Generation of vascular smooth muscle cells from human embryonic stem cells

This protocol describes the generation of coronary-like vascular smooth muscle cells (VSMCs) and cardiogenic progenitor cells from human embryonic stem cells (hESC). Differentiation into VSMCs is achieved in a 4-stage process. In the first and second stages, after 8 days in suspension culture, cells differentiate, in embryoid bodies (EBs), into primitive-streak and cardiac mesoderm-like cells, respectively. At the end of the third stage, the embryoid bodies contain a mixture of endothelial, cardiogenic, and smooth muscle progenitor cells. When EBs are disrupted and cells are cultured in low-density monolayer cultures for several passages, cells continue to express smooth muscle markers, whereas the expression of both cardiogenic and endothelial markers is reduced. The functional potential of the hESC-derived smooth muscle cells (SMCs) was tested in two in-vivo experiments. In the first one, matrigel plugs containing hESC-derived SMCs, were transplanted in a NOD-SCID mouse dorsal skin fold angiogenesis model. In the second experiment, cells were subcutaneously injected into the hind limb of NOD-SCID mice. In both cases, the cells integrated into the vascular network of the host animal model. Differentiation into cardiogenic progenitor cells is achieved by treating the 8 day EB suspension culture with Dkk1, bFGF, and VEGF for 6 days. The resulting cells express cardiovascular-specific markers.