Pdx1 Levels Research Articles

Gene expression analysis of embryonic pancreas development master regulators and terminal cell fate markers in resected pancreatic cancer: A correlation with clinical outcome

BackgroundDespite the recent introduction of new drugs and the development of innovative multi-target treatments, the prognosis of pancreatic ductal adenocarcinoma (PDAC) remains very poor. Even when PDAC is resectable, the rate of local or widespread disease recurrence remains particularly high. Currently, reliable prognostic biomarkers of recurrence are lacking. We decided to explore the potential usefulness of pancreatic developmental regulators as biomarkers of PDAC relapse. MethodsWe analyzed by quantitative real-time PCR the mRNA of selected factors involved either in pancreatic organogenesis (ISL1, NEUROD1, NGN3, NKX2.2, NKX6.1, PAX4, PAX6, PDX1 and PTF1α) or associated with terminally committed pancreatic cells (CHGA, CHGB, GAD2, GCG, HNF6α, INS, KRT19, SYP) in 17 PDAC cell lines and in frozen tumor samples from 41 PDAC patients. ResultsHigh baseline levels of the ISL1, KRT19, PAX6 and PDX1 mRNAs in PDAC cell lines, were risk factors for time-dependent xenograft appearance after subcutaneous injection in CD1-Nude mice. Consistently, in human PDAC samples, high levels of KRT19 mRNA were associated with reduced overall survival and earlier recurrence. Higher levels of PDX1 or PAX6 mRNAs were instead associated with a higher frequency of local recurrence. ConclusionsOur findings suggest that selected factors associated with pancreas development or its terminal differentiation might be implicated in mechanisms of PDAC progression and/or metastatic spread and that the measurement of their mRNA in tumors might be potentially used to improve patient prognostic stratification and prediction of the relapse site.

Maintenance of ß-Cell Heterogeneity Is Required for Normal Islet Function

Aim: Recent studies have shown the existence of discrete β-cell subpopulations with differing levels of maturity and function. We sought here to understand how this diversity may influence islet function and insulin secretion using a ‘loss of heterogeneity‘ model. Material and Methods: Overexpression was achieved using an adenoviral polycistronic construct for Pdx1, MafA, Ngn3 and mCherry (Ad3-NPM). Pdx1 was silenced using short hairpin RNAs (shRNA). Gene and protein levels were detected by qPCR and immunohistochemistry (IHC), respectively. Ca2+ fluxes, ATP/ADP ratios and cAMP levels were analysed using dyes/biosensors and high-speed spinning disk microscopy. Glucose- (GSIS) and incretin-stimulated insulin secretion (ISIS) were measured using HTRF assays. Results: Ad3-NPM increased Pdx1 (12-fold vs. CT; P<0.01) and MafA levels (2-fold vs. CT) without affecting Ngn3. IHC showed that Pdx1 overexpression occurred predominantly in Pdx1low β-cells, forcing higher uniformity throughout the β-cell complement. The α/β-cell ratio did not change, indicating absence of transdifferentiation. Ca2+ responses to glucose were markedly blunted (ΔF=0.81 vs. 0.44 AU, CT vs. Ad3-NPM; P<0.01), and this was accompanied by a decrease in β-cell-β-cell cell connectivity and hub number (12.6 vs. 5.6 % hubs, CT vs. Ad3-NPM; P<0.05), the latter previously shown to be relatively immature cells with pacemaker features. ATP/ADP ratios and cAMP levels were unaffected. Glucose- and incretin-stimulated insulin secretion was impaired (% content secreted = 8.7 vs. 5.2 for GSIS and 56.4 vs. 29.02 for ISIS, CT vs. Ad3-NPM; both P<0.01). Similar results for Ca2+ dynamics, cellular connectivity and insulin secretion were obtained in islets treated with Pdx1 shRNA to increase the size of the Pdx1low population. Conclusion: Loss of β-cell heterogeneity, whether due to increased or decreased maturity, leads to islet failure and impaired insulin secretion. Thus, cellular diversity appears necessary for normal islet function. Disclosure D. Nasteska: None. G.A. Rutter: Research Support; Self; Servier. Q. Zhou: None. D. Hodson: None.

