Generation of iPS-derived endothelial cells and use in organoids vascularizationin

Abstract

The use of human pluripotent stem cells (iPS) for in vitro disease modeling and clinical applications requires protocols that convert these cells into relevant adult cell types with an efficiency rate. The major challenge in the organoids formation is the vascularization of these. Here, we report the rapid and efficient differentiation of human iPS into mature vascular endothelial cells capable of forming vessels in collagen gels. iPS cells were committed to a mesodermal fate and subsequent exposure to VEGF resulted in the differentiation of endothelial cells. The protocol produced mature cells with efficiencies over 80% within six days. Upon purification to 99% via VECadherin as a surface marker, endothelial cells maintained their identity until passage 5, as assessed by marker gene expression and immunostainings (CD31, VECadherin, vWF). Functional tests have been carried out. The capability of endothelials cells to form capillaries in matrigel was tested and confirmed. IPS-derived endothelial cells are also able to capture oxidized LDL and form a tight monolayer that is destabilized by thrombin treatment. Tissue factor-induced thrombin generation in platelet free plasma is reduced in the presence of iPS-derived vascular endothelial cells showing an antithrombotic phenotype. Endothelial cells were cultivated in collagen gel during one week and form structured vessels with lumen. All together these results showed relevant in vitro functionality of iPS-derived vascular endothelial cells. Our results suggest that these endothelial cells could be used to vascularized organoids structure.