Development of an In Vitro Model to Target HIF1-alpha protein in Pancreatic beta Cells to Improve Engraftment in Patients with Type I Diabetes Mellitus

Type I Diabetes Mellitus is characterized by autoimmune-mediated destruction of insulin secreting beta cell and it affects approximately 2.3 million people in the United States. Long standing disease may lead to multi-organ complications due to poorly controlled levels of blood glucose. Transplantation of pancreatic islets from cadaveric donors is a promising therapy since it offers tight glucose control without major surgery. Currently, the donor pool cannot meet the demand and islets derived from embryonic stem cells can serve as a donor independent source for transplantation. A major setback of islet transplantation is insufficient blood supply which leads to low oxygen environment (hypoxia) and eventually to poor islet engraftment. HIF1-alpha protein is a master regulator of the cells’ response to low oxygen environment. When HIF1-alpha protein is stabilized, it up-regulates genes which induce the secretion of soluble factors, such as Vascular Endothelial Growth Factor (VEGF), that promote connection to blood supply (vascularization). We hypothesize that in-vitro transient down-modulation of HIF1-alpha key de-stabilizers pre-transplantation will promote vascularization and therefore improve post-transplantation engraftment of embryonic stem cell derived pancreatic beta cells.

An in-vitro model was developed to target key de-stabilizers of the HIF1-alpha protein (e.g. VHL, Prolyl Hydroxylases and FIH proteins) under hypoxic transplantation conditions. Using INS-1 cells, which serve as a model for stem cell derived beta cells, it was demonstrated that the HIF1-alpha protein was stabilized, as indicated by elevated levels of HIF1-alpha protein and secretion of the VEGF. Enhanced endothelial cell tube formation was observed due to soluble factors, such as VEGF, an indicator of higher vascularization potential. However, reduction in levels of PDX1 protein may suggest that insulin production was diminished. This may reduce graft ability to regulate blood glucose levels. Therefore, to improve transplantation outcome, it is important to understand the effects which transient down-modulating de-stabilizers of HIF1-alpha protein have on beta cells engraftment potential, as well as on insulin production.