Generation and Functional Characterization of Human Pluripotent Stem Cell Lines That Report on INSULIN and GLUCAGON Expression

Abstract

Diabetes is characterized by high levels of blood glucose due to malfunction (Type II) or destruction (Type I) of the INSULIN producing beta-cells (β-) in the pancreatic Islets of Langerhans. INSULIN action in muscle and fat allows for rapid blood clearing after a meal. The INSULIN producing β-cells are one of many endocrine cell types present in the Islets. Most, notably, the product of alpha-cells (α-), GLUCAGON, has been shown to work as a secreted repressor of INSULIN action. Since Type I diabetic patients can benefit from transplants of cadaveric Islets, novel cell replacement therapies have focus on generating stem cell derived β-cells from human embryonic stem cells. Previous research has shown the feedback cycle of α- and β-cells is important for adequate glucose control and there have been issues with obtaining adequate numbers of Islets for cell replacement therapy, making stem cell derived pancreatic cells an attractive option for treatment. This study focused on a way to enhance the function of stem cell derived pancreatic β-cells through the addition of pancreatic α-cells for the treatment of Type I diabetes. This idea hinged on the creation of transgenic human embryonic stem cell lines that targeted the mCherry red fluorescent protein to the INSULIN and GLUCAGON genomic loci. These cells lines would then be differentiated individually toward the β- or α-cell lineage and sorted by mCherry fluorescence so that they could be reaggregated together in Islet-like organoids (miniature 3D in-vitro organ) and tested for changes in glucose response. The goal of the study was to test if the generation of α- and β-Islet organoids would enhance glucose stimulated INSULIN secretion (GSIS) in stem cell-derived β-cells.