A mouse embryonic stem cell line was generated which stably expressed the ngn3 transcription factor under the control of the Tet-On inducible system using knock-ins in the ROSA26 and the HPRT loci. The undifferentiated mouse embryonic stem cells were then differentiated into Embryoid Bodies in suspension culture and were either treated with Doxycycline to induce NGN3 expression or left untreated as a contol. Cells were harvested at 12 hours, 24 hours and 48 hours.

Identify direct targets of Ngn3 in ESC differentiation and identify genetic networks for endocrine pancreas specification.

Used directed mouse ESC differentiation combining the sequential induction of endoderm character, simultaneous induction of Ptf1a and Pdx1 expression, and Ngn3-inducible expression. A short protocol (4 stages) was used to generate pancreas-progenitor-like cells for Ngn3 target screening. Using Affymetrix 430A arrays, RNA from 3 biological replicates were compared from Ngn3-induced and un-induced cells at 12, 24 and 48 hours. A long protocol (5 stages) was used to evaluate the induction of Ngn3 expression on differentiation into pancreas endocrine progenitors based on RT-PCR, immunohistochemistry, and insulin release assays of differentiated embryoid bodies.

Induction of Ngn3 was rapid and reached saturation by the first (12hr) time point assayed. Known targets were up-regulated including NeuroD, Pax4, Nkx2.2, Ia1, and Dll1. Over 200 probe sets were regulated at all 3 time points. Specific signaling pathways identified were Wnt, integrin-mediated, and Notch. Known and novel targets (18) were validated by semi-quantitative RT-PCR.

Several components of the Notch pathway are directly regulated by Ngn3. Ngn3 may also regulate the formation of interstitial clusters of cells based on changes in cell adhesion, cell motility, cell membrane, and cytoskeleton correlating with Ngn3 induction. The combination of in vivo patterning signals and Ngn3 induction led to strong up-regulation of terminal endocrine markers, strong expression of hormones, and glucose-dependent insulin release suggesting that bona fide pancreas endocrine cells were generated.

• ES cell differentiation: 2800, 3365, 3425, 3668, 4216, 4314, 4574,
• Ngn3: 1110, 1470, 2520, 2800, 3021, 3100, 3425, 3733, 3795, 4195, 4374,