A Transgenic tdTomato Rat for Cell Migration and Tissue Engineering Applications

Abstract

The growing deficit in suitable tissues for patients awaiting organ transplants demonstrates the clinical need for engineered tissues as alternative graft sources. Demonstrating safety and efficacy by tracking the migration and fate of implanted cells is a key consideration required for approval of promising engineered tissues. Cells from transgenic animals that express green fluorescent protein (GFP) are commonly used for this purpose. However, GFP can create difficulties in practice due to high levels of green autofluorescence in many musculoskeletal tissues. Tandem-dimer tomato (tdTomato) is a stable, robust red fluorescent protein that is nearly threefold brighter than GFP. Our objective was to create a line of transgenic rats that ubiquitously express tdTomato in all cells, driven by the human ubiquitin C promoter. We sought to determine the rats' utility in tissue engineering applications by fabricating engineered skeletal muscle units (SMUs) from isolated muscle-derived tdTomato cells. These tdTomato SMUs were implanted into a volumetric muscle loss (VML) defect of the tibialis anterior muscle in a rat ubiquitously expressing GFP. We also evaluated a novel method for modularly combining individual SMUs to create a larger engineered tissue. Following a recovery period of 28 days, we found that implantation of the modular SMU led to a significant decrease in the size of the remaining VML deficit. Histological analysis of explanted tissues demonstrated both tdTomato and GFP expression in the repair site, indicating involvement of both implanted and host cells in the regeneration process. These results demonstrate the successful generation of a tdTomato transgenic rat, and the use of these rats in tissue engineering and cell migration applications. Furthermore, this study successfully validated a method for scaling engineered tissues to larger sizes, a factor that will be important for repairing volumetric injuries in more clinically relevant models.