Generation of islet-like cells from mouse gall bladder by direct ex vivo reprogramming

Cell replacement is an emerging therapy for type 1 diabetes. Pluripotent stem cells have received a lot of attention as a potential source of transplantable beta-cells, but their ability to form teratomas poses significant risks. Here, we evaluated the potential of primary mouse gall bladder epithelial cells (GBCs) as targets for ex vivo genetic reprogramming to the beta-cell fate. Conditions for robust expansion and genetic transduction of primary GBCs by adenoviral vectors were developed. Using a GFP reporter for insulin, conditions for reprogramming were then optimized. Global expression analysis by RNA-sequencing was used to quantitatively compare reprogrammed GBCs (rGBCs) to true beta-cells, revealing both similarities and differences. Adenoviral-mediated expression of NEUROG3, Pdx1, and MafA in GBCs resulted in robust induction of pancreatic endocrine genes, including Ins1, Ins2, Neurod1, Nkx2-2 and Isl1. Furthermore, expression of GBC-specific genes was repressed, including Sox17 and Hest Reprogramming was also enhanced by addition of retinoic acid and inhibition of Notch signaling. Importantly, rGBCs were able to engraft long term in vivo and remained insulin-positive for 15 weeks. We conclude that GBCs are a viable source for autologous cell replacement in diabetes, but that complete reprogramming will require further manipulations. (C) 2013 Elsevier B.V. All rights reserved.