Furthermore, a decrease in the markers expressed in other germ layers would suggest that a particular protocol is able to enrich the cell population with definitive endodermal cells. In this study, RA and DBcAMP appeared to induce production of definitive endoderm from ES cells when they were cultured with serum and bFGF for an extended period of time as evidenced by an expression of Sox 17 and Foxa 2. Also, there were minimal levels of Sox 1 and brachyury suggesting that there was no significant production of neuroectodermal and mesodermal tissues. This observation is in keeping with previous studies indicating that RA plays an important role in the development of the murine dorsal pancreas and induces production of PDX-1 positive endoderm in an ES cell differentiation model that involves EB formation. Further evidence that RA facilitates this differentiation process is the observation that Fibroblast growth factor 2 binds transmembrane fibroblast growth factor receptors and plays a key role in mouse embryogenesis, including development of the pancreas and liver. FGF2 signaling has also been suggested to play an important role in development of the exocrine pancreas and in Moexipril HCl maintenance of adult beta cell function. In liver development, FGF2 signaling from the cardiac mesoderm induces hepatogenesis, and furthermore, FGF2 signaling appears to stimulate expression of sonic hedgehog which in turn inhibits pancreatic development. FGF2 has been shown to be important in pancreatic development and maintenance of beta cell function, suggesting that FGF2 may promote pancreatic differentiation. In a separate experiment, exposing the cells to FGF2 for a shorter period of time resulted in lower insulin content in cells. During normal development, mature beta cells develop after formation of definitive endoderm, and the glucose responsive insulin release is a feature of mature beta cells. The expression of definitive endoderm markers increased throughout the four steps of the protocol. In addition,Miglustat the expression levels of several markers indicative of pancreas differentiation, including Nkx6.1, Pax6 and insulin I, also progressively increased whereas the expression of Pdx-1, a transcriptional regulator that plays critical roles in pancreas specification and development, as well as beta cell function appeared to peak at step 3 and then decreased during step 4. Collectively, these data suggest that the differentiation protocol promoted the progressive maturation of ES cells towards definitive endoderm and pancreatic lineage. The expression of Nkx6.1, PAX 6, Ngn3, IAPP, IGRP, CPE, Insulin I, C-peptide and PDX-1 in the cell population suggests that a subpopulation of cells in Step 4 were endocrine cell precursors. Furthermore, Step 4 cells also contained a small subset of mature glucose responsive insulin producing cells as supported by the immunohistochemistry and insulin release data. In summary, the ES cell differentiation protocol described in this study resulted in the generation of cells that displayed characteristics of definitive endoderm. There is a production of a small number of heterogeneous population of cells that express characteristics of pancreatic tissues; however, most cells appear to be in the pancreatic endocrine precursor stage. The insulin production from the cells in this protocol is not significant and further analysis is required to elucidate the nature and the origin of the insulin producing cells. Studies are underway to assess the efficiency of the protocol for endoderm production.