Engineering surfaces for site-specific vascular differentiation of mouse embryonic stem cells

Differentiation of stem and progenitor cells routinely relies on the application of soluble growth factors, an approach that enables temporal control of cell fate but enables no spatial control of the differentiation process. Angiogenic progenitor cells derived from mouse embryonic stem cells (ESCs) were differentiated here according to the pattern of immobilized vascular endothelial growth factor-A (VEGF). Mouse ESCs engineered to express green fluorescent protein (eGFP) under control of promoter for the receptor tyrosine kinase Flk1 were used. The Flk1+ angiogenic progenitors were selected from day 3 differentiating embryoid bodies based on their expression of eGFP using fluorescence activated cell sorting. Mouse VEGF165 was covalently immobilized onto collagen IV (ColIV) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) chemistry. A non-cell adhesive layer of photocrosslinkable chitosan was first created, after which VEGF–ColIV was stamped as 100 μm wide lanes on top of the chitosan layer and the Flk1+ angiogenic progenitors were seeded for site-specific differentiation. Lanes stamped with only ColIV served as controls. The results presented here demonstrate that the cultivation of Flk1+ progenitors on surfaces with immobilized VEGF yielded primarily endothelial cells (53 ± 13% CD31 positive and 17 ± 2% smooth muscle actin positive), whereas surfaces without VEGF favored vascular smooth muscle-like cell differentiation (26 ± 17% CD31 positive and 38 ± 9% smooth muscle actin positive).