The shortage of pancreatic Beta-cells exhibiting glucose-responsive insulin secretion has been a major obstacle to the development of widely applicable cell transplantation therapies for diabetes. To address this precursors. This would provide a large quantity of cells, grown in vitro, that retain glucose-responsive insulin secretion or the ability to differentiate and acquire this property. Such cells then be used for cell transplantation therapies for diabetes. Expanded populations of human Beta-cells and endocrine cell precursors can be grown in vitro using two distinct approaches. The first is to grow primary cells on extracellular matrix (ECM) in the presence of hepatocyte growth factor/scatter factor (HGF/SF). The second is to express dominant oncogenes in the cells, resulting in matrix and growth factor independent growth in vitro. However, upon growth stimulation by either method, the cells lose differentiated function, particularly insulin gene expression. This is not surprising given the inverse relationship between growth and differentiation that exists in many cell types, including the endocrine pancreas. Interestingly, the pattern of changes in differentiated function is very similar, regardless of whether growth is stimulated with oncogenes or with growth factor and extracellular matrix. We hypothesize that there are common signal transduction pathways that are activated by the oncogenes that we have used to create human Beta-cells to divide in culture. Furthermore, we hypothesize that these pathways converge to act on the transcription factors that determine the pattern of gene expression in Beta-cells, especially those that play a role in insulin gene expression. Therefore, the focus of this proposal is to determine the pattern of expression and to use retroviral vectors to express those factors in a series of human pancreatic endocrine cell lines and expanded populations of primary human Beta-cells.
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