Project Summary Tissue engineered vascular grafts (TEVGs) produced through seeding human induced pluripotent stem cell (hiPSC)-derived vascular smooth muscle cells (VSMCs) onto polyglycolic acid (PGA) meshes have been developed to repair patients’ vascular injuries. These hiPSC- TEVGs can be made further applicable through decellularization, allowing them to be stored for long periods and used off the shelf. For large diameter vessels (>6mm inner diameter), these decellularized TEVGs can be implanted directly, with host cells sufficiently able to recellularized the graft over time. However, small diameter TEVGs (2-4mm) are prone to thrombosis if implanted directly, requiring a layer of endothelial cells (ECs) to be coated in the lumen before these procedures. Significant research has been done to create hiPSC-ECs sufficient for endothelialization of small diameter TEVGs, as many patients have autologous ECs unable to be used for this process due to age or disease. For this reason, it is vital for the development of this technology for researchers to know how these coated hiPSC-ECs behave in vivo non- invasively. In this supplemental proposal, this need will be addressed by producing a stable hiPSC line expressing human sodium iodide symporter (hNIS), a solute carrier that confers the ability to uptake radioisotopes into the cell. These hNIS-hiPSCs will be used to derive ECs that can be tracked in vivo non-invasively using SPECT/CT imaging, providing key data to the behavior and function of these cells after implantation. This will be accomplished through the use of transcription activator-like effector nuclease (TALEN) gene editing to insert a constitutively active hNIS cassette into the AAVS1 “safe harbor” locus in stable hiPSCs. hiPSC- TEVGs will then be decellularized and coated with hNIS-ECs derived from the hNIS-hiPSCs, and these grafts will be trained for implantation by undergoing shear stress in a flow bioreactor, to enhance hNIS-EC maturity. These matured hNIS-EC TEVGs will then be used as a aortic interposition grafts in an immunocompromised rat model, which allow for the validation of the functionality of these hNIS-ECs to allow in vivo visualization over time using SPECT/CT imaging. This innovative technology will allow for non-invasive assessment of the health and extent of implanted hiPSC-EC coating in decellularized grafts, providing vital information into the ability of the endothelium to prevent thrombosis in patients who require vascular injury repair.