Conventional methods for vascular endothelial differentiation of human embryonic stem (hES) cells had suffered from subculture incompetence of the final products due to dominant expansion of contaminating pericytic components. We have overcome this problem by adding a “hematopoietic cytokine cocktail” to the differentiation medium. No pericytes are produced by our method, and the vascular endothelial cells are expanded purely by repetitive subculture up to 10–20 passages, depending on the lines of hES cells. The hES-derived vascular endothelial cells undergo senescence thereafter as in the case of primary cultured human vascular endothelial cells. During the course of subculture, vascular endothelial functions are well preserved. Because our system is entirely feeder-free, including the step to maintain undifferentiated hES cells, contamination by xenogeneic cells is completely excluded. In this chapter, we describe our method in detail with troubleshooting guidelines.
