Toward Engineering a Human Neoendothelium with Circulating Progenitor Cells

Abstract

Tissue-engineered vascular grafts may one day provide a solution to many of the limitations associated with using synthetic vascular grafts. However, identifying a suitable cell source and polymer scaffold to recreate the properties of a native blood vessel remains a challenge. In this work, we assess the feasibility of using endothelial progenitor cells (EPCs) found in circulating blood to generate a functional endothelium on poly(1,8-octanediol-co-citrate) (POC), a biodegradable elastomeric polyester. EPCs were isolated from human blood and biochemically differentiated into endothelial-like cells (HE-like) in vitro. The differentiated cell phenotype and function was confirmed by the appearance of the characteristic endothelial cell (EC) cobblestone morphology and positive staining for EC markers, von Willebrand factor, vascular endothelial cadherin, flk-1, and CD31. In addition, HE-like cells cultured on POC express endothelial nitric oxide synthase at levels comparable to aortic ECs. Furthermore, as with mature endothelial cells, HE-like cell populations show negligible expression of tissue factor. Similarly, HE-like cells produce and secrete prostacyclin and tissue plasminogen activator at levels comparable to venous and aortic ECs. When compared to fibroblast cells, HE-like cells cultured on POC show a decrease in the rate of plasma and wholeblood clot formation as well as a decrease in platelet adhesion. Finally, the data show that HE-like cells can withstand physiological shear stress of 10 dynes/cm 2 when cultured on POC-modified expanded poly(tetrafluoroethylene) vascular grafts. Collectively, these data are the foundation for future clinical studies in the creation of an autologous endothelial cell-seeded vascular graft. STEM CELLS 2010;28:318-328 Disclosure of potential conflicts of interest is found at the end of this article.

Endothelial Outgrowth Cells: Function and Performance in Vascular Grafts

“…TNF-a stimulation of EOCs results in tissue factor protein up-regulation similar to ECs and shortens the time to tissue factor/FVII-dependent thrombin generation and plasma clotting. 36,59 In summary, EOCs demonstrate an inflammatory response in vitro to TNF-a and oscillatory shear stress similar to ECs.…”

“…Thorough cell characterization is necessary to confirm the EOC phenotype due to the multiple cell types that may be generated using the current standard derivation protocols. 15,16,19,35,36 Further, our group and others have noted that colonies of outgrowth cells occasionally assume a large, irregular shape with slow proliferation and limited expansion potential. 33,35,37 In the absence of any definitive markers that distinguish these various EPCderived populations, screening each colony is currently necessary to validate the EOC phenotype.…”

“…40,41 EOCs express the fundamental proteins used by ECs to regulate thrombosis, including tissue factor, thrombomodulin, endothelial Protein C receptor, tPA, and von Willebrand factor. 26,27,35,36,[42][43][44][45][46][47][48] Regulation of thrombus formation by EOCs has been suggested in vitro by studies demonstrating reduced platelet adhesion on EOC-lined materials and the generation of the anticoagulant activated Protein C. 45,46,48 In addition, in response to pro-thrombotic stimuli, EOCs increase their expression of tissue factor, decrease plasma clotting time, and increase tPA expression. 36 A number of strategies have been used in an attempt to improve EOCs' thromboprotective function before graft implantation.…”

“…27 The majority of groups 17,26,35,46,58 though some groups have found no difference between cell types. 36 However, eNOS expression does not directly correlate with NO production, as evidenced by studies demonstrating that EOCs have lower eNOS expression but similar NO production as aortic ECs when stimulated by calcium 17 or fluid flow. 58 Intimal hyperplasia after vascular injury can also be reduced by activated Protein C. 62,63 Activated Protein C acts to inhibit inflammation-induced EC apoptosis and permeability, thereby maintaining functional EC regulation of underlying smooth muscle cells.…”

“…To ensure complete coverage of the tubular graft lumen, seeding is generally performed using multiple inoculations with manual or automated rotation, or by perfusing a cell suspension through the graft wall. 35,36,42,66,[79][80][81] …”