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

ABSTRACT: In embryonic development, the pancreas and liver share developmental history up to the stage of bud formation. Therefore, we postulated that direct reprogramming of liver to pancreatic cells can occur when suitable transcription factors are overexpressed. Using a polycistronic vector we misexpress Pdx1, Ngn3, and MafA in the livers of NOD-SCID mice rendered diabetic by treatment with streptozotocin (STZ). The diabetes is relieved long term. Many ectopic duct-like structures appear that express a variety of ?-cell markers, including dense core granules visible by electron microscopy (EM). Use of a vector also expressing GFP shows that the ducts persist long after the viral gene expression has ceased, indicating that this is a true irreversible cell reprogramming event. We have recovered the insulin(+) cells by cell sorting and shown that they display glucose-sensitive insulin secretion. The early formed insulin(+) cells can be seen to coexpress SOX9 and are also labeled in mice lineage labeled for Sox9 expression. SOX9(+) cells are normally found associated with small bile ducts in the periportal region, indicating that the duct-like structures arise from this source. This work confirms that developmentally related cells can be reprogrammed by suitable transcription factors and also suggests a unique therapy for diabetes.

SUBMITTER: Banga A

PROVIDER: S-EPMC3458366 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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

Banga Anannya A Akinci Ersin E Greder Lucas V LV Dutton James R JR Slack Jonathan M W JM

Proceedings of the National Academy of Sciences of the United States of America 20120904 38

In embryonic development, the pancreas and liver share developmental history up to the stage of bud formation. Therefore, we postulated that direct reprogramming of liver to pancreatic cells can occur when suitable transcription factors are overexpressed. Using a polycistronic vector we misexpress Pdx1, Ngn3, and MafA in the livers of NOD-SCID mice rendered diabetic by treatment with streptozotocin (STZ). The diabetes is relieved long term. Many ectopic duct-like structures appear that express a ...[more]

PMID: 22949652

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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

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

Publications

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

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