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Plasticity of adult human pancreatic duct cells by neurogenin3-mediated reprogramming.

ABSTRACT: AIMS/HYPOTHESIS: Duct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3). In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it. METHODS: The extent of the Ngn3-mediated duct-to-endocrine cell reprogramming was measured employing genome wide mRNA profiling. By modulation of the Delta-Notch signaling or addition of pancreatic endocrine transcription factors Myt1, MafA and Pdx1 we intended to improve the reprogramming. RESULTS: Ngn3 stimulates duct cells to express a focused set of genes that are characteristic for islet endocrine cells and/or neural tissues. This neuro-endocrine shift however, is incomplete with less than 10% of full duct-to-endocrine reprogramming achieved. Transduction of exogenous Ngn3 activates endogenous Ngn3 suggesting auto-activation of this gene. Furthermore, pancreatic endocrine reprogramming of human duct cells can be moderately enhanced by inhibition of Delta-Notch signaling as well as by co-expressing the transcription factor Myt1, but not MafA and Pdx1. CONCLUSIONS/INTERPRETATION: The results provide further insight into the plasticity of adult human duct cells and suggest measurable routes to enhance Ngn3-mediated in vitro reprogramming protocols for regenerative beta cell therapy in diabetes.

SUBMITTER: Swales N

PROVIDER: S-EPMC3351393 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Plasticity of adult human pancreatic duct cells by neurogenin3-mediated reprogramming.

Swales Nathalie N Martens Geert A GA Bonné Stefan S Heremans Yves Y Borup Rehannah R Van de Casteele Mark M Ling Zhidong Z Pipeleers Daniel D Ravassard Philippe P Nielsen Finn F Ferrer Jorge J Heimberg Harry H

PloS one 20120514 5

<h4>Aims/hypothesis</h4>Duct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3). In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it.<h4>Methods</h4>The extent of the Ngn3-mediated duct-to-endocrine cell reprogramming was measured employing genome wide mRNA profiling. By modulation of the Delta-Notch signaling or a ...[more]

PMID: 22606327

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Project description:Aims/hypothesis: Duct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3). In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it. Methods: The extent of the Ngn3-mediated duct-to-endocrine cell reprogramming was measured employing genome wide mRNA profiling. By modulation of the Delta-Notch signaling or addition of pancreatic endocrine transcription factors Myt1, MafA and Pdx1 we intended to improve the reprogramming. Results: Ngn3 stimulates duct cells to express a focused set of genes that are abundant in islet endocrine cells and/or neural tissues. This neuro-endocrine shift, however, covers a minor fraction (5%) of the estimated genome-wide transcriptome difference between duct and islet endocrine cells. Interestingly, transduction of exogenous Ngn3 activates endogenous Ngn3 suggesting auto-activation of this gene. Furthermore, pancreatic endocrine reprogramming of human duct cells can be moderately enhanced by inhibition of Delta-Notch signaling as well as by co-expressing the transcription factor Myt1, but not MafA and Pdx1. Conclusions/interpretation: The results provide further insight into the plasticity of adult human duct cells and suggest measurable routes to enhance Ngn3-mediated in vitro reprogramming protocols for regenerative beta cell therapy in diabetes. We used Affymetrix HG133A and HG133B to get a comprehensive view on the reprogramming potential in vitro of human pancreatic duct cell cultures (n=3-4) at 3 and 14/20 days after ectopic adenoviral expression of murine neurogenin 3 as compared to GFP-expressing control vectors. The microarray analysis was performed on 3 independent samples that each contained RNA extracted from a pool of 3 independent donor pancreata. The total number of non-selected donor organs is 9. Transcripts were considered as differentially regulated by Ngn3 when 1.5 fold (LCB, unpaired P < 0.05) up- or down-regulated in AdGFP-Ngn3 versus AdGFP controls, at 3 and/or 14 dpi. Transcripts that showed differential expression between day 3 and day 14 in AdGFP-Ngn3 duct cells but not in AdGFP control cells, were also considered Ngn3-regulated

Project description:: β-Cell replacement therapy represents the most promising approach to restore β-cell mass and glucose homeostasis in patients with type 1 diabetes. Safety and ethical issues associated with pluripotent stem cells stimulated the search for adult progenitor cells with endocrine differentiation capacities. We have already described a model for expansion and differentiation of human pancreatic duct-derived cells (HDDCs) into insulin-producing cells. Here we show an innovative and robust in vitro system for large-scale production of β-like cells from HDDCs using a nonintegrative RNA-based reprogramming technique. Synthetic modified RNAs for pancreatic transcription factors (pancreatic duodenal homeobox 1, neurogenin3, and V-Maf musculoaponeurotic fibrosarcoma oncogene homolog A [MAFA]) were manufactured and daily transfected in HDDCs without strongly affecting immune response and cell viability. MAFA overexpression was efficient and sufficient to induce β-cell differentiation of HDDCs, which acquired a broad repertoire of mature β-cell markers while downregulating characteristic epithelial-mesenchymal transition markers. Within 7 days, MAFA-reprogrammed HDDC populations contained 37% insulin-positive cells and a proportion of endocrine cells expressing somatostatin and pancreatic polypeptide. Ultrastructure analysis of differentiated HDDCs showed both immature and mature insulin granules with light-backscattering properties. Furthermore, in vitro HDDC-derived β cells (called β-HDDCs) secreted human insulin and C-peptide in response to glucose, KCl, 3-isobutyl-1-methylxanthine, and tolbutamide stimulation. Transplantation of β-HDDCs into diabetic SCID-beige mice confirmed their functional glucose-responsive insulin secretion and their capacity to mitigate hyperglycemia. Our data describe a new, reliable, and fast procedure in adult human pancreatic cells to generate clinically relevant amounts of new β cells with potential to reverse diabetes.Significanceβ-Cell replacement therapy represents the most promising approach to restore glucose homeostasis in patients with type 1 diabetes. This study shows an innovative and robust in vitro system for large-scale production of β-like cells from human pancreatic duct-derived cells (HDDCs) using a nonintegrative RNA-based reprogramming technique. V-Maf musculoaponeurotic fibrosarcoma oncogene homolog A overexpression was efficient and sufficient to induce β-cell differentiation and insulin secretion from HDDCs in response to glucose stimulation, allowing the cells to mitigate hyperglycemia in diabetic SCID-beige mice. The data describe a new, reliable, and fast procedure in adult human pancreatic cells to generate clinically relevant amounts of new β cells with the potential to reverse diabetes.

Dataset Information

Plasticity of adult human pancreatic duct cells by neurogenin3-mediated reprogramming.

Publications

Plasticity of adult human pancreatic duct cells by neurogenin3-mediated reprogramming.

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