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Insulin-producing cells derived from embryonic stem cells could be surrogates for beta cells in diabetes therapy. However, their derivation remains hard to achieve with current protocols which rely on initial embryoid body formation. We assume that factors known to inhibit pancreas development contribute to this limitation in vitro. To evaluate this hypothesis, embryoid bodies were examined after different culture periods by real time RT-PCR to profile the expression of genes known to regulate embryonic pancreas development. Our data indicate that transcripts for pancreas markers (insulin, glucagon and amylase ) were expressed during differentiation, but the highest levels achieved were at least 10(5) times lower than in the adult mouse pancreas. Notch signalling was activated as suggested by Delta, Jagged, Ngn3 and NeuroD1 profiles. However, Sonic hedgehog, a known inhibitor of pancreas induction in vivo drastically increased in day 6 embryoid bodies, while Inhibin betaA and betaB were down-regulated and follistatin up-regulated. Members of the Fibroblast- and Transforming Growth Factor families which pattern the endoderm were expressed at low levels, while those that inhibit pancreas development were highly transcribed. The profile of pancreas regulators expressed in embryoid bodies is therefore not compatible with differentiation of pancreatic and insulin-producing cells. These findings provide an explanation for the limited derivation of such cells to date, in addition to basic information for establishing novel differentiation protocols.

Expression of regulatory genes for pancreas development during murine embryonic stem cell differentiation