Endothelial Progenitor and Mesenchymal Stem Cell–Derived Cells Persist in Tissue-Engineered Patch In Vivo: Application of Green and Red Fluorescent Protein–Expressing Retroviral Vector VIRNA L. SALES, M.D., 1 BRET A. METTLER, M.D., 1 MARCO LOPEZ-ILASACA, M.D., Ph.D., 2 JOHN A. JOHNSON, Jr., B.S., 1 AND JOHN E. MAYER, Jr., M.D. 1 ABSTRACT An unresolved question regarding tissue-engineered (TE) cardiac valves and vessels is the fate of the transplanted cells in vivo. We have developed a strategy to track the anatomic location of seeded cells within TE constructs and neighboring tissues using a retroviral vector system encoding green and red fluorescent proteins (GFPs and RFPs, respectively) in ovine circulating endothelial progenitor cells (EPCs) and bone marrow–derived mesenchymal stem cells (BMSCs). We demonstrate that stable transduction ex vivo with high-titer Moloney murine leukemia virus–based retroviral vector yields transduction effi- ciency of greater than 97% GFP + EPC– and RFP + mesenchymal stem cell (MSC)-derived cells. Cellular phenotype and transgene expression were also maintained through 25 subsequent passages. Using a ret- roviral vector system to distinguish our pre-seeded cells from tissue-resident progenitor cells and circu- lating endothelial and marrow-derived precursors, we simultaneously co-seeded 210 6 GFP + EPCs and 210 5 RFP + MSCs onto the TE patches. In a series of ovine pulmonary artery patch augmentation studies, transplanted GFP + EPC– and RFP + MSC-derived cells persisted within the TE patch 7 to 14 days after implantation, as identified using immunofluorescence. Analysis showed 81% luminal coverage of the TE patches before implantation with transduced cells, increasing to 96% at day 7 and decreasing to 67% at day 14 post-implantation. This suggests a temporal association between retroviral expression of progenitor cells and mediating effects of these cells on the physiological remodeling and maturation of the TE con- structs. To our knowledge, this is the first cardiovascular tissue-engineering in vivo study using a double- labeling method to demonstrate a direct evidence of the source, persistence, and incorporation into a TE vascular patch of co-cultured and simultaneously pre-seeded adult progenitor cells. INTRODUCTION R EPAIR OF MANY CONGENITAL CARDIAC DEFECTS is now possible early in life, but many defects involve mal- formations or absence of the pulmonary valve and main pulmonary artery (PA), which must be replaced. 1 Currently available replacement options include allografts, prosthetic valves and vascular grafts, and bioprosthetic valves. These replacements, however, suffer from limited durability and are subject to calcification, thrombosis, and lack of growth potential. 1 These limitations have led us to investigate the use of a TE approach to constructing living cardiac valves and blood vessels. The approach presented is based on seeding autologous cells onto biodegradable scaffolds. 1 Department of Cardiovascular Surgery, Children’s Hospital, Boston, Massachusetts. 2 Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts. Presented at the Joint Meeting of the Tissue Engineering Society International and European Tissue Engineering, Lausanne, Swit- zerland, October 10–13, 2004. TISSUE ENGINEERING Volume 13, Number 3, 2007 # Mary Ann Liebert, Inc. DOI: 10.1089/ten.2006.0128 525