Differentiation of mouse embryonic stem cells toward functional pancreatic β-cell surrogates through epigenetic regulation of Pdx1 by nitric oxide

Abstract Pdx1 (Pancreatic and duodenal homeobox 1) is a transcription factor that regulates the embryonic development of the pancreas and the differentiation towards beta cells. Previously, we have shown that exposure of mouse embryonic stem cells (mESC) to high concentrations of NO donor diethylenetriamine nitric oxide adduct (DETA-NO) triggers differentiation events and promotes the expression of Pdx1. Here we report evidence that Pdx1 expression is associated with release of Polycomb Repressive Complex 2 (PRC2) and P300 from its promoter region. These events are accompanied by epigenetic changes in bivalent marks of histone H3K27me3 and H3K4me3, site specific changes in DNA methylation, and no change in H3 acetylation. Based on these findings, we developed a protocol to differentiate mESC towards insulin producing cells consisting of sequential exposure to DETA-NO, valproic acid and P300 inhibitor (C646) to enhance Pdx1 expression and a final maturation step of culture in suspension to form cell aggregates. This small molecule- based protocol succeeds in obtaining cells that express pancreatic beta cell markers such as PDX1, INS1, GCK and GLUT2 and respond in vitro to high-glucose and KCl.