GLP-1 Controls Pancreatic Endocrine Development

Glucagon-like peptide 1 (GLP-1) has been successfully leveraged as a treatment of type 2 diabetes mellitus. Its beneficial effects on β-cell growth and function are documented in the adult rodents but its role during pancreatic organogenesis remains unexplored. GLP-1 has numerous targets in vivo and has been shown to regulate key signaling pathways in β-cells. In the mature islets, GLP-1 modulates the MAPK and Akt signaling pathways, controls glucose sensing and responsiveness and increases Pdx-1 levels. All these processes are also known to be instrumental in the regulation of the proper growth, differentiation and maturation of the early endocrine progenitors. A few studies indirectly implied that GLP-1 could also have some impact on pancreatic organogenesis, promoting endocrine development in in vitro models. However, adult GLP-1 receptor (GLP-1R) knock out mice only display mild metabolic impairments mostly triggered by exposure to high-fat diet, but the phenotype of these animals had not been studied during early development. Using our total body knockout of the GLP-1 receptor, we found that total size of the pancreas of the newborn knocked-out animals appears to be reduced at birth and the β-cell mass is diminished by 89%. These results support that GLP-1R signaling is crucial for normal pancreatic development but that catch up growth can takes place between birth and adulthood to compensate for reduced GLP-1R signaling. These data illustrate that in addition to its insulinotropic effects in mature islets, GLP-1 signaling plays a central role during pancreatic development and the acquisition of a normal β-cell mass. Disclosure C.M.H. Cras-Méneur: None. R.J. Seeley: Research Support; Self; Novo Nordisk Inc., Novo Nordisk Inc., Ethicon US, LLC.. Consultant; Self; Janssen Pharmaceuticals, Inc.. Research Support; Self; Zafgen, MedImmune. Consultant; Self; Kallyope, Scohia.

Combination of Obestatin and Bone Marrow Mesenchymal Stem Cells Prevents Aggravation of Endocrine Pancreatic Damage in Type II Diabetic Rats.

One of the new promising therapies in treatment of diabetes mellitus is mesenchymal stem cells (MSCs) which have an interesting therapeutic potentiality based on their paracrine effect and transdifferentiation potentiality. Also obestatin improves the generation of functional β cells/islet-like cell clusters in vitro, suggesting implications for cell-based replacement therapy in diabetes. So the aim of this study was to evaluate the effect of combination of both MSCs and obestatin on an experimental model of type II diabetes mellitus (T2DM). Sixty male rats were divided into; group I (control group), group II (T2DM group) induced by administration of high fat diet (HFD) and injection of streptozotocin (STZ) in low dose, group III (T2DM treated with MSCs), group IV (T2DM treated with obestatin), group V (T2DM treated with MSCs and obestatin). Fasting blood glucose, C-peptide, insulin and lipid profile were measured. HOMA-IR and HOMA-β were calculated. Pancreatic expression of insulin, glucagon like peptide -1 (GLP-1) and pancreatic duodenal homeobox 1 (Pdx1) mRNA levels were measured. In addition pancreatic histological changes, insulin and Bax were analyzed by immunohistochemical examination of islets of Langerhans. Diabetic rats showed significant increase in HOMA-IR, serum glucose and lipid profile levels with significant decrease in insulin, HOMA-β, GLP-1 and Pdx1 levels. MSCs and obestatin caused significant improvement in all parameters with more significant improvement in combined therapy. The protective effects afforded by MSCs and obestatin may derive from improvement of the metabolic profile, antiapoptosis and by increase in pancreatic GLP-1and Pdx1 gene expression.

Adiponectin protects against development of metabolic disturbances in a PCOS mouse model

Adiponectin, together with adipocyte size, is the strongest factor associated with insulin resistance in women with polycystic ovary syndrome (PCOS). This study investigates the causal relationship between adiponectin levels and metabolic and reproductive functions in PCOS. Prepubertal mice overexpressing adiponectin from adipose tissue (APNtg), adiponectin knockouts (APNko), and their wild-type (WT) littermate mice were continuously exposed to placebo or dihydrotestosterone (DHT) to induce PCOS-like traits. As expected, DHT exposure led to reproductive dysfunction, as judged by continuous anestrus, smaller ovaries with a decreased number of corpus luteum, and an increased number of cystic/atretic follicles. A two-way between-groups analysis showed that there was a significant main effect for DHT exposure, but not for genotype, indicating adiponectin does not influence follicle development. Adiponectin had, however, some protective effects on ovarian function. Similar to in many women with PCOS, DHT exposure led to reduced adiponectin levels, larger adipocyte size, and reduced insulin sensitivity in WTs. APNtg mice remained metabolically healthy despite DHT exposure, while APNko-DHT mice were even more insulin resistant than their DHT-exposed littermate WTs. DHT exposure also reduced the mRNA expression of genes involved in metabolic pathways in gonadal adipose tissue of WT and APNko, but this effect of DHT was not observed in APNtg mice. Moreover, APNtg-DHT mice displayed increased pancreatic mRNA levels of insulin receptors, Pdx1 and Igf1R, suggesting adiponectin stimulates beta cell viability/hyperplasia in the context of PCOS. In conclusion, adiponectin improves metabolic health but has only minor effects on reproductive functions in this PCOS-like mouse model.

