Biliary atresia is associated with polygenic susceptibility in ciliogenesis and planar polarity effector genes.

BACKGROUND & AIMS: Biliary atresia (BA) is poorly understood and leads to liver transplantation (LT), with the requirement for and associated risks of lifelong immunosuppression, in most children. We performed a genome-wide association study (GWAS) to determine the genetic basis of BA. METHODS: We performed a GWAS in 811 European BA cases treated with LT in US, Canadian and UK centers, and 4,654 genetically matched controls. Whole-genome sequencing of 100 cases evaluated synthetic association with rare variants. Functional studies included whole liver transcriptome analysis of 64 BA cases and perturbations in experimental models. RESULTS: A GWAS of common single nucleotide polymorphisms (SNPs), i.e. allele frequencies >1%, identified intronic SNPs rs6446628 in AFAP1 with genome-wide significance (p = 3.93E-8) and rs34599046 in TUSC3 at sub-threshold genome-wide significance (p = 1.34E-7), both supported by credible peaks of neighboring SNPs. Like other previously reported BA-associated genes, AFAP1 and TUSC3 are ciliogenesis and planar polarity effectors (CPLANE). In gene-set-based GWAS, BA was associated with 6,005 SNPs in 102 CPLANE genes (p = 5.84E-15). Compared with non-CPLANE genes, more CPLANE genes harbored rare variants (allele frequency <1%) that were assigned Human Phenotype Ontology terms related to hepatobiliary anomalies by predictive algorithms, 87% vs. 40%, p <0.0001. Rare variants were present in multiple genes distinct from those with BA-associated common variants in most BA cases. AFAP1 and TUSC3 knockdown blocked ciliogenesis in mouse tracheal cells. Inhibition of ciliogenesis caused biliary dysgenesis in zebrafish. AFAP1 and TUSC3 were expressed in fetal liver organoids, as well as fetal and BA livers, but not in normal or disease-control livers. Integrative analysis of BA-associated variants and liver transcripts revealed abnormal vasculogenesis and epithelial tube formation, explaining portal vein anomalies that co-exist with BA. CONCLUSIONS: BA is associated with polygenic susceptibility in CPLANE genes. Rare variants contribute to polygenic risk in vulnerable pathways via unique genes. IMPACT AND IMPLICATIONS: Liver transplantation is needed to cure most children born with biliary atresia, a poorly understood rare disease. Transplant immunosuppression increases the likelihood of life-threatening infections and cancers. To improve care by preventing this disease and its progression to transplantation, we examined its genetic basis. We find that this disease is associated with both common and rare mutations in highly specialized genes which maintain normal communication and movement of cells, and their organization into bile ducts and blood vessels during early development of the human embryo. Because defects in these genes also cause other birth defects, our findings could lead to preventive strategies to lower the incidence of biliary atresia and potentially other birth defects.

ß-cell Smad2 null mice have improved ß-cell function and are protected from diet-induced hyperglycemia.

Understanding signaling pathways that regulate pancreatic ß-cell function to produce, store, and release insulin, as well as pathways that control ß-cell proliferation, is vital to find new treatments for diabetes mellitus. Transforming growth factor-beta (TGF-ß) signaling is involved in a broad range of ß-cell functions. The canonical TGF-ß signaling pathway functions through intracellular smads, including smad2 and smad3, to regulate cell development, proliferation, differentiation, and function in many organs. Here, we demonstrate the role of TGF-ß/smad2 signaling in regulating mature ß-cell proliferation and function using ß-cell-specific smad2 null mutant mice. ß-cell-specific smad2-deficient mice exhibited improved glucose clearance as demonstrated by glucose tolerance testing, enhanced in vivo and ex vivo glucose-stimulated insulin secretion, and increased ß-cell mass and proliferation. Furthermore, when these mice were fed a high-fat diet to induce hyperglycemia, they again showed improved glucose tolerance, insulin secretion, and insulin sensitivity. In addition, ex vivo analysis of smad2-deficient islets showed that they displayed increased glucose-stimulated insulin secretion and upregulation of genes involved in insulin synthesis and insulin secretion. Thus, we conclude that smad2 could represent an attractive therapeutic target for type 2 diabetes mellitus.

