EXCEED THE SPACE PROVIDED. Diabetes mellitus, whether IDDM or NIDDM results from an inadequate mass of functional pancreatic (3-cells. New therapies of beta cell replacement may come from knowledge of how to stimulate growth and differentiation of islet cells. Since adult ductal epithelium retains the ability to differentiate and form new acini and islets of Langerhans, many assume that stem cells remain in the adult ducts. However, based on our data from the partial pancreatectomy (Px) rat model of pancreatic regeneration, we hypothesize that most or all the duct cells are capable of serving as precursor cells and are thus facultative stem cells, that by replication a mature duct cell can revert to a less differentiated cell that can then respond to external signals and differentiate into islet cells, acinar or, again, ducts. Indeed, we have found that islet cells can be cultivated in vitro from expanded adult human duct epithelium by manipulating the culture conditions. Over the 3-4 weeks culture, there is a 10 -15 fold increase of insulin content per flask, 3 -7 fold increase in DNA content and the formation of 3 dimensional structures of ductal cysts from which 50-150 (J.mdiameter islet-like clusters of pancreatic endocrine cells budded. This in vitro expansion and differentiation of duct cells allows manipulation and analysis of the molecular mechanisms involved in the differentiation process. Here strategies to optimize both the expansion and differentiation of human ductal epithelium will be tested. Gene expression profiles of the stages of differentiation from mature duct to pluripotent cell to islet cell will be defined using multiplex RT- PCR for known markers (genes) of pancreatic differentiation and differential expression techniques (e.g. differential display and cDNA microarrays) for unknown genes. The external signals most epithelial cells rely on for initiation and maintenance of their differentiated phenotype are cell-cell and cell matrix interactions and growth factors/cytokines. We hypothesize that changes in the expression of matrix binding proteins and matrix proteins drive the ductal expansion and redifferentiation in vivo.Using the in vivorat Px model, hypotheses based on changes on the components of the extracellular matrix surrounding the ducts and their regulation by HGF and TGF (3 will be tested. These complementary in vitro human and in vivo rat systems will allow biologically relevant characterization of regulation of the duct precursor cells in the adult pancreas and thus should advance our long term goal of being able to generate more P cells. 'ERFORMANCE SITE(S) (organization, city, state) Elliot P Joslin Research Laboratories, Joslin Diabetes Center, Boston MA. KEY PERSONNEL ========================================Section End===========================================
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