A promising direction in cell therapeutic treatment of diabetes is the production of sufficient numbers of pancreatic endocrine cells that are functionally identical to primary islets. Mouse embryonic stem (ES) cells, having virtually unlimited replicative capacity and the potential to produce most of the differentiated cell types, have the promise of providing a viable solution. Many studies have reported the differentiation of insulin-producing cells from mouse ES cells and, more recently, from human ES cells. However, inefficiency of differentiation, low insulin content of the insulin-producing cells, a reliance on non-directed ES cell differentiation limits most of these methods. The current promising approach, which is also the focus of this work, is to direct the ES cell differentiation through a process mimicking normal pancreatic development.

The mouse ES cells are cultured on a monolayer, and the pancreatic differentiation path is traced by first inducing endoderm formation. The upregulation of the Pdx-1 gene marks an initial step in pancreatic commitment, which was induced by replating the endoderms on a thin layer of Matrigel. Retinoic Acid (RA), an inducer of pancreas development, when added in the in-vitro culture system in the early differentiation stage was found to enhance Pdx1 expression. Furthermore, inhibition of sonic hedgehog gene was found to have a synergistic effect in the upregulation of Pdx1 expression. The next phase of pancreatic development is endocrine cell specification, which is initiated by the expression of the pro-endocrine gene Ngn3. The Pdx1 positive progenitors, on further maturation, were found to strongly express Ngn3, hence confirming their endocrine commitment. The Ngn3 gene, which is expressed in all endocrine progenitors, initiates a cascade of transcription factors, including Nkx2-2, NeuroD, Nkx6-1, Pax4 and Isl1. All these genes were positively expressed in the ES cell derived endocrine progenitor population. The final stage of the maturation of pancreatic cells is in the formation of islets of Langerhans, consisting of α, β, δ and PP cells, which produce the hormones glucagons, insulin, somatostatin and pancreatic polypeptide, respectively. With further maturation of the ES cell derived endocrine progenitor cells in an appropriate media, gene expression of all these hormones were detected, with the exception of insulin. Strong insulin expression was induced by coculturing the pancreatic progenitor cells with endothelial cells. Although there has been some evidence of the role of endothelial cells during the in-vivo maturation of pancreatic islets, this is the first report demonstrating the effect of endothelial cells in in-vitro differentiating pancreatic β cells. This culture system will be useful in obtaining a renewable source of pancreatic β cells for diabetes treatment.