Establishing of a pure entothelial cell culture from differentiating murine embryonic stem cells

Endothelium is of essential importance for the maintenance of vessel function. Endothelial dysfunction is considered as a key event in the pathology of cardiovascular diseases. Newer studies have proven that progressive ischemic dysfunction and organ damage can be prevented and restored by new therapeutic strategies like cell-based therapy. These attempts encompass application of autologous progenitor cells derived from peripheral blood or bone marrow. The accurate definition and origin of progenitor cells and their function during vessel recovery still remain as unsolved issues. Peripheral blood contains only a low fraction of endothelial progenitor cells and extraction of these cells from bone marrow is based on invasive methods. Embryonic stem (ES) cells can develop into any functional cell type, and may serve as a source for cell therapy. Nevertheless, because of ethical concerns about and immunogenicity of ES cells, their clinical use is still controversial. These problems can be avoided by application of induced pluripotent stem cells. The present work focuses on development of a method to routinely derive large numbers of endothelial cells from differentiating murine ES cells. Transgenic cell lines expressing a reporter and a resistance gene under the control of endothelial-specific promoters such as Flk-1 and VE-Cadherin were established, allowing for antibiotic selection of desired cells as a solution to the problems linked with mechanical isolation. Gene transfer in ES cells was carried out by lentiviral transduction, followed by antibiotic selection of stably transfected clones. Taking advantage of this approach yielded robustly positive results with a very high efficiency, leading to the establishment of several ES cell clones, expressing GFP and zeocin under the control of the Flk-1 and VE-Cadherin promoter, respectively. The generated ES cell lines will later allow for the antibiotic-supported isolation of Flk-1 or VE-Cadherin positive cells. In addition, the cell-specific GFP expression in these cell lines, will also be useful for other applications such as enhancement of differentiation towards endothelial cells, as well as developmental studies.