Induced pluripotent stem cells (iPSCs) are the most expected cellular sources for regenerative medicine and hold tremendous therapeutic potential. Many groups have shown that adult fibroblasts and epithelial cells can be reprogrammed to a pluripotent, ESC-like state by viral or plasmid vector-mediated delivery of a small number of reprogramming factors. However, the use of viral vectors or plasmid DNA poses the danger of tumorigenesis through potential DNA integration. Therefore, it is necessary to develop new delivery methods for the transient expression of relevant reprogramming factors. Here, we propose to develop alternative methods for iPSC generation without using virus or plasmid DNA, thus making it safer for medical application. In the first aim, we will test whether we can generate iPSCs using bacteria-mediated protein injection. We will utilize a highly efficient protein delivery machinery, called type III secretion system (T3SS), encoded by a non-cytotoxic strain of Pseudomonas aeruginosa. In the second aim, we will test whether chemicals inducing site-specific HDAC inhibition can trigger transient expression of endogenous reprogramming factors and thus facilitate iPSC generation. Here we explore this possibility by utilizing a class of small molecules called Pyrrole imidazole polyamides (PIPs), which can bind to DNA in a sequence specific manner. Development of such safer iPSC induction methods will make the clinical application of iPSCs one step closer to reality. The University of Florida contributes substantially to the local and regional economy. In 2008, UF created 2,525 jobs and recent studies have shown that UF contributes nearly $6 billion annually to Florida's economy. The university employs about 34,000 people directly on its main campus and via UF organizations, such as the Institute of Food and Agricultural Sciences, is responsible for the creation of 74,894 jobs statewide. The current proposal will create 2 new jobs and retain 4 existing jobs. PUBLIC HEALTH RELEVANCE: Induced pluripotent stem cells (iPSCs) are a highly anticipated cellular source for regenerative medicine and hold tremendous therapeutic potential. Presently, medical application of iPSCs is hampered by the danger of tumor formation. The current protocols utilizing viral or plasmid gene delivery methods, have a potential to damage the cellular genome permanently, and pose a risk for cancer initiation. Therefore, it is necessary to develop new delivery methods for the transient expression of appropriate reprogramming factors. Here we will attempt to overcome current limitations using (1) a highly efficient protein injection system and (2) chemicals to induce sequence specific gene reactivation. Development of such safer iPSC induction methods will make the clinical application of iPSCs one step closer to reality.