To harness the therapeutic potential of stem cells for the treatment of diabetes novel technologies will need to be developed. First, stem cells will be have to be isolated and expanded. Second, the stem cells will have to undergo directed and controlled differentiation toward the beta-cell lineage. Finally, fully functional beta-cells must be isolated from the resulting complex mixes of cells. In this application, we propose to develop reagents that will assist in the further development of all three of these steps. We will generate a comprehensive panel of monoclonal antibodies directed against cell surface epitopes of murine, primate and human pancreatic cells. These antibodies will permit the fractionation of pancreatic cell suspensions using fluorescence activated cell sorting and/or magnetic bead panning. This approach can be used to isolate pancreatic stem cells (step 1) as well as for the purification of differentiated progeny before therapeutic transplantation (step 3). In addition, this proposal is designed to identify the network of genes controlled by the pancreatic transcription factor, PDX-1. Evidence suggests that the family of protein-coding genes under the control of PDX-1 defines many of the phenotypic characteristics of differentiated beta cells. Although a few of these genes have been identified, the full constellation of PDX-1 responsive targets is unknown. We propose that PDX-1 also drives the expression of non-coding genes that are equally important for regulating beta cell function (steps 2&3). Definition of both families of targets is required for a complete understanding of beta cell biology and pathophysiology
