Colony Forming Unit-Endothelial Cells (CFU-EC) Are Derived from CD14+ Cells and Need Support by CD14− Cells.

Abstract

Since endothelial progenitor cells (EPC) were found in adult blood, active interferences of vascular diseases by cellular therapy with EPC were initiated. However, controversies exist on the identity of the EPC, e.g. concerning phenotype before and after culture, in vitro and in vivo behavior, as well as on quantities and differentiation. EPC can be quantified in vitro by culturing CFU-EC. In order to clarify which cells are essential and which are facilitating the formation of CFU-EC, we determined the contribution of several cell types to the formation of colonies. Peripheral blood mononuclear cells were isolated and were either positively selected or depleted for several cell populations (CD34+/KDR+/CD146+, CD3+, CD14+, CD19+ or CD56+). Subsequently the cells were labeled with PKH2 or irradiated (40Gy) to determine the contribution to colony formation. The purity of the cell populations was determined by flow cytometry. From these cell populations, CFU-EC were cultured on fibronectin-coated tissue culture plates, in endothelial selective medium EndoCultTM (Stem Cell Technologies). After 2 days of culture, non-adherent cells were subsequently cultured for an additional 3 days to form colonies. To determine the contribution of soluble factors to colony formation, transwells (0.8 μm pore size) were used to separate the cell populations during culture. CFU-EC are not derived from mature endothelial cells nor from immature EPC, as similar number of colonies per well were found when the starting population was depleted for CD146+/CD34+/KDR+ cells (18±9 vs. 20±14 in controls; n=4). In contrast, when CD14+ cells were depleted from the starting population, no CFU-EC were formed (0±0 vs. 29±23 in controls; n=7, p<0.02). After depletion of CD3+, CD19+ or CD56+ cells, CFU-EC were formed similarly as compared to controls. To investigate whether the CD14+ cells were the clonogenic cells, or whether they are facilitating the formation of colonies, monocytes were labeled with phagocyte specific PKH2 or irradiated and combined with untreated CD14− cells. Labeled cells were found in the colonies and among the spindle shaped cells surrounding the colonies. Hardly any colony formation was found after irradiating CD14+ cells (4.5±1 vs. 34±9 in controls), indicating that proliferating monocytes are necessary for CFU-EC formation. Culturing CD14+ cells alone did not result in a similar number of colonies as compared to the control (4±4 vs. 29±23, in controls; n=7, p<0.03). This indicates that CD14− cells are important in outgrowth of CFU-EC from monocytes. To test if this is due to paracrine factors, CD14− cells were cultured in a transwell insert above the >95% CD14+ fraction. This resulted in an increase in colony formation (>95% monocytes: 2±1; with insert: 10±9, n=2). In conclusion, CFU-EC are derived from CD14+ cells, but need the presence of CD14− cells, which is in part due to paracrine factors secreted by CD14− cells. This study further clarifies the identity of the EPC as determined by CFU-EC, and may give more insight to the role of several cell populations in vascular repair.