The Role of Fibroblast Growth Factor-2 in the Expansion of Pancreatic Islets IN VITRO

Ettinger, Eve Sari, The Role of Fibroblast Growth Factor-2 in the Expansion of Pancreatic Islets In Vitro. Doctor of Philosophy (Biomedical Sciences, Biochemistry and Molecular Biology), May 2004, 181 pp., 7 tables, 46 figures, 109 references. The focus of these studies was to identify a pool of progenitor cells within the adult islet, which can be expanded in vitro and aggregated into glucose responsive neoislets as a potential source for transplantation in diabetic patients. We hypothesize that normal adult islets contain an unspecialized proliferative progenitor cell population that can be expanded, aggregated by FGF-2 via PKC mechanism in 2-D culture or in a Rotating Wall Vessel Cell Culture System and matured into glucose-sensitive neoislets through the differential expression or activation of one or more PKC isoform(s). It was shown by immunofluorescence that normal adult islets from several species (rat, porcine, human and canine) contain proliferative endocrine precursor (endocrine epithelial) cells, which coexpress insulin, glucagon and somatostatin. These cells also express PDX-1, a marker of mature B cells and endocrine precursors as well as glut 2 and glucokinase, which are necessary components of glucose metabolism. Endocrine epithelial cells were shown to aggregate into neoislets either spontaneously in 2-D culture or via a rotating wall vessel cell culture system in the presence of fibroblast growth factor-2. FGF2 has been implicated in the aggregation of endocrine epithelial cells into neoislets via the FGFR-cadherin-catenin complex or through PKC signal transduction. Due to the variations in islet isolation, Ins-1 cells were used as a model of B cell aggregation. PKC profiles of endocrine epithelial cells, neoislets, native islets, Ins-1 cells and Ins-1 neoislets were determined. PKC-α, BII, ε and ζ were the only isoforms expressed in these cells and are potential targets for modulation in the differentiation of endocrine epithelial cells. Insulin secretion in response to a glucose challenge was examined in normal islets, neoislets, endocrine epithelial cells, and Ins-1 cells. Although endocrine epithelial cells express insulin, show dense secretory granules and contain two critical proteins in the glucose-sensing cascade, they are immature with respect to glucose-responsive insulin secretion. Neoislets show insignificant insulin secretion in response to elevated glucose when compared to normal islets. Aggregation of endocrine epithelial cells may be the first step in differentiation into mature β cells, however glucose-responsive insulin secretion must be achieved in vitro for use as an alternate source of islets for transplantation in diabetic patients.