Single Cell Analysis Of Embryonic Stem Cell-Derived Vascular Endothelial Subphenotypes

A well-formed and robust vasculature is critical to the health of most organ systems in the body. To that end, our laboratory has directed the differentiation of vascular subphenotypes from stem cells for a number of applications. Our step-wise methodology outlining vascular endothelial cell (EC) differentiation from embryonic stem cells (ESC) in sera-free conditions demonstrates a range of endothelial heterogeneity and vascular functionality in these cultures. Vascular endothelial subphenotypes analogous those EC in angiogenic (tip and stalk EC) and quiescent blood vessels (phalanx EC) were in our 2D in vitro cultures. Here, distinct “tip/stalk-like” and “phalanx-like” EC subphenotypes were analyzed for cell surface marker expression using FACS analysis and immunohistochemistry. We used a nine-color antibody surface marker expression panel to physical separate various putative populations of tip and stalk ESC-derived EC (ESC-EC) and replate as single cells. Some of the subpopulations of tip and stalk ESC-EC were clonogenic with distinct proliferation rates, and morphological, functional, and genetic subphenotypes. Successful clonogenic tip and stalk ESC-EC were isolated as Dll4+/VE-cad+/Notch1- and Dll4-/VE-cad+/Notch1+, respectively. Moreover, greater numbers of tip cells (51%) were clonogenic compared with stalk cells (4%), suggesting that stalk cells need tip cells for survival and/or maintenance. Most importantly, these studies show that we can direct stem cells into specialized EC subphenotypes in vitro to address a range of therapeutic applications for these cells. Research funding is provided by CIRM grant Basic Biology V.