This application addresses broad challenge area (14) Stem Cells and the specific challenge topic 14-EB-101 Synthetic Delivery Systems for Generating Pluripotent Stem Cells The recent advent of cell reprogramming as a means of producing induced pluripotent stem (iPS) cells from somatic cells has produced great excitement in the biological and medical communities due to its potential to circumvent the immunological and ethical issues surrounding traditional embryonic stem cells. For this technology to prove useful as a therapeutic and/or experimental tool one must first develop a technique that can reprogram cells in a rapid, efficient and repeatable manner. Most importantly, one must find alternatives to the potentially cancer-causing retroviruses that are currently used for this process. In this proposal, we request funding to further develop a current, proof of concept, cell injection prototype and to utilize it as an enabling tool to conduct reprogramming studies. The device is capable of delivering pico liters of materials across the cell membrane in a high throughput, efficient manner. The microfluidic system utilizes a micron-scale nozzle to inject a jet of liquid into cells passing through a channel. This jet pierces the cell membrane, without causing cell lysis, and is capable of delivering the factors necessary to reprogram somatic cells. By further developing this device, we hope to use the system to produce iPS cells at a rate and efficiency comparable to viral transfection, while avoiding the issues of mutagenesis and toxicity. The efficacy of micro-injector based cell reprogramming will be thoroughly tested using published methods of inspecting iPS cells. Tissue cultures produced by the system will also be used in animal studies for verification of pluripotency in reprogrammed cells. Eventually we will use the device in tandem with our novel polymeric delivery mechanism as a means of further enhancing performance. In addition, the quantitative, high throughput nature of the device will allow us to conduct studies on the biological aspects of the reprogramming process itself. More specifically, we can determine what optimal combination of genes and factors will result in maximum reprogramming efficiency as well as the specific role of individual genes in the overall process. Due to the physical nature of the delivery system, our device can also explore the use of proteins as a means of enhancing reprogramming (or even replacing DNA) in the production of iPS cells. Over the past years, embryonic stem cells have revolutionized the field of regenerative medicine;their potential for producing entire organs and tackling genetic disorders, such as Multiple Sclerosis (MS), has made them a favorite among researchers. The cell injection device described herein will have the capability of producing stem cells in a rapid, safe and efficient manner by reprogramming a patient's existing adult cells. The system can thus not only circumvent the ethical and technical issues that surround embryonic stem cells, but also avoids many of the toxicity and cancer risks associated with existing virus-based reprogramming techniques.