Differentiation and dynamic analysis of primitive vessels from embryonic stem cells.

Embryonic stem (ES) cells, which are derived from developing mouse blastocysts, have the ability to differentiate into various cell types in vitro. When placed in basal medium with added serum, mouse ES cells undergo a programmed differentiation favoring formation of cell types that are found in the embryonic yolk sac, including vascular endothelial cells. These in vitro differentiated endothelial cells form primitive blood vessels, analogous to the first vessels that form in the embryo and the yolk sac. This differentiation model is ideal for both genetic and pharmacological manipulation of early vascular development. We have made mouse ES cell lines that express endothelial-specific GFP or H2B-GFP and used these lines to study the processes of mammalian vessel development by real-time imaging. Here we describe protocols for making transgenic ES cells and imaging the processes of blood vessel development. We also provide methods for ES cell maintenance and differentiation, and methods for analysis of vascular marker expression.