Isolation of CD34+AC133+Flk1+ Cells Does Not Identify the Most Primitive Endothelial Progenitor Cells.

Human EPCs are currently defined as a CD133+/Flk1+ subpopulation of CD34+ cells that can be isolated from peripheral blood, bone marrow, and fetal liver (Nature Medicine, 2003). While these cells are widely considered to be EPCs and subsequently used for angiogenic therapies and biomarkers of cardiovascular disease, their proliferative and clonogenic potential has not been compared to other populations of EPCs, which do not express the cell surface antigens, CD34 and AC133. Analogous to a paradigm established in the hematopoietic cell system, we recently developed a single cell clonogenic assay to reproducibly identify the following EPCs: (1) high proliferative potential - endothelial colony forming cells (HPP-ECFC), which form macroscopic colonies that form secondary and tertiary colonies upon replating, (2) low proliferative potential - endothelial colony forming cells (LPP-ECFC), which form colonies greater than 50 cells, but do not form secondary colonies upon replating, (3) endothelial cell clusters (EC-clusters) that contain less than 50 cells, and (4) mature terminally differentiated endothelial cells (EC), which do not divide (Blood, 2004). Utilizing this assay and EPCs derived from human umbilical cord blood mononuclear cells, we compared the clonogenic and proliferative potential of 1,000 single CD34+AC133+Flk1+ and CD34-AC133−Flk1+ umbilical cord blood derived endothelial cells. We conducted four independent experiments. We demonstrate that a complete hierarchy of EPCs can be identified in a population of CD34−AC133−Flk1+ cord blood derived ECs (Table I, n=4). Remarkably, we further show that CD34−AC133−Flk1+ cord blood derived ECs contain more proliferative EPCs (LPP-ECFCs and HPP-ECFCs) compared to CD134+AC133+Flk1+ cord blood derived ECs (Table I, n=4, *p<0.05). In fact, preliminary experiments suggest that CD34−AC133−Flk1+ cells mature into CD34+AC133+Flk1+ cells. Finally, some individual single CD34−AC133−Flk1+ HPP-ECFCs can expand rapidly to 107 cells in ex vivo culture. Retroviral marking of the cell progeny derived from the single cell confirmed that they were derived from the parent cell. Thus, these studies describe the use of a clonogenic assay to identify a novel population of CD34−AC133−Flk1+ EPCs, and preliminary experiments demonstrate that CD34 and AC133 are not universal cell surface markers of the most primitive EPCs. Percent of 1,000 Single Cells Plated Mature EC EC-Cluster LPP-ECFC HPP-ECFC CD34+AC133+Flk1+ ECs 70±11 16±5 10±4 3±1 CD34-AC133-Flk1+ ECs 52±12 17±6 20±3* 10±4*