Epac agonist improves barrier function in iPSC-derived endothelial colony forming cells for whole organ tissue engineering

Abstract

Whole organ engineering paradigms typically involve repopulating acellular organ scaffolds with recipient-compatible cells, to generate a neo-organ that may provide key physiological functions. In the case of whole lung engineering, functionally endothelialized pulmonary vasculature is critical for establishing a fluid-tight barrier at the level of the alveolus, so that oxygen and carbon dioxide can be exchanged in the organ. We have previously developed a protocol to efficiently seed endothelial cells into the microvascular channels of decellularized lung scaffolds, but fully functional endothelial coverage, in terms of barrier function and resistance to thrombosis, was not achieved. In this study, we investigated whether various small molecules could favorably impact endothelial functionality after seeding into decellularized lung scaffolds. We demonstrated that the Epac-selective cAMP analog 8CPT-2Me-cAMP improves endothelial barrier function in repopulated lung scaffolds. When treated with the Epac agonist, barrier function of human umbilical vein endothelial cells (HUVECs) improved, and was maintained for at least three days, whereas the effect of other tested molecules lasted for only 5 h. Treatment with the Epac agonist re-organized actin structure, and appeared to increase the continuity of junction proteins such as VE-cadherin and ZO1. Blockade of actin polymerization abolished the effect of the Epac agonist on barrier function and actin reorganization, confirming a strong actin-mediated effect. Similarly, after treatment with Epac agonist, the barrier function in iPSC-derived endothelial colony forming cells (ECFCs) was increased and the enhanced barrier was maintained for at least 60 h. After culture in lung scaffolds for 5 days, iPSC-ECFCs maintained their phenotype by expressing CD31, eNOS, vWF, and VE-Cadherin. Treatment with the Epac agonist significantly improved the barrier function of iPSC-ECFC-repopulated lung for at least 6 h. Taken together, these findings demonstrated that Epac-selective 8CPT-2Me-cAMP activation enhanced vascular barrier in iPSC-ECFC-engineered lungs, and may be useful to improve endothelial functionality for whole organ tissue engineering.