Autologous venous or arterial grafts remain the gold standard therapy for patients with coronary heart disease; however, suitable replacement vessels require multiple surgical sites for harvesting and are often already compromised. Synthetic grafts and tissue-engineered blood vessels (TEBVs) have overcome some of these limitations and have also sought to avoid issues with thrombosis or occlusion. Previous studies have used primary cells and adult stem cells to mimic the native blood vessel structure in TEBVs; however, immunogenicity is still a problem. The recent breakthrough of reprogramming adult somatic cells to induced pluripotent stem cells (iPSCs) provides an alternative cell source, where non-immunogenic cells of multiple lineages can be derived from patient-specific iPSCs. We propose to construct an entirely cellular TEBV using cell sheets of differentiated iPSCs for replacing diseased or damaged arteries. We will develop an iPSC line with a dual reporter for pure populations of differentiated endothelial cells (ECs) and vascular smooth muscle cells (vSMCs). To further our understanding of developmental stages, iPSCs will be differentiated from posterior primitive streak to Flk-1-positive vascular cell precursors to either ECs (using VEGF) or vSMCs (using platelet derived growth factor BB (PDGF-BB) and transforming growth factor beta (TGF-)). ECs can be sorted using CD31 (PECAM) and CD144 (VE-Cadherin), and we can use our double reporter system to isolate vSMCs, which are characterized by expression of red fluorescent protein (DsRed) for mural cell marker neural/glial antigen 2 (NG2) and green fluorescent protein (GFP) for smooth muscle actin (SMA). Using a thermoresponsive system, differentiated iPSCs can be formed into cell sheets and detached enzymatically without affecting cell sheet integrity. To study how SMC tissue heterogeneity arises, cell sheets of SMCs will be co-cultured with cell sheets of different tissue types (e.g., airway, cardiomyocytes, or ECs) and evaluated for gene expression (SMA, myosin heavy chain, and SM22a) and functional properties (contractility and calcium uptake). Cell sheets will also be evaluated for mechanical strength and biochemical composition for comparison to native vessel mechanics. With this proposed study, we will lay the groundwork for in vivo evaluation of the functional characteristics of our fabricated TEBV implant to replace damaged blood vessels.
Smooth muscle cells (SMCs) are found in blood vessels, airways, and other organs, where their primary role is contraction; however, little is known if SMCs found in these different anatomical locations have characteristic differences that may change their functionality. This project could shed light on these differences, as well as what type of SMC is derived from induced pluripotent stem cells (iPSCs). Understanding how we can obtain large quantities of functional vascular SMCs differentiated from iPSCs will help development of a cellular vascular graft that better mimics the native blood vessel for bypass surgeries.
