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The adult pancreatic ductal system was suggested to harbor facultative beta-cell progenitors similar to the embryonic pancreas, and the appearance of insulin-positive duct cells has been used as evidence for natural duct-to-beta-cell reprogramming. Nevertheless, the phenotype and fate of these insulin-positive cells in ducts have not been determined. Here, we used a cell-tagging dye, CFDA-SE, to permanently label pancreatic duct cells through an intraductal infusion technique. Representing a time when significant increases in beta-cell mass occur, pregnancy was later induced in these CFDA-SE-treated mice to assess the phenotype and fate of the insulin-positive cells in ducts. We found that a small portion of CFDA-SE-labeled duct cells became insulin-positive, but they were not fully functional beta-cells based on the in vitro glucose response and the expression levels of key beta-cell genes. Moreover, these insulin-positive cells in ducts expressed significantly lower levels of genes associated with extracellular matrix degradation and cell migration, which may thus prevent their budding and migration into preexisting islets. A similar conclusion was reached through analysis of the Gene Expression Omnibus database for both mice and humans. Together, our data suggest that the contribution of duct cells to normal beta-cells in adult islets is minimal at best.

Chronic pancreatitis affects over 250,000 people in the US and millions worldwide. It is associated with chronic debilitating pain, pancreatic exocrine failure, and high risk of pancreatic cancer and usually progresses to diabetes. Treatment options are limited and ineffective. We developed a new potential therapy, wherein a pancreatic ductal infusion of 1%-2% acetic acid in mice and nonhuman primates resulted in a nonregenerative, near-complete ablation of the exocrine pancreas, with complete preservation of the islets. Pancreatic ductal infusion of acetic acid in a mouse model of chronic pancreatitis led to resolution of chronic inflammation and pancreatitis-associated pain. Furthermore, acetic acid-treated animals showed improved glucose tolerance and insulin secretion. The loss of exocrine tissue in this procedure would not typically require further management in patients with chronic pancreatitis because they usually have pancreatic exocrine failure requiring dietary enzyme supplements. Thus, this procedure, which should be readily translatable to humans through an endoscopic retrograde cholangiopancreatography (ERCP), may offer a potential innovative nonsurgical therapy for chronic pancreatitis that relieves pain and prevents the progression of pancreatic diabetes.

A key question in diabetes research is whether new β-cells can be derived from endogenous, nonendocrine cells. The potential for pancreatic ductal cells to convert into β-cells is a highly debated issue. To date, it remains unclear what anatomical process would result in duct-derived cells coming to exist within preexisting islets. We used a whole-mount technique to directly visualize the pancreatic ductal network in young wild-type mice, young humans, and wild-type and transgenic mice after partial pancreatectomy. Pancreatic ductal networks, originating from the main ductal tree, were found to reside deep within islets in young mice and humans but not in mature mice or humans. These networks were also not present in normal adult mice after partial pancreatectomy, but TGF-β receptor mutant mice demonstrated formation of these intraislet duct structures after partial pancreatectomy. Genetic and viral lineage tracings were used to determine whether endocrine cells were derived from pancreatic ducts. Lineage tracing confirmed that pancreatic ductal cells can typically convert into new β-cells in normal young developing mice as well as in adult TGF-β signaling mutant mice after partial pancreatectomy. Here the direct visual evidence of ducts growing into islets, along with lineage tracing, not only represents strong evidence for duct cells giving rise to β-cells in the postnatal pancreas but also importantly implicates TGF-β signaling in this process.

Genetic manipulations, with or without lineage tracing for specific pancreatic cell types, are very powerful tools for studying diabetes, pancreatitis and pancreatic cancer. Nevertheless, the use of Cre/loxP systems to conditionally activate or inactivate the expression of genes in a cell type- and/or temporal-specific manner is not applicable to cell tracing and/or gene manipulations in more than one lineage at a time. Here we report a technique that allows efficient delivery of dyes for cell tagging into the mouse pancreas through the duct system, and that also delivers viruses carrying transgenes or siRNA under a specific promoter. When this technique is applied in genetically modified mice, it enables the investigator to perform either double lineage tracing or cell lineage tracing combined with gene manipulation in a second lineage. The technique requires <40 min.

Aims/hypothesis: Vascular endothelial growth factor (VEGF) is essential for proper pancreatic development, islet vascularisation and insulin secretion. In the adult pancreas, VEGF is thought to be predominantly secreted by beta cells. Although human duct cells have previously been shown to secrete VEGF at angiogenic levels in culture, an analysis of the kinetics of VEGF synthesis and secretion, as well as elucidation of an in vivo role for this ductal VEGF in affecting islet function and physiology, has been lacking., Methods: We analysed purified duct cells independently prepared by flow cytometry, surgical isolation or laser-capture microdissection. We infected duct cells in vivo with Vegf (also known as Vegfa) short hairpin RNA (shRNA) in an intrapancreatic ductal infusion system and examined the effect of VEGF knockdown in duct cells in vitro and in vivo., Results: Pancreatic duct cells express high levels of Vegf mRNA. Compared with beta cells, duct cells had a much higher ratio of secreted to intracellular VEGF. As a bioassay, formation of tubular structures by human umbilical vein endothelial cells was essentially undetectable when cultured alone and was substantially increased when co-cultured with pancreatic duct cells but significantly reduced when co-cultured with duct cells pretreated with Vegf shRNA. Compared with islets transplanted alone, improved vascularisation and function was detected in the islets co-transplanted with duct cells but not in islets co-transplanted with duct cells pretreated with Vegf shRNA., Conclusions/interpretation: Human islet preparations for transplantation typically contain some contaminating duct cells and our findings suggest that the presence of duct cells in the islet preparation may improve transplantation outcomes.

Specific labeling of pancreatic ducts has proven to be quite difficult. Such labeling has been highly sought after because of the power it would confer to studies of pancreatic ductal carcinogenesis, as well as studies of the source of new insulin-producing β-cells. Cre-loxp recombination could, in theory, lineage-tag pancreatic ducts, but results have been conflicting, mainly due to low labeling efficiencies. Here, we achieved a high pancreatic duct labeling efficiency using a recombinant adeno-associated virus (rAAV) with a duct-specific sox9 promoter infused into the mouse common biliary/pancreatic duct. We saw rapid, diffuse duct-specific labeling, with 50 and 89% labeling in the pancreatic tail and head region, respectively. This highly specific labeling of ducts should greatly enhance our ability to study the role of pancreatic ducts in numerous aspects of pancreatic growth, development and function.

It remains controversial whether adult pancreatic ducts harbor facultative beta cell progenitors. Because neurogenin3 (Ngn3) is a key determinant of pancreatic endocrine cell neogenesis during embryogenesis, many studies have also relied upon Ngn3 expression as evidence of beta cell neogenesis in adults. Recently, however, Ngn3 as a marker of adult beta cell neogenesis has been called into question by reports of Ngn3 expression in fully-developed beta cells. Nevertheless, direct evidence as to whether Ngn3 activation in adult pancreatic duct cells may lead to duct-to-beta cell transdifferentiation is lacking. Here we studied two models of Ngn3 activation in adult pancreatic duct cells (low-dose alloxan treatment and pancreatic duct ligation) and lineage-traced Ngn3-activated duct cells by labeling them through intraductal infusion with a cell-tagging dye, CFDA-SE No dye-labeled beta cells were found during the follow-up in either model, suggesting that activation of Ngn3 in duct cells is not sufficient to direct their transdifferentiation into beta cells. Therefore, Ngn3 activation in duct cells is not a signature for adult beta cell neogenesis.

Confocal microscopy in combination with commercial software is frequently used to generate three-dimensional images of tissue architecture. Here we report a novel, whole-mount imaging protocol technique that allows detailed three-dimensional imaging of adult pancreatic structures. This technique provides an improved appreciation of the anatomical detail of pancreatic structures and of the relationship between the pancreatic ducts and islets. In addition, imaging of the pancreatic ducts revealed a previously unappreciated high degree of hypervascularity., (Copyright © 2012 Wiley Periodicals, Inc.)

Background & Aims: There have been conflicting results on a cell of origin in pancreatic regeneration. These discrepancies predominantly stem from lack of specific markers for the pancreatic precursors/stem cells, as well as differences in the targeted cells and severity of tissue injury in the experimental models so far proposed. We attempted to create a model that used diphtheria toxin receptor (DTR) to ablate specific cell populations, control the extent of injury, and avoid induction of the inflammatory response., Methods: To target specific types of pancreatic cells, we crossed R26DTR or R26DTR/lacZ mice with transgenic mice that express the Cre recombinase in the pancreas, under control of the Pdx1 (global pancreatic) or elastase (acinar-specific) promoters., Results: Exposure of PdxCre;R26DTR mice to diphtheria toxin resulted in extensive ablation of acinar and endocrine tissues but not ductal cells. Surviving cells within the ductal compartment contributed to regeneration of endocrine and acinar cells via recapitulation of the embryonic pancreatic developmental program. However, following selective ablation of acinar tissue in ElaCreERT2;R26DTR mice, regeneration likely occurred by reprogramming of ductal cells to acinar lineage., Conclusions: In the pancreas of adult mice, epithelial cells within the ductal compartment contribute to regeneration of endocrine and acinar cells. The severity of injury determines the regenerative mechanisms and cell types that contribute to this process., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Epithelial-mesenchymal interactions are crucial for the proper development of many organs, including the pancreas. Within the pancreas, the ducts are thought to harbor stem/progenitor cells, and possibly to give rise to pancreatic ductal carcinoma. Little is known about the mechanism of formation of pancreatic ducts in the embryo. Pancreatic mesenchyme contains numerous soluble factors which help to sustain the growth and differentiation of exocrine and endocrine structures. Here, we report that one such morphoregulatory mesenchymal protein, epimorphin, plays an important role during pancreatic ductal proliferation and differentiation. We found that epimorphin is expressed in pancreatic mesenchyme during early stages of development, and at mesenchymal-epithelial interfaces surrounding the ducts at later stages. Strong upregulation of epimorphin expression was seen during in vitro pancreatic duct differentiation. Similarly, in vitro pancreatic duct formation was inhibited by a neutralizing antibody against epimorphin, whereas addition of recombinant epimorphin partially rescued duct formation. Together, our study demonstrates the role of epimorphin in pancreatic ductal morphogenesis.