Temporal Restriction of Pancreatic Branching Competence During Embryogenesis Is Mirrored In Differentiating Embryonic Stem Cells

To develop methods for the generation of insulin-producing β-cells for the treatment of diabetes, we have used GFP-tagged embryonic stem cells (ESCs) to elucidate the process of pancreas development. Using the reporter Pdx1GFP/w ESC line, we have previously described a serum-free differentiation protocol in which Pdx1-GFP+ cells formed GFP bright (GFPbr) epithelial buds that resembled those present in the developing mouse pancreas. In this study we extend these findings to demonstrate that these cells can undergo a process of branching morphogenesis, similar to that seen during pancreatic development of the mid-gestation embryo. These partially disaggregated embryoid bodies containing GFPbr buds initially form epithelial ring-like structures when cultured in Matrigel. After several days in culture, these rings undergo a process of proliferation and form a ramified network of epithelial branches. Comparative analysis of explanted dissociated pancreatic buds from E13.5 Pdx1GFP/w embryos and ESC-derived GFPb...