O2	Early differentiation of human endothelial progenitor cells.
L.Bellik, M.C.Vinci, F.Ledda, A.Parenti
Pharmacology Department, University of Florence, Florence, IT.

Although several preclinical and clinical studies have exploited the potential of circulating endothelial progenitor cells (EPCs) to restore organ revascularization, the exact phenotype and lineage of these cells is still a matter of debate and different expansion protocols are used to obtain them. In the current study, EPC expansion from peripheral blood mononuclear cells (PBMCs) was analyzed within the first week of culture by flow cytometry. Both the adherent and suspended cells, of which the latter usually discarded, were considered. Cultured PBMCs displayed, between the 2nd and 3rd day, a shift in their light scatter characteristics thus suggesting the sequential acquisition of mature endothelial cell morphology. Consistently changes of endothelial markers were also observed, which decreased in the lymphomonocytic cells and significantly increased in the newly appearing cell population. For instance, VEGFR2 was almost undetectable (1%) and increased up to 50% in both adherent and suspended cells. VE-cadherin expression was almost superimposible on that of VEGFR2. Immunostaining performed after 3 days showed that cells were strongly positive for the markers UEA-1 and acLDL, demonstrating the endothelial precursor cell phenotype. Both cellular fractions grown for 2 days in presence of angiogenic stimuli were functionally characterized. Adherent and suspended cells significantly migrated in response to VEGF and were able to form cord and tubular- like structures when seeded in Matrigel. The present data provide for the first time, a systematic study of EPC phenotype and functional features within the first 3 days of culture and we demonstrate that PBMC fraction in suspension, which is usually discarded or seeded again after a few days, displays comparable behaviour and may constitute a non-negligible target.

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