The long term goal of the studies in this proposal is to understand the biology of pancreatic ? cell like progenitors derived from embryonic stem (ES) cells, with the intent of potentially using these cells for clinical applications such as islet transplantation for type I diabetics. Of particular relevance to this application, we have developed a novel colony assay for ? like progenitors in vitro. This colony assay or culture system allows dissociated single cells to develop into insulin-expressing colonies after 8 days of culture in a semi-solid media. Such assay measures the function, rather than phenotype, of ? like progenitors. In addition, this assay can be used to quantitate the number of ? like progenitors or the insulin-expressing colony forming units (ICFUs) in a given population. With this tool available, we propose to study fundamental properties of ICFUs derived from murine ES cells. We hypothesize that ICFUs can be enriched by cell surface markers, are expandable in number, and can be further matured in vitro. The studies outlines in the proposal will 1) sort and enrich ICFUs using cell surface markers, 2) expand ICFUs using exogenous growth factors, and 3) derive mature insulin-expressing colonies from ICFUs. These studies may lead to the development of novel methods for producing large numbers of functional insulin-secreting cells for potential therapeutic applications, and the possible identification of multipotential pancreatic endocrine progenitors or stem cells in vitro. PROJECT NARRATIVE A novel in vitro clonogenic assay that measures the activities of pancreatic beta cell like progenitors will be used to study the biological properties of these cells. We will test whether the murine embryonic stem cell derived beta cell like progenitors can be enriched by cell surface markers, expanded by exogenous factors and matured into functional glucose-sensing insulin-secreting cells. These studies may lead to the development of efficient methods for cell replacement therapy for Type I diabetics, and the possible identification of multipotential pancreatic endocrine progenitors or stem cells in vitro.
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