Evidence of a developmental origin for ß-cell heterogeneity using a dual lineage-tracing technology.

The Cre/loxP system has been used extensively in mouse models with a limitation of one lineage at a time. Differences in function and other properties among populations of adult ß-cells is termed ß-cell heterogeneity, which was recently associated with diabetic phenotypes. Nevertheless, the presence of a developmentally derived ß-cell heterogeneity is unclear. Here, we have developed a novel dual lineage-tracing technology, using a combination of two recombinase systems, Dre/RoxP and Cre/LoxP, to independently trace green fluorescent Pdx1-lineage cells and red fluorescent Ptf1a-lineage cells in the developing and adult mouse pancreas. We detected a few Pdx1+/Ptf1a- lineage cells in addition to the vast majority of Pdx1+/Ptf1a+ lineage cells in the pancreas. Moreover, Pdx1+/Ptf1a+ lineage ß-cells had fewer Ki-67+ proliferating ß-cells, and expressed higher mRNA levels of insulin, Glut2, Pdx1, MafA and Nkx6.1, but lower CCND1 and CDK4 levels, compared with Pdx1+/Ptf1a- lineage ß-cells. Furthermore, more TSQ-high, SSC-high cells were detected in the Pdx1+Ptf1a+ lineage population than in the Pdx1+Ptf1a- lineage population. Together, these data suggest that differential activation of Ptf1a in the developing pancreas may correlate with this ß-cell heterogeneity.

Polarized macrophages promote gestational beta cell growth through extracellular signal-regulated kinase 5 signalling.

AIM: To show that depletion of pancreatic macrophages impairs gestational beta cell proliferation and leads to glucose intolerance. MATERIALS AND METHODS: Genetic animal models were applied to study the effects of depletion of pancreatic macrophges on gestational beta-cell proliferaiton and glucose response. The crosstalk between macrophages and beta-cells was studied in vivo using beta-cell-specific extracellular-signal-regulated kinase 5 (ERK5) knockout and epidermal growth receptor (EGFR) knockout mice, and in vitro using a co-culture system. RESULTS: Beta cell-derived placental growth factor (PlGF) recruited naïve macrophages and polarized them towards an M2-like phenotype. These macrophages then secreted epidermal growth factor (EGF), which activated extracellular signal-regulated kinase 5 (ERK5) signalling in beta cells to promote gestational beta cell proliferation. On the other hand, activation of ERK5 signalling in beta cells likely, in turn, enhanced the production and secretion of PlGF by beta cells. CONCLUSIONS: Our study shows a regulatory loop between macrophages and beta cells through PlGF/EGF/ERK5 signalling cascades to regulate gestational beta cell growth.

SMAD7 enhances adult ß-cell proliferation without significantly affecting ß-cell function in mice.

The interplay between the transforming growth factor ß (TGF-ß) signaling proteins, SMAD family member 2 (SMAD2) and 3 (SMAD3), and the TGF-ß-inhibiting SMAD, SMAD7, seems to play a vital role in proper pancreatic endocrine development and also in normal ß-cell function in adult pancreatic islets. Here, we generated conditional SMAD7 knockout mice by crossing insulin1Cre mice with SMAD7fx/fx mice. We also created a ß cell-specific SMAD7-overexpressing mouse line by crossing insulin1Dre mice with HPRT-SMAD7/RosaGFP mice. We analyzed ß-cell function in adult islets when SMAD7 was either absent or overexpressed in ß cells. Loss of SMAD7 in ß cells inhibited proliferation, and SMAD7 overexpression enhanced cell proliferation. However, alterations in basic glucose homeostasis were not detectable following either SMAD7 deletion or overexpression in ß cells. Our results show that both the absence and overexpression of SMAD7 affect TGF-ß signaling and modulates ß-cell proliferation but does not appear to alter ß-cell function. Reversible SMAD7 overexpression may represent an attractive therapeutic option to enhance ß-cell proliferation without negative effects on ß-cell function.

Alpha-to-beta cell trans-differentiation for treatment of diabetes.

