Type I diabetes is marked by a deficiency of endocrine β cells in the pancreatic islets of Langerhans. Daily injection of insulin is the current treatment for the disease. Because insulin injection cannot match the precise timing and dosing of physiological secretion of insulin by islets in response to hyperglycemia, severe side effects develop over time. Transplantation of islets represents a potential cure; however, limitations on the availability of cadaveric organs restrict this procedure to only a small percentage of patients. Pancreatic endocrine stem cells exist in the developing embryonic pancreas. The isolation, expansion, and differentiation of pancreatic endocrine stem cells would provide an unlimited source of islet cells for transplantation and treatment for type I diabetes. A potential source for endocrine stem cells is embryonic stem (ES) cells. Because of their unlimited proliferation and differentiation potentials, ES cells are considered an important source for cell therapies targeted to several diseases, including diabetes.There are two nonallelic insulin genes (insulin I and II) expressed in multiple sites in mice during development [1][2][3][4][5][6][7]
Abstract
A panel of genetic markers was used to assess the in vitro commitment of murine embryonic stem (ES) cells toward the endoderm-derived pancreas and to distinguish insulin-expressing cells of this lineage from other lineagessuch as neuron, liver, and yolk sac. There are two nonallelic insulin genes in mice. Neuronal cells express only insulin II, whereas the pancreas expresses both insulin I and II. Yolk sac and fetal liver express predominately insulin II, small amounts of insulin I, and no glucagon. We found that ES-derived embryoid bodies cultured in the presence of stage-specific concentrations of monothioglycerol and 15% fetal calf serum, followed by serum-free conditions, give rise to a population that expresses insulin I, insulin II, pdx-1 (a pancreas marker), and Sox17 (an endoderm marker). Immunohistochemical staining shows intracellular insulin particles, and its de novo production was confirmed by staining for C-peptide. Most, but not all, of the insulin + or C-peptide + cells coexpress glucagon, demonstrating a differentiation pathway to pancreas rather than yolk sac or fetal liver. Addition of β-cell specification and differentiation factors activin βB, nicotinamide, and exendin-4 to later-stage culture increased insulin-positive cells to 2.73% of the total population, compared with the control culture, which gave rise to less than 1% insulin-staining cells. These findings suggest that stepwise culture manipulations can direct ES cells to become early endocrine pancreas.