In Vitro Two-Dimensional Endothelial Differentiation of Human Embryonic Stem Cells

Pluripotent human embryonic stem cells (hESCs) comprise of cells from all three germ layers in vivo. They have been proved to differentiate in vitro into a variety of cell lineages. Endothelial cells derived from hESCs could potentially contribute to cellular treatment of vascular diseases and regenerative development of engineered vessels. As different germ cells are intimately linked during hESC differentiation, embryonic endothelial cells can be isolated based on specific cell surface markers and further be identified by distinct properties, including expression of endothelial markers, LDL uptake, Matrigel tubular formation, and in vivo microvessel generation. Because of inefficient yield from three-dimensional (3D) embryoid body (EB) differentiation, we sought to develop a simple two-dimensional (2D) culture system for differentiating hESCs into endothelial lineages that bypasses EB formation. In this chapter, we describe a procedure for the 2D differentiation of hES cells toward endothelial cells. It is an efficient and reproducible method with potential for large-scale cultures in future applications.