Towards Developing a Reproducible Method for Coaxing Embryonic Stem Cells to Pancreatic Islet Cells

Lye T. Lock, Chemical and Biological Engineering, State University of New York at Buffalo, 1015 Furnas Hall, Buffalo, NY 14260 and Emmanuel (Manolis) S. Tzanakakis, Department of Chemical and Biological Engineering, State University of New York at Buffalo, 907 Furnas Hall, Buffalo, NY 14260.

Diabetes mellitus is the sixth leading cause of death in the US. Current pharmacological treatments and insulin administration can only control the progression of this disease. Islet transplantation has afforded significant benefits including prolonged liberation from frequent insulin injections. Yet, islet transplantation cannot be used widely for diabetes treatment due to the extremely limited availability of donor islets. Embryonic stem cells (ESCs) can be an inexhaustible source of islet cells for transplantation. Previously published protocols have been characterized by low differentiation efficiency and results have been difficult to reproduce.

In this study, we describe an ongoing effort to develop a robust and reproducible method for directing the fate of ESCs towards pancreatic islets. Although we have used mouse ESCs as a model system, our findings translate to hESCs without significant modifications. At the first stage of differentiation, cells are coaxed towards definitive endoderm (DE). To that end, we have used medium containing either low concentration of serum or no serum, along with ligands of the TGF-β and Wnt families. Mouse and hESCs undergo morphological and biochemical changes over 4-6 days in culture. Cells in the resulting population are positive for Sox17, Foxa2 and CXCR4 whereas the expression of pluripotency markers such as Oct3/4 and Nanog is significantly reduced. Marker expression is assessed by quantitative PCR, immunocytochemistry and flow cytometry. A protocol for differentiation of DE cells to posterior foregut and beyond will also be presented. Selection of the islet cells derived from ESCs will be performed via adenoviral delivery of transgenes encoding for fluorescing proteins (e.g. green fluorescent protein; GFP) under an islet-cell specific promoter. To that end, we have generated an adenovirus carrying the red fluorescence protein gene flanked by the insulin gene promoter. We also explore the application of our differentiation scheme in conjunction with large scale expansion of ESCs. Our system will contribute towards developing large-scale bioprocesses for production of islet cells from ESCs for diabetes therapies.