6 to 12 hours (group 1) and from 13 to 36 hours (group 2) were cultured in endothelial cell culture medium (EGM-2 Bulletkit, Lonza) on fibronectin-coated cell culture plates (Becton Dickinson) for maximum 20 days monitoring the appearance of EPC cobblestone-shaped colonies. The colony-derived cells (CDCs) were enumerated and studied by immunofluorescence and flow cytometry for typical endothelial markers as well as for capillary-like structures formation using the Matrigel assay (Becton Dickinson). RESULTS. MNCs from group 1 CBs gave rise to EPC colonies with a mean generation rate (ratio of CDCs / CD34+ seeded cells) of 29.3% (range, 12.1 – 38.9), while MNCs from group 2 CBs showed a 0% generation rate. Consistently with an EPC phenotype, CDCs were able to form vascular networks on extracellular matrix (Matrigel), showed Ac-LDL uptake and were positive for CD34, CD31, CD146, vWF, KDR and negative for CD45 markers. A positive correlation between EPCs generation rate and MNCs CD34+ cell content was observed; while the other parameters (including MNCs viability) seemed not to influence EPCs generation. CONCLUSIONS. Even if our data need to be confirmed in a larger study, our preliminary experience indicates that CBs may be an attractive source of EPCs and shows that EPCs generation is highly influenced by CB age. These biological features should be considered in planning regenerative medicine protocols.