Flow-cytometric identification, enumeration, purification, and expansion of CD133+ and VEGF-R2+ endothelial...

A flow cytometric method for identifying, purifying, and expanding endothelial progenitor cells (EPC) derived from peripheral blood is reported. During our experiments, we found that isolation of viable EPC is not possible by using the standard flow cytometric protocols, since erythrocyte lysing influences cell survival. Furthermore, erythrocyte lysing has a high impact on quantative analysis of EPC with 20% lower numbers compared to no-lyse data. The viability of EPCs was tested with a colony forming test after both lysis based FACS of EPCs and without lysing. CD133 and VEGF-R2 revealed as positive markers for EPC selection and 7-amino actinomycin D (7-AAD) to eliminate dead cells. The few purified CD133+ and VEGF-R2+ cells showed strong colony-forming capacity in a human stem cell methylcellulose based medium (colony assay) when isolated by the no-lyse protocol. The colonies showed the typical shape of early EPC-colonies with round immature cells in the centre and dendritic or spindelcell-shaped peripheral cells, which were also immunologically identified as EPC-derived. Compared to this, erythrocyte lysing reagents destroyed even all sorted EPCs. Summarizing the presented data suggest that the use of erythrocyte lysing reagents is neither suitable for cloning nor for counting of endothelial progenitor cells, and no-lyse protocols should be used.