Growth regulation and differentiation in the human pancreatic beta cell

This dissertation is an exploration of mechanisms controlling cell proliferation and the relationship between growth and differentiation in the human endocrine pancreas. A major impetus for my interest in this field is the need for large numbers of beta cells or beta cell precursors that can be used for cell transplantation for diabetes. My work focuses on the study and application of three distinct approaches to generating transplantable material for this purpose: (1) Expansion of primary endocrine cells harvested from cadaveric pancreases, (2) characterization and modification of an existing transformed human pancreatic cell line, and (3) design and implementation of new strategies to develop novel cell lines from pancreatic islet cells. My results can be summarized as follows:; Primary human fetal and adult islet cells can be stimulated to divide in vitro by hepatocyte growth factor (HGF), but their proliferative capacity is severely limited by rapid progression to senescence and is not extended by the induction of telomerase activity. SV40 large T (SVLT)/Rasval12 -transformed pancreatic cell lines escape senescence and can be immortalized by telomerase. However, these cell lines lack differentiated functions and are highly tumorigenic in nude mice. Implementation of the Cre/lox system resulted in the generation of transformed cell lines from which the oncogenes can be removed with close to 99% efficiency. Oncogene excision abrogated tumor formation and increased responsiveness to differentiating stimuli. Most importantly, transplantation of aggregates of oncogene-deleted cells into nude mice resulted in glucose-responsive insulin secretion. Unfortunately, some transformation-associated changes are irreversible, such as aneuploidy and chromosomal rearrangements. Although telomere stabilization by induction of telomerase activity improves genomic stability in non-transformed cells, telomere elongation does not prevent the development of aneuploidy in SVLT-expressing cells. Therefore, the last part of my dissertation focuses on alternative strategies to generate cell lines with fewer or less disruptive oncogenic activities. The most successful cell lines were generated by expressing the human papilloma virus (HPV) 16 E7 gene in combination with RasVal12 and telomerase. E7RT cells retained more primary cell-like characteristics and more endocrine markers than SVLT/Ras lines. Genomic stability and transplantation studies are ongoing.