Reprogramming of pancreatic exocrine cells towards a beta (β) cell character using Pdx1, Ngn3 and MafA

Abstract

Pdx1 (pancreatic and duodenal homeobox 1), Ngn3 (neurogenin 3) and MafA (v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A) have been reported to bring about the transdifferentiation of pancreatic exocrine cells to beta (β) cells in vivo. We have investigated the mechanism of this process using a standard in vitro model of pancreatic exocrine cells, the rat AR42j-B13 cell line. We constructed a new adenoviral vector encoding all three genes, called Ad-PNM (adenoviral Pdx1, Ngn3, MafA construct). When introduced into AR42j-B13 cells, Ad-PNM caused a rapid change to a flattened morphology and a cessation of cell division. The expression of exocrine markers is suppressed. Both insulin genes are up-regulated as well as a number of transcription factors normally characteristic of beta cells. At the chromatin level, histone tail modifications of the Pdx1, Ins1 (insulin 1) and Ins2 (insulin 2) gene promoters are shifted in a direction associated with gene activity, and the level of DNA CpG methylation is reduced at the Ins1 promoter. The transformed cells secrete insulin and are capable of relieving diabetes in streptozotocin-treated NOD-SCID (non-obese diabetic severe combined immunodeficiency) mice. However the transformation is not complete. The cells lack expression of several genes important for beta cell function and they do not show glucose-sensitive insulin secretion. We conclude that, for this exocrine cell model, although the transformation is dramatic, the reprogramming is not complete and lacks critical aspects of the beta cell phenotype.

In vivo reprogramming of Sox9 + cells in the liver to insulin-secreting ducts

“…Construction of the polycistronic construct Ad-PNM was described in ref. 42. Properties of the 2A sequence are described in ref.…”

Rejuvenating liver and pancreas through cell transdifferentiation

“…More recently, the same strategy has been employed in an in vitro model. Akinci et al [11] investigated the conversion of pancreatic exocrine cells into β cells in vitro using adenovirus mediated Ngn3, Pdx1 and Mafa. The authors successfully generated induced β cell-like cells with many shared properties.…”

Improved insulin-secreting properties of pancreatic islet mesenchymalstem cells by constitutive expression of Pax4 and MafA

“…Pdx1 determines the region of the primitive gut that will form the pancreas; Ngn3 provides endocrine lineage progression; and expression of Pax4, NeuroD1/beta2, Nkx2.2, and MafA leads to differentiation into mature beta cells (Bernardo, 2008). Various types of studies have aimed to reprogram different cell types into a beta-like state (Tang, 2006;Nishimura, 2009;Akinci, 2012).…”