Abstract
Endothelial progenitor cells (EPCs) can be purified from peripheral blood, bone marrow or cord blood and are typically defined by a limited number of cell surface markers and a few functional tests. A detailed in vitro characterization is often restricted by the low cell numbers of circulating EPCs. Therefore in vitro culturing and expansion methods are applied, which allow at least distinguishing two different types of EPCs, early and late EPCs. Herein, we describe an in vitro culture technique with the aim to generate high numbers of phenotypically, functionally and genetically defined early EPCs from human cord blood. Characterization of EPCs was done by flow cytometry, immunofluorescence microscopy, colony forming unit (CFU) assay and endothelial tube formation assay. There was an average 48-fold increase in EPC numbers. EPCs expressed VEGFR-2, CD144, CD18, and CD61, and were positive for acetylated LDL uptake and ulex lectin binding. The cells stimulated endothelial tube formation only in co-cultures with mature endothelial cells and formed CFUs. Microarray analysis revealed highly up-regulated genes, including LL-37 (CAMP), PDK4, and alpha-2-macroglobulin. In addition, genes known to be associated with cardioprotective (GDF15) or pro-angiogenic (galectin-3) properties were also significantly up-regulated after a 72 h differentiation period on fibronectin. We present a novel method that allows to generate high numbers of phenotypically, functionally and genetically characterized early EPCs. Furthermore, we identified several genes newly linked to EPC differentiation, among them LL-37 (CAMP) was the most up-regulated gene.
Highlights
Endothelial progenitor cells (EPCs) represent a group of circulating cells derived from CD34+ hematopoietic stem cells (HPC)
Single staining flow cytometry revealed that approximately 90% of the cells expressed CD34 after the positive selection process
We describe a method that allows the generation of a high number of putative early EPCs after in vitro differentiation of expanded cord blood derived CD34+ cells
Summary
Endothelial progenitor cells (EPCs) represent a group of circulating cells derived from CD34+ hematopoietic stem cells (HPC). They are thought to stimulate angiogenesis either by their ability to differentiate into mature endothelial cells or by stimulating the formation and repair of the endothelium and vessel formation via paracrine stimuli [1,2,3,4,5,6]. The use of EPCs as a potential therapeutical tool for treatment of cardiovascular disease (CVD) has drawn much interest [5,7,8]. The most widely used phenotypic characterization for EPCs includes expression of CD34 and VEGFR-2 (KDR, CD309)
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