Endothelial Progenitor Populations in Differentiating Embryonic Stem Cells I: Identification and Differentiation Kinetics

Abstract

Embryonic stem cells (ESCs) have enormous potential in tissue engineering and cell therapies. However, the therapeutic use of ESCs has been restricted because of the presence of undifferentiated cells or cells with undesired phenotypes. We have explored identifying and selecting endothelial cells (ECs) using green fluorescent protein (GFP) under the control of different endothelial promoters. This method can result in progenitor populations that differ based on promoter activity; however, there have not been rigorous studies comparing differentiation kinetics and selection using these promoters as well as the resulting phenotype. In this study, we examined differentiation profiles of ESCs selected using three different endothelial promoters (Flk1, PECAM, and Tie1) that correspond to endothelial proteins expressed at different time points (early, middle, and late) in ESC differentiation. All three promoters yielded cells with EC-specific protein expression and DiI-Ac-LDL uptake when sorted for GFP⁺ population; however, Flk1-driven GFP⁺ cells yielded both smooth muscle cells and ECs or progenitors, whereas Tie1-driven GFP⁺ cells yielded mostly endothelial phenotype. Both Flk1 and PECAM promoters showed a noticeable level of GFP expression while in the undifferentiated state, making the elimination of undifferentiated cells difficult. Our findings show the differentiation kinetics of the various EC promoters and how different endothelial promoters can be used to select distinct subpopulations of ECs and endothelial precursors across a spectrum of differentiation.