Abstract 1795: Hypermethylation status of PDX1 has potential of diagnostic and predictive biomarker for risk stratification in colorectal cancer patients

Abstract Background Abnormal DNA methylation is an important epigenetic mechanism that has been strongly implicated in the pathogenesis and progression of colorectal neoplasia. In contrast to other potential biomarker species such as mRNA and protein levels, measurement of DNA methylation has been shown to be stable, reproducible and consistent between individuals. Based on these standpoints, detecting aberrant methylation of cancer-related genes helps for the development of diagnostic and prognostic biomarkers in colorectal cancer (CRC). In this study, we comprehensively evaluated whole-genome methylation analysis using colorectal cancer tissues and normal colonic mucosa to discover candidate CRC-specific DNA methylation as potential diagnostic and predictive biomarkers for risk stratification in CRC patients. Patients and Method The study design included an initial discovery phases, and followed by a subsequent clinical validation phase. In discovery phase, we performed whole-genome methylation analysis using CRC tissues and normal colonic mucosae from total fourteen patients to identify CRC-specific methylated CpG cites. Furthermore, we evaluated methylation levels of candidate methylated CpG cites by quantitative bisulfite pyrosequencing using one hundred six colorectal specimens from fifty-three CRC patients to validate the findings from discovery phase. Result In discovery phase, we successfully identified several candidates of CRC-specific hypermethylated- and hypomethylated-CpG cites in CRC tissues compared with normal colonic mucosa. Promoter lesion of PDX1 is one of candidate methylated CpG cites in discovery phase, and we focus a specific CpG cite of PDX1 promoter lesion as a target cite to progress in validation phase. We quantified methylation status of PDX1 using 106 colonic tissues, and demonstrated that PDX1 were significantly hyper-methylated in CRC tissues compared with normal mucosa (p<0.0001, Wilcoxon rank correlation test). Receiver operating characteristic (ROC) curves analysis revealed that methylation levels of PDX1 remarkably discriminated CRC tissues from normal colonic mucosa (AUC:0.9, Sensitivity:85.4%, Specificity:91.5%). Interestingly, methylation levels of PDX1 tend to increase in stage-dependent manner, and hypermethylated status of PDX1 was significantly correlated with presence of distant metastasis in validation cohort. Conclusion Assessment of the PDX1 methylation status could be used as potential of diagnostic and predictive biomarker for risk stratification in CRC patients. Citation Format: Takashi Ichikawa, Yuji Toiyama, Yoshinaga Okugawa, Hiromi Yasuda, Hiroyuki Fujikawa, Yoshiki Okita, Shigeyuki Yoshiyama, Junichiro Hiro, Minako Kobayashi, Toshimitsu Araki, Masaki Ohi, Yasuhiro Inoue, Keiichi Uchida, Yasuhiko Mohri, Masato Kusunoki. Hypermethylation status of PDX1 has potential of diagnostic and predictive biomarker for risk stratification in colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1795. doi:10.1158/1538-7445.AM2017-1795

The Role of Epigenetic Regulation and Pluripotency-Related MicroRNAs in Differentiation of Pancreatic Stem Cells to Beta Cells.

In this study, we aimed to research the effects of class-I HDACs and glucose on differentiation of pancreatic islet derived mesenchymal stem cells (PI-MSCs) to beta cells. Beta cell differentiation determined by flow cytometric analysis and gene expression levels of PDX1, PAX4, PAX6, NKX6.1, NGN3, INS2, and GLUT2. As a result the valproic acid, is an inhibitor of class-I HDACs, caused the highest beta cell differentiation in PI-MSCs. However, the cells in this group were at early stages of differentiation. Glucose co-administration to this group carried the differentiation to higher levels, but these newly formed beta cells were not functional. Moreover, reduction in the levels of pluripotency factors that Oct3/4, c-Myc, and Nanog were parallel to beta cell differentiation. Also, the levels of HDAC1 and acetylated H3/H4 were increased and methylated H3 was decreased by VPA treatment. In addition, we have detected over expression in genes of miR-18a-5p, miR-19b-5p, miR-30d-3p, miR-124, miR-146a-5p, miR-184, miR-335, and miR-433-5p in parallel to beta cell differentiation. As the conclusion, this study is important for understanding the epigenetic mechanism that controls the beta cell differentation and it suggests new molecules that can be used for diagnosis, and treatment of diabetes. J. Cell. Biochem. 119: 455-467, 2018. © 2017 Wiley Periodicals, Inc.

Overexpression of Kinase-Dead mTOR Impairs Glucose Homeostasis by Regulating Insulin Secretion and Not β-Cell Mass.

Regulation of glucose homeostasis by insulin depends on β-cell growth and function. Nutrients and growth factor stimuli converge on the conserved protein kinase mechanistic target of rapamycin (mTOR), existing in two complexes, mTORC1 and mTORC2. To understand the functional relevance of mTOR enzymatic activity in β-cell development and glucose homeostasis, we generated mice overexpressing either one or two copies of a kinase-dead mTOR mutant (KD-mTOR) transgene exclusively in β-cells. We examined glucose homeostasis and β-cell function of these mice fed a control chow or high-fat diet. Mice with two copies of the transgene [RIPCre;KD-mTOR (Homozygous)] develop glucose intolerance due to a defect in β-cell function without alterations in β-cell mass with control chow. Islets from RIPCre;KD-mTOR (Homozygous) mice showed reduced mTORC1 and mTORC2 signaling along with transcripts and protein levels of Pdx-1. Islets with reduced mTORC2 signaling in their β-cells (RIPCre;Rictorfl/fl) also showed reduced Pdx-1. When challenged with a high-fat diet, mice carrying one copy of KD-mTOR mutant transgene developed glucose intolerance and β-cell insulin secretion defect but showed no changes in β-cell mass. These findings suggest that the mTOR-mediated signaling pathway is not essential to β-cell growth but is involved in regulating β-cell function in normal and diabetogenic conditions.

Immunomodulatory and protective effects of adipose tissue-derived mesenchymal stem cells in an allograft islet composite transplantation for experimental autoimmune type 1 diabetes

BackgroundAllogeneic islet transplantation could be an ideal alternative therapy for Type 1 Diabetes Mellitus (T1DM). Adipose Tissue-derived Mesenchymal Stem Cells (AT-MSCs) characterized by immunomodulatory and protective effects may have the potential to improve the outcome of this highly immunogenic transplant. MethodsSyngenic AT-MSCs along with allograft islets embedded in hydrogelic composite and transplanted intraperitoneally in Streptozotocin (STZ) induced diabetic C57BL/6 mice. ResultsIn vitro experiments of co-imbedded islets and AT-MSCs in a hydrogel revealed AT-MSCs are able to significantly increase insulin secretion. During a 32 days of post-transplant period, blood glucose monitoring showed a decrease from over 400mg/dl to less than 150mg/dl and at the end of 32 days, mice have been dissected and assessed. Graft histopathology demonstrated that hydrogel makes an artificial immune isolation site and AT-MSCs contribute greatly to the reduction of the immune cells infiltration. Analyses of mononuclear cells isolated from Mesenteric Lymph Nodes (MLNs) and spleen showed that AT-MSCs co-transplanted with allograft decreased pro-inflammatory cytokines and increased regulatory cytokines (for both MLNs and spleen) and regulatory T cells (Treg) population (only for MLNs). In addition, real time-PCR assays revealed that transcript levels of IDO, iNOS, and PDX1, significantly increased in allograft islets in the presence of AT-MSCs. Conclusionsaccording to results, this investigation indicates that AT-MSCs can be regarded as promising complementary candidates for engineered-cell therapy using hydrogel composites in islet transplantation.

Plant-Produced Asialo-Erythropoietin Restores Pancreatic Beta-Cell Function by Suppressing Mammalian Sterile-20-like Kinase (MST1) and Caspase-3 Activation.

Pancreatic beta-cell death adversely contributes to the progression of both type I and II diabetes by undermining beta-cell mass and subsequently diminishing endogenous insulin production. Therapeutics to impede or even reverse the apoptosis and dysfunction of beta-cells are urgently needed. Asialo-rhuEPO, an enzymatically desialylated form of recombinant human erythropoietin (rhuEPