During human embryogenesis, the Islets of Langerhans develop from pancreatic ductules. This same developmental pattern is seen in other mammals as well. Loss of beta cells from this initial pancreatic endocrine cell population leads to insulin- dependent diabetes mellitus (IDDM, juvenile-onset, Ketosis- prone, Type I) which is normally managed by insulin treatment. One approach to the treatment of Type I diabetes could involve stimulation of beta cell regeneration and differentiation in adult pancreatic tissue. Although endocrine cell differentiation from pancreatic ducts can occur in the neonate, this developmental process is normally surpressed in adult mammals. Recent studies indicate that endocrine cells (alpha and beta) can be induced to differentiate under the appropriate in vitro conditions from pancreatic ducts isolated from adult male rats. The studies proposed in this grant application are designed to structurally and functionally characterize endocrine cell neogenesis and differentiation from pancreatic ducts using this in vitro model. In the present proposal, studies involve isolation and culture of rat and human pancreatic ducts. Initial experiments involving rat ducts, focus on the endocrine progenitor cells which reside within the ductal epithelium. These studies will characterize the initial stages in the formation of pancreatic islets using 1) autoradiography, 2) immunocytochemistry, 3) in situ hybridization of insulin and glucagon genetic probes, and 4) electron microscopy. Later studies will determine 1) if regenerated endocrine cells of rat ducts discharge hormones in response to secretagogues, and 2) if endocrine cell neogenesis from pancreatic ducts can be induced in vivo in adult male rats. Additional studies will determine if in vitro neogenesis of endocrine cells can be induced from pancreatic ducts isolated from the adult human pancreas. Final studies will screen a variety of proposed """"""""inducer"""""""" molecules for their ability to stimulate this process in cultured rat pancreatic ducts. Although beyond the scope of this three-year grant proposal, the long term goal of this work is to develop procedures by which endocrine cell neogenesis can be induced in Type I diabetics who have lost their initial beta-cell population.
