Functional arterial and venous fate is determined by graded VEGF signaling and notch status during embryonic stem cell differentiation

Objective-The aim of this work was to develop a mouse embryonic stem (ES) cell system addressing the early specification of the developing vasculature into functional arteries and veins. Methods and Results-ES cells were differentiated 4 days on collagen-type IV coated dishes to obtain Flk1(+) endothelial precursors. Sub-culture of these precursors for additional 4 days robustly generated, in a VEGF dose-dependent manner, mature endothelial cells. Arterial marker genes were specifically expressed in cultures differentiated with high VEGF concentration whereas the venous marker gene COUP-TFII was upregulated in endothelial cells induced through low and intermediate VEGF concentrations. This VEGF-dependent arterialization could be blocked by inhibition of Notch resulting in an arterial to venous fate switch. Functional and morphological studies, ie, measurement of sprout length, pericyte recruitment, and interleukin-I-induced leukocyte adhesion, further confirmed their arterial and venous identity. Conclusions-We conclude that endothelial cells with distinct molecular, morphological, and functional characteristics of arteries and veins can be derived through in vitro differentiation of ES cells in a VEGF dose-dependent and Notch-regulated manner.