The Patterning of Pancreatic Progenitors Is Regulated by the Temporal Control of NOTCH Signaling

Pancreatic development occurs through the specification of three main lineages from a single progenitor cell. Previous studies have shown the importance of NOTCH signaling in both endocrine and exocrine differentiation during pancreatic development. Here we use the directed differentiation of pluripotent cells to demonstrate that the duration a culture remains in a NOTCH engaged progenitor state contributes to downstream lineage specification. Short-duration Pancreatic Progenitor (PP) cultures that were exposed to NOTCH inhibition showed significant up regulation of the Pro-endocrine genes NGN3, NKX2.2 and NEUROD with a corresponding activation of the endocrine genes INS, GCG and SST. The expression levels of these genes were greatly blunted when NOTCH inhibition of the PP culture was delayed. Cultures that were kept at the PP state lost endocrine competence when challenged by NOTCH inhibition, and displayed patterning towards exocrine lineages (DUCT/ACINAR) as evident through the up-regulation of CPB1, MIST1 and ONECUT1. Terminal differentiation of prolonged PP culture, adopted a ductal phenotype as evident through increased expression of F3, HNF1B and PROM1. Using the PP population in subsequent DoE based computer modeling experiments to optimize for either INS, MIST1 or F3 expression demonstrated that NKX6.1 was significantly up-regulated in short term PP cultures optimized for insulin expression as compared to the delayed cultures. Acinar conditions achieved through optimization for MIST1 expression displayed highly increased PTF1A expression. Finally, PP cultures that were maintained in the absence NOTCH inhibition and subsequently optimized for F3 expression displayed increased HNF1B and DBA expression. Altogether this suggests that the lineage specification within the pancreatic field of pluripotent derived cells is partially dictated by the duration a cell spends in a NOTCH active state, confirming findings of the embryonic murine developing organ.