The murine embryonic stem (ES) cell line, ES-D3 was used in this project as a model for (a) investigating controversial aspects of current protocols used for the derivation of (3 cell types from ES cells and (b) investigating the potential to regulate the differentiation of ES cells into endodermal derivatives 1 e 0-1 ike cells, using extra-cellular factors previously associated with various aspects of pancreatic development. Several groups have reported the derivation of insulin-expressing cell types from ES cells. Many believe that these insulin-positive cells were a result of insulin uptake from supplements used in the culture medium Others have suggested that the ES-denved insulin-expressing cells may be of a neuronal or extraembryonic lineage, rather than an endocrine lineage Using an adapted version of the Lumelsky protocol (Lumelsky et al, 2001), we demonstrated the induction of 3-dimensional clusters similar to those described in the original study, which expressed a range of pancreatic, extra-embryonic and neuronal transcripts. Insulin protein was detected in the differentiated clusters by ELISA. A novel method developed to detect insulin biosynthesis by the cells, and analysis of Cpeptide expression, indicated that this insulin is probably derived from the culture medium, not from the differentiated ES cell. Culture of ES cells in suspension leads to the formation of embryoid bodies (EBs). Treatment of these aggregates with retinoic acid (RA) was previously shown to induce endodermal differentiation We exposed RA_EBs to secondary treatment with agents associated with pancreatic endocrine differentiation, e g Activin A (AA), Sodium Butyrate (SB) and Betacellulin (BTC), with the aim of developing a novel protocol for the regulated derivation of insulin-expressing cell types. The resulting differentiated populations were analysed for transcript expression of a range of multi-lineage markers i e neuronal, myogenic, exocrine and endocrine pancreas, extra-embryonic and apoptotic markers. Immunohistochemistry methods were established and applied for the analysis of a selection of the chosen markers, following formal in-fixing and paraffinembedding. Applying 2-stage regulated differentiation protocols developedduring the course of the study, we demonstrated the derivation of an intermediate multi-potential population (RAJEBs) from undifferentiated ES cells that preferentially gave rise to pancreatic endocrine insulin-expressing cell types in the presence of SB and neuronal-like cell types in the presence of AA or BTC. Low passages of the MIN-6 insulinoma cell line are glucose-responsive whereas at high passage they become glucose non-responsive. This loss in the glucose stimulated insulin secretion (GSIS) phenotype correlates with changes in morphology, increased proliferation and increased alkaline phosphatase (AP) activity, suggesting that the loss of GSIS may be due to de-differentiation of the high passage MIN6 cells. To investigate this hypothesis further, gene expression differences between low passage and high passage MIN6 cells were investigated by microarray and real-time PCR (qPCR) analysis.