Diabetes mellitus is a significant cause of morbidity and mortality in the United States and worldwide. According to the CDC, in 2017, ∼34.2 million of the American population had diabetes. Also, in 2017, diabetes was the seventh leading cause of death and has become the number one biomedical financial burden in the United States. Insulin replacement therapy and medications that increase insulin secretion and improve insulin sensitivity are the main therapies used to treat diabetes. Unfortunately, there is currently no radical cure for the different types of diabetes. Loss of ß cell mass is the end result that leads to both type 1 and type 2 diabetes. In the past decade, there has been an increased effort to develop therapeutic strategies to replace the lost ß cell mass and restore insulin secretion. α cells have recently become an attractive target for replacing the lost ß cell mass, which could eventually be a potential strategy to cure diabetes. This review highlights the advantages of using α cells as a source for generating new ß cells, the various investigative approaches to convert α cells into insulin-producing cells, and the future prospects and problems of this promising diabetes therapeutic strategy.

Forkhead Box Protein 1 (FoxO1) Inhibits Accelerated ß Cell Aging in Pancreas-specific SMAD7 Mutant Mice.

The mechanisms underlying the effects of exocrine dysfunction on the development of diabetes remain largely unknown. Here we show that pancreatic depletion of SMAD7 resulted in age-dependent increases in ß cell dysfunction with accelerated glucose intolerance, followed by overt diabetes. The accelerated ß cell dysfunction and loss of proliferation capacity, two features of ß cell aging, appeared to be non-cell-autonomous, secondary to the adjacent exocrine failure as a "bystander effect." Increased Forkhead box protein 1 (FoxO1) acetylation and nuclear retention was followed by progressive FoxO1 loss in ß cells that marked the onset of diabetes. Moreover, forced FoxO1 expression in ß cells prevented ß cell dysfunction and loss in this model. Thus, we present a model of accelerated ß cell aging that may be useful for studying the mechanisms underlying ß cell failure in diabetes. Moreover, we provide evidence highlighting a critical role of FoxO1 in maintaining ß cell identity in the context of SMAD7 failure.

Gcg CreERT2 knockin mice as a tool for genetic manipulation in pancreatic alpha cells.

AIMS/HYPOTHESIS: The Cre/loxP system, which enables tissue-specific manipulation of genes, is widely used in mice for diabetes research. Our aim was to develop a new Cre-driver mouse line for the specific and efficient manipulation of genes in pancreatic alpha cells. METHODS: A Gcg CreERT2 knockin mouse, which expresses a tamoxifen-inducible form of Cre from the endogenous preproglucagon (Gcg) gene locus, was generated by homologous recombination. The new Gcg CreERT2 mouse line was crossed to the Rosa26 tdTomato (R26 tdTomato ) Cre reporter mouse line in order to evaluate the tissue specificity, efficiency and tamoxifen dependency of Gcg CreERT2 -mediated recombination. Cell types of pancreatic islets were identified using immunohistochemistry. Biochemical and physiological data, including blood glucose levels, plasma glucagon and glucagon-like peptide (GLP)-1 levels, and pancreatic glucagon content, were collected and used to assess the overall effect of Gcg gene targeting on Gcg CreERT2/w heterozygous mice. RESULTS: Tamoxifen-treated Gcg CreERT2/w ;R26 tdTomato/w mice displayed Cre reporter activity, i.e. expression of tdTomato red fluorescent protein (RFP) in all known cells that produce proglucagon-derived peptides. In the adult pancreas, RFP was detected in 94-97% of alpha cells, whereas it was detected in a negligible (~ 0.2%) proportion of beta cells. While more than 98% of cells labelled with tamoxifen-induced RFP were glucagon-positive cells, 14-25% of pancreatic polypeptide (PP)-positive cells were also positive for RFP, indicating the presence of glucagon/PP bihormonal cell population. Tamoxifen-independent expression of RFP occurred in approximately 6% of alpha cells. In contrast to alpha cells and GLP-1-producing neurons, in which RFP expression persisted for at least 5 months after tamoxifen administration (presumably due to rare neogenesis in these cell types in adulthood), nearly half of RFP-positive intestinal L cells were replaced with RFP-negative L cells over the first 2 weeks after tamoxifen administration. Heterozygous Gcg CreERT2/w mice showed reduced Gcg mRNA levels in islets, but maintained normal levels of pancreatic and plasma glucagon. The mice did not exhibit any detectable baseline physiological abnormalities, at least in young adulthood. CONCLUSIONS/INTERPRETATION: The newly developed Gcg CreERT2 knockin mouse shows faithful expression of CreERT2 in pancreatic alpha cells, intestinal L cells and GLP-1-producing neurons. This mouse line will be particularly useful for manipulating genes in alpha cells, due to highly specific and efficient CreERT2-mediated recombination in this cell type in the pancreas.

Epidermal Growth Factor Receptor Signaling Regulates ß Cell Proliferation in Adult Mice.

A thorough understanding of the signaling pathways involved in the regulation of ß cell proliferation is an important initial step in restoring ß cell mass in the diabetic patient. Here, we show that epidermal growth factor receptor 1 (EGFR) was significantly up-regulated in the islets of C57BL/6 mice after 50% partial pancreatectomy (PPx), a model for workload-induced ß cell proliferation. Specific deletion of EGFR in the ß cells of adult mice impaired ß cell proliferation at baseline and after 50% PPx, suggesting that the EGFR signaling pathway plays an essential role in adult ß cell proliferation. Further analyses showed that ß cell-specific depletion of EGFR resulted in impaired expression of cyclin D1 and impaired suppression of p27 after PPx, both of which enhance ß cell proliferation. These data highlight the importance of EGFR signaling and its downstream signaling cascade in postnatal ß cell growth.

A synopsis of factors regulating beta cell development and beta cell mass.

The insulin-secreting beta cells in the endocrine pancreas regulate blood glucose levels, and loss of functional beta cells leads to insulin deficiency, hyperglycemia (high blood glucose) and diabetes mellitus. Current treatment strategies for type-1 (autoimmune) diabetes are islet transplantation, which has significant risks and limitations, or normalization of blood glucose with insulin injections, which is clearly not ideal. The type-1 patients can lack insulin counter-regulatory mechanism; therefore, hypoglycemia is a potential risk. Hence, a cell-based therapy offers a better alternative for the treatment of diabetes. Past research was focused on attempting to generate replacement beta cells from stem cells; however, recently there has been an increasing interest in identifying mechanisms that will lead to the conversion of pre-existing differentiated endocrine cells into beta cells. The goal of this review is to provide an overview of several of the key factors that regulate new beta cell formation (neogenesis) and beta cell proliferation.

M2 macrophages promote beta-cell proliferation by up-regulation of SMAD7.

Determination of signaling pathways that regulate beta-cell replication is critical for beta-cell therapy. Here, we show that blocking pancreatic macrophage infiltration after pancreatic duct ligation (PDL) completely inhibits beta-cell proliferation. The TGFß superfamily signaling inhibitor SMAD7 was significantly up-regulated in beta cells after PDL. Beta cells failed to proliferate in response to PDL in beta-cell-specific SMAD7 mutant mice. Forced expression of SMAD7 in beta cells by itself was sufficient to promote beta-cell proliferation in vivo. M2, rather than M1 macrophages, seem to be the inducers of SMAD7-mediated beta-cell proliferation. M2 macrophages not only release TGFß1 to directly induce up-regulation of SMAD7 in beta cells but also release EGF to activate EGF receptor signaling that inhibits TGFß1-activated SMAD2 nuclear translocation, resulting in TGFß signaling inhibition. SMAD7 promotes beta-cell proliferation by increasing CyclinD1 and CyclinD2, and by inducing nuclear exclusion of p27. Our study thus reveals a molecular pathway to potentially increase beta-cell mass through enhanced SMAD7 activity induced by extracellular stimuli.

Dynamic imaging of pancreatic nuclear factor κB (NF-κB) activation in live mice using adeno-associated virus (AAV) infusion and bioluminescence.

Nuclear factor κB (NF-κB) is an important signaling molecule that plays a critical role in the development of acute pancreatitis. Current methods for examining NF-κB activation involve infection of an adenoviral NF-κB-luciferase reporter into cell lines or electrophoretic mobility shift assay of lysate. The use of adeno-associated viruses (AAVs) has proven to be an effective method of transfecting whole organs in live animals. We examined whether intrapancreatic duct infusion of AAV containing an NF-κB-luciferase reporter (AAV-NF-κB-luciferase) can reliably measure pancreatic NF-κB activation. We confirmed the infectivity of the AAV-NF-κB-luciferase reporter in HEK293 cells using a traditional luciferase readout. Mice were infused with AAV-NF-κB-luciferase 5 weeks before induction of pancreatitis (caerulein, 50 µg/kg). Unlike transgenic mice that globally express NF-κB-luciferase, AAV-infused mice showed a 15-fold increase in pancreas-specific NF-κB bioluminescence following 12 h of caerulein compared with baseline luminescence (p < 0.05). The specificity of the NF-κB-luciferase signal to the pancreas was confirmed by isolating the pancreas and adjacent organs and observing a predominant bioluminescent signal in the pancreas compared with liver, spleen, and stomach. A complementary mouse model of post-ERCP-pancreatitis also induced pancreatic NF-κB signals. Taken together these data provide the first demonstration that NF-κB activation can be examined in a live, dynamic fashion during pancreatic inflammation. We believe this technique offers a valuable tool to study real-time activation of NF-κB in vivo.

Pancreatic duct cells as a source of VEGF in mice.

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.

Smad signaling pathways regulate pancreatic endocrine development.

Expansion of the pancreatic endocrine cell population occurs during both embryonic development and during post-natal pancreatic growth and regeneration. Mechanisms of the expansion of endocrine cells during embryonic development are not completely understood, and no clear mechanistic link has been established between growth of the embryonic endocrine pancreas and the islet cell replication that occurs in an adult animal. We found that transforming growth factor-beta (TGF-ß) superfamily signaling, which has been implicated in many developmental processes, plays a key role in regulating pancreatic endocrine maturation and development. Specifically, the intracellular mediators of TGF-ß signaling, smad2 and smad3, along with their inhibitor smad7, appear to mediate this process. Smad2, smad3 and smad7 were all broadly expressed throughout the early embryonic pancreatic epithelium. However, during later stages of development, smad2 and smad3 became strongly localized to the nuclei of the endocrine positive cells, whereas the inhibitory smad7 became absent in the endocrine component. Genetic inactivation of smad2 and smad3 led to a significant expansion of the embryonic endocrine compartment, whereas genetic inactivation of smad7 led to a significant decrease in the endocrine compartment. In vitro antisense studies further corroborated these results and supported the possibility that interplay between the inhibitory smad7 and the intracellular mediators smad2/3 is a control point for pancreatic endocrine development. These results should provide a better understanding of the key control mechanisms for ß-cell development.

Hypoglycemia reduces vascular endothelial growth factor A production by pancreatic beta cells as a regulator of beta cell mass.

VEGF-A expression in beta cells is critical for pancreatic development, formation of islet-specific vasculature, and Insulin secretion. However, two key questions remain. First, is VEGF-A release from beta cells coupled to VEGF-A production in beta cells? Second, how is the VEGF-A response by beta cells affected by metabolic signals? Here, we show that VEGF-A secretion, but not gene transcription, in either cultured islets or purified pancreatic beta cells, was significantly reduced early on during low glucose conditions. In vivo, a sustained hypoglycemia in mice was induced with Insulin pellets, resulting in a significant reduction in beta cell mass. This loss of beta cell mass could be significantly rescued with continuous delivery of exogenous VEGF-A, which had no effect on beta cell mass in normoglycemic mice. In addition, an increase in apoptotic endothelial cells during hypoglycemia preceded an increase in apoptotic beta cells. Both endothelial and beta cell apoptosis were prevented by exogenous VEGF-A, suggesting a possible causative relationship between reduced VEGF-A and the loss of islet vasculature and beta cells. Furthermore, in none of these experimental groups did beta cell proliferation and islet vessel density change, suggesting a tightly regulated balance between these two cellular compartments. The average islet size decreased in hypoglycemia, which was also prevented by exogenous VEGF-A. Taken together, our data suggest that VEGF-A release in beta cells is independent of VEGF-A synthesis. Beta cell mass can be regulated through modulated release of VEGF-A from beta cells based on physiological need.

Neurogenin3 activation is not sufficient to direct duct-to-beta cell transdifferentiation in the adult pancreas.

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.

Acinar to ß-like cell conversion through inhibition of focal adhesion kinase.

Insufficient functional ß-cell mass causes diabetes; however, an effective cell replacement therapy for curing diabetes is currently not available. Reprogramming of acinar cells toward functional insulin-producing cells would offer an abundant and autologous source of insulin-producing cells. Our lineage tracing studies along with transcriptomic characterization demonstrate that treatment of adult mice with a small molecule that specifically inhibits kinase activity of focal adhesion kinase results in trans-differentiation of a subset of peri-islet acinar cells into insulin producing ß-like cells. The acinar-derived insulin-producing cells infiltrate the pre-existing endocrine islets, partially restore ß-cell mass, and significantly improve glucose homeostasis in diabetic mice. These findings provide evidence that inhibition of the kinase activity of focal adhesion kinase can convert acinar cells into insulin-producing cells and could offer a promising strategy for treating diabetes.

α-Cells are dispensable in postnatal morphogenesis and maturation of mouse pancreatic islets.

Glucagon-producing α-cells are the second-most abundant cell type in the islet. Whereas α-cells make up less than 20% of the cells in a mature mouse islet, they occupy a much larger proportion of the pancreatic endocrine cell population during the early postnatal period, the time when morphological and functional maturation occurs to form adult islets. To determine whether α-cells have a role in postnatal islet development, a diphtheria toxin-mediated α-cell ablation mouse model was established. Rapid and persistent depletion of α-cells was achieved by daily injection of the toxin for 2 wk starting at postnatal day 1 (P1). Total pancreatic glucagon content in the α-cell-ablated mice was undetectable at P14 and still less than 0.3% of that of the control mice at 4 mo of age. Histological analyses revealed that formation of spherical islets occurred normally, and the islet size distribution was not changed despite the near-total lack of α-cells. Furthermore, there were no differences in expression of ß-cell maturation marker proteins, including urocortin 3 and glucose transporter 2, in the α-cell-ablated islets at P14. Mice lacking α-cells grew normally and appeared healthy. Both glucose and insulin tolerance tests demonstrated that the α-cell-ablated mice had normal glucose homeostasis. These results indicate that α-cells do not play a critical role in postnatal islet morphogenesis or functional maturation of ß-cells.

Specific transduction and labeling of pancreatic ducts by targeted recombinant viral infusion into mouse pancreatic ducts.

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 ß-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.

Bone-marrow derived cells do not contribute to new beta-cells in the inflamed pancreas.

The contribution of bone-marrow derived cells (BMCs) to a newly formed beta-cell population in adults is controversial. Previous studies have only used models of bone marrow transplantation from sex-mismatched donors (or other models of genetic labeling) into recipient animals that had undergone irradiation. This approach suffers from the significant shortcoming of the off-target effects of irradiation. Partial pancreatic duct ligation (PDL) is a mouse model of acute pancreatitis with a modest increase in beta-cell number. However, the possibility that recruited BMCs in the inflamed pancreas may convert into beta-cells has not been examined. Here, we used an irradiation-free model to track the fate of the BMCs from the donor mice. A ROSA-mTmG red fluorescent mouse was surgically joined to an INS1Cre knock-in mouse by parabiosis to establish a mixed circulation. PDL was then performed in the INS1Cre mice 2 weeks after parabiosis, which was one week after establishment of the stable blood chimera. The contribution of red cells from ROSA-mTmG mice to beta-cells in INS1Cre mouse was evaluated based on red fluorescence, while cell fusion was evaluated by the presence of green fluorescence in beta-cells. We did not detect any red or green insulin+ cells in the INS1Cre mice, suggesting that there was no contribution of BMCs to the newly formed beta-cells, either by direct differentiation, or by cell fusion. Thus, the contribution of BMCs to beta-cells in the inflamed pancreas should be minimal, if any.