657. Delivery of Genetically Engineered Adipose-Derived Cell Sheets for Treatment of Ischemic Disorders – Development of Application in Animal Models

Abstract

Cell sheet (CS) approach is extensively developed in recent years for a number of applications in regenerative medicine including lesion treatment, tissue-engineered constructs and organoplastics. In this study we have targeted ischemic diseases – myocardial infarction (MI) and peripheral artery disease (PAD) using CS from differetnt sources transplanted as CS to the impaired region.We established a protocol for generation of CS from c-kit+ cardiac primitive cells and cardiosphere cells for delivery to rats with induced MI using clinical-grade fibrin glue for CS adhesion subepicardially. We found CS to be vascularized after transplantation and used ultrasound assessment to detect improvement of left ventricle function and significant positive changes of tissue status in histology studies: capillary density and reduction of fibrosis). Moreover, we conducted a comparative analysis of this approach vs. CS from adipose-derived stomal cells (ADSC) and VEGF-expressing ADSC. The latter were obtained via baculovirus transduction of CS, which enabled significant production of human VEGF165 by murine cells and, thus, enhanced their therapeutic potential.Our previous application of ADSC for therapeutic angiogenesis and established methods for genetic modification of cells allowed us to develop a protocol in limb ischemia model. We have demonstrated, that subcutaneous implantation of CS or VEGF-expressing CS from mouse ADSC resulted in significant improvement of limb perfusion with modified cells to be superior to mock-transduced. Increased blood flow was supported by higher CD31+ capillary density, reduction of necrosis and detection of viable CS subcutaneously with focal proliferation of mADSC and minimal (app. 10%) prevalence of apoptotic cells at 14 days post transplantation. Paracrine mechanism for CS mode of action is suggested due to the fact, that ww found vessel growth within CS mass, which indicates possibility of cells’ nutrition and uptake of secreted proteins from the site of transplantation besides diffusion.Thus, our findings indicate, that CS-based delivery for treatment of MI or PAD is a potential method in regenerative medicine in this field and furthermore genetic modification of cells used for CS generation may be a way to “tune up” the cells’ paracrine activity and efficacy of their application.