Characterization of ‘Early’ vs ‘Late’ Endothelial Progenitor Cells (EPCs) Which Are Derived from Human Umbilical Cord Blood (HCB) during Ex Vivo Expansion.

EPCs have been isolated from adult peripheral blood and bone marrow. Recently, several groups reported that two types (‘early’ & ‘late’) of EPC could be isolated from peripheral blood and bone marrow when pertinent cocktails of cytokines were used. Interestingly, early and late EPCs are different in terms of expression of surface markers, the abilities of tube formation in vitro and the capabilities of re-vascularization on hind limb ischemia models in mice. We found EPC formation during ex vivo expansion of HCB and one EPC could be found from 314 CD34+ cells from HCB based on limiting dilutional assay (ref. Stem Cells; 2003, Yoo et al). However, little is known about the characteristics of ‘early’ and ‘late’ EPCs that are derived from HCB. In this study, our aims are to isolate the ‘early’ and ‘late’ EPCs from HCB during ex-vivo HCB expansion period and to characterize the biologic properties between ‘early’ and ‘late’ EPCs. 1 x 108 mononuclear cells were plated on a 100mm culture dish coated with 50ug/ml of human fibronectin (Calbiochem) and cultured in EGM-2 BulletKit system (Clonetics). Endothelial cells were assessed by colony counts, flow cytometry, proliferation assay, RT-PCR and in vitro tube formation in Matrigel plate. Migration of EPCs were also measured by in vitro transmigration assay in the presence of VEGF and SDF-1. In results, early spindle-shaped cells (‘early’ EPCs) which were grown at first week of culture were positive for CD31, CD14 and CXCR-4. Cobblestone shaped cells (‘late’ EPC) were in peak growth at second and third weeks of culture and were also positive using above antibodies except CD14. Early EPCs had not expressed mRNA of KDR, vWF and VE-Cadherin by RT-PCR. However, late EPCs expressed high level of mRNA of those endothelial marker genes. Both early and late EPCs expressed mRNA of eNOS. Late EPC produced more nitric oxide and formed more capillary tubes than those of early spindle-shaped cells. Early EPCs were readily migrated by VEGF and SDF-1 compared with those of late EPCs. In conclusions, we have found two different types of EPCs with different biologic properties during HCB ex vivo expansion. These findings may have potential clinical applications for “cell therapy” on vascular injuries (ie, hindlimb ischemia and myocardial infacrtion). Murine models for vascular injuries are being established to test the efficacy of different types of EPCs from HCB in our Lab.