Differentiation of Mouse Embryonic Stem Cells Toward Functional Pancreatic beta-Cell Surrogates Through Epigenetic Regulation of Pdx1 by Nitric Oxide

Abstract

Pancreatic and duodenal homeobox 1 (Pdx1) is a transcription factor that regulates the embryonic development of the pancreas and the differentiation toward beta cells. Previously, we have shown that exposure of mouse embryonic stem cells (mESCs) to high concentrations of diethylenetriamine nitric oxide adduct (DETA-NO) triggers differentiation events and promotes the expression of Pdxl. Here we report evidence that Pdxl 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 markers of histones trimethylated histone H3 lysine 27 (H3K27me3) and H3K4me3, site-specific changes in DNA methylation, and no change in H3 acetylation. On the basis of these findings, we developed a protocol to differentiate mESCs toward insulin-producing cells consisting of sequential exposure to DETA-NO, valproic acid, and P300 inhibitor (C646) to enhance Pdxl 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.