Hedgehog signaling is operative in differentiating ES cells and is involved in the blockade of pancreatic fate acquisition and maintenance

Transplantation of pancreatic islets of Langerhans has proven effective in setting diabetics insulin-free for periods as long as 2 years. Although very promising, this treatment cannot be generally applied because of islet donor shortage. In view of finding alternatives to shortened cadaveric islets, several attempts to generate pancreatic epithelial cells from other sources including embryonic stem (ES) cells were initiated, but have remained far from producing mature and functional beta-cells in vitro. Relying on known developmental features of the pancreas, we recently showed that the transcriptome of differentiating ES cells in embryoid bodies (EBs) is incompatible with derivation of pancreatic cells. Our present data confirm this hypothesis, indicating that exposure of mouse embryonic pancreas explants to soluble factors from EBs inhibits growth, morphogenesis, endocrine and exocrine differentiation as evaluated by explant size, mRNA and protein expression. Shh, an established pancreas repressor both at early and late developmental stages was produced and secreted by EBs, and participated in this effect by inducing hedgehog targets Ptc1 and Gli1 in the explants. This effect of hedgehog signaling could be only partially blocked by high concentrations of hedgehog interacting protein. In addition, definitive endoderm induction from ES cells further increased hedgehog production and activity in EBs, leading to down-regulation of the pancreas marker Pdx1. These data strongly suggest that hedgehog production in EBs limits pancreatic fate acquisition and forms a major obstacle in differentiating ES-derived definitive endoderm cells to pancreatic cells.