Fig. 3. Stiffness-primed mesoderm induction in the presence of serum enhances EC differentiation.

(A) Schematic of stiffness-primed mesoderm induction followed by EC differentiation on E ~ 3 GPa substrates. α-MEM, α-minimum essential medium; FBS, fetal bovine serum; EGM, endothelial growth medium. (B) Left: Gene expression of mesodermal markers for cells differentiated on soft 3-kPa substrates, normalized to expression from E ~ 3 GPa surfaces. Right: Gene expression analysis of cells differentiated on stiff 1.7-MPa substrates, normalized to expression from E ~ 3 GPa surfaces. Color key is presented in log₁₀ scale. (C) Bright-field images of cobblestone endothelial colonies (white arrows) on day 12 EVCs. (D) Day 12 EVC flow cytometry plots of VECad expression in red, with corresponding HUVEC VECad expression in green. Black font, VECad⁺ cells; green font, highly expressing VECad⁺ cells. Data are presented as means ± SEM. (E) Representative immunofluorescence images of VECad expression on day 12 EVCs: Low-magnification (top) and high-magnification (bottom) images are shown (green, VECad; red, phalloidin; blue, nuclei). At least three biological replicates were performed.