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

Pancreatic and duodenal homeobox 1 (Pdx1) is a transcription factor that regulates the embryonic development of the pancreas and the differentiation toward β 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 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 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 Pdx1 expression and a final maturation step of culture in suspension to form cell aggregates. This small moleculebased protocol succeeds in obtaining cells that express pancreatic b-cell markers such as PDX1, INS1, GCK, and GLUT2 and respond in vitro to high glucose and KCl.This study was supported by grants from Consejería de Igualdad, Salud y Políticas Sociales, Junta de Andalucía (PI105/2010), and Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía (CTS7127/2011) to F. J. Bedoya; from Consejería de Igualdad, Salud y Políticas Sociales, Junta de Andalucía, ISCIII cofunded by Fondos FEDER (RED-TERCEL; Nos. RD06/0010/0025, RD12/0019/0028, and PI10/00964), Consejería de Economía, Innovación, Ciencia y Empleo (P10.CTS.6505), and the Ministry of Health and Consumer Affairs (Advanced Therapies Program Grant TRA-120) to B. Soria; from Consejería de Igualdad, Salud y Políticas Sociales (PI0022/2008) and from Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía (PAI, BIO311) to F. Martín; and from Servicio Andaluz de Salud (SAS 11245) and Ministerio de Economía y Competitividad Secretaría de Estado de Investigación Desarrollo e Innovación (IPT-2011-1615-900000) to J. R. Tejedo.Peer Reviewe