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 problem, the ultimate goal of our research is to develop expanded populations of human beta cells or beta-cell 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 could then be used for cell transplantation therapies for diabetes. Two approaches are being pursued to expand human beta-cells and endocrine cell precursors in vitro. 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. Following the expansion of primary cells, they must be removed from the ECM and aggregated into islet-like cell clusters (ICCs). However, detachment of the expanded primary cells from the matrix results in a form of apoptosis known as anoikis. For the beta-cell line expressing the SV40 T antigen and H-ras Val12 dominant oncogenes, removal of the oncogenes using the cre-lox recombinase system, prior to aggregation into ICCs, results in rapid and efficient apoptosis. Thus, apoptosis has been encountered in two distinct situations in attempts to expand human beta cells and beta-cell precursors in culture. In this proposal, approaches to inhibition apoptosis using inhibitors of nitric oxide and adenoviral vectors expressing anti-apoptotic genes will be explored. The effects of the anti-apoptotic genes on transplantation of primary, non-expanded cultures of human pancreatic endocrine cells will also be studied. These studies are important to the development of expanded populations of human beta cells and beta-cell precursors for cell transplantation therapies for diabetes.
