Of paramount importance for the development of cell therapies to treat 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, generation of insulin-producing neural lineages and uptake of exogenous insulin from medium limit most of these methods. These issues elucidate the difficulty in producing functional â-cells from ES cells. A more plausible approach is undertaken in this work, where the ES cell differentiation is directed through a process mimicking normal pancreatic development.

The pancreatic differentiation pathway was traced by first inducing endoderm formation by culturing the mouse ES cells on fibronectin-coated collagen gels. An initial step towards pancreatic commitment is the expression of the Pdx1 gene. The Pdx1 expression was induced to the endodermal cells by replating them 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 &alpha, &beta, &delta 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 could only be observed on inhibition of the notch signaling, which has been reported to shunt the pancreatic progenitors cells toward a duct fate (1) and prevent their maturation to endocrine hormone expressing cells. Further characterization of the ES cell derived pancreatic cells and optimization of the differentiation protocol is underway. This culture system will be useful in obtaining a renewable source of pancreatic &beta cells for diabetes treatment.

Reference (1)Notch signaling reveals developmental plasticity of Pax4(+) pancreatic endocrine progenitors and shunts them to a duct fate, Greenwood AL, Li S, Jones K, Melton DA., Mech Dev. 2007, 124 (2), 97.