Endothelial Progenitor Cells (EPCs) Isolated from the Rhesus Macaque but Not the Mouse Are Phenotypically and Functionally Similar to Human EPCs. (2004)

Abstract We recently identified a novel hierarchy of human endothelial progenitor cells (EPCs), which are functionally defined by their proliferative and clonogenic potential (Blood, 2004). Emerging evidence suggests that EPCs may be used as angiogenic therapies, or as biomarkers to assess cardiovascular disease risk. Thus, identification of animal models, which phenocopy the human EPC hierarchy, is an important priority for preclinical testing of experimental therapeutics. Given the importance of the Rhesus Macaque as a preclinical model, we tested whether EPCs could be isolated from the peripheral blood of the Rhesus Macaque and compared to EPCs isolated from human adult peripheral blood. Mononuclear cells were isolated from 20 ml of Rhesus peripheral blood and cultured in EGM-2 medium, which promotes the formation of EPC colonies. After 7 days in culture, we identified approximately 20 endothelial cell colonies (n=9), which appeared identical to human EPC colonies. We subcultured the endothelial cell colonies into monolayers for immunophenotyping and functional analysis. Endothelial cells (ECs) derived from the Rhesus EPC colonies formed vessels in matrigel, and demonstrated uptake of acetylated LDL, which are characteristics of ECs. Similar to ECs derived from human EPCs, Rhesus ECs expressed the endothelial cell antigens, CD31, CD144, CD105, CD146, and Flk1. Importantly, Rhesus ECs did not express the hematopoietic cell specific antigens, CD45 and CD14. Similar to ECs derived from human peripheral blood EPC colonies, Rhesus ECs could be serially passaged for at least 40 population doublings without signs of cellular senescence. A hallmark of stem and progenitor cells is their ability to proliferate and give rise to functional progeny. Analogous to a paradigm established in the hematopoietic cell system, we recently developed a single cell deposition assay to reproducibly identify the following human 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). To determine whether these different populations of EPCs could be identified in the ECs derived from Rhesus EPCs, we performed single cells deposition assays on 1,000 cells. All types of EPCs could be identified in the Rhesus ECs (Table I). Further, ECs derived from the Rhesus EPCs rapidly form chimeric vessels with human ECs derived from adult blood, implying that the molecular mechanisms critical for vessel formation are conserved between the two species. Finally, while the murine model is an animal model widely used for studying EPCs, a similar hierarchy of EPCs could not be established from the peripheral blood of mice. Thus, given the diversity of therapeutic applications of EPCs for treating a variety of human diseases, these studies establish the Rhesus Macaque as an important preclinical model. Percent of 1,000 Single Cells Plated Mature EC EC-Cluster LPP-ECFC HPP-ECFC Rhesus ECs 85.8±2.1 4.2±1.1 7.8±0.5 1.3±0.5 Human ECs 80.8±9.6 8.6±1.4 12.4±8.1 0.2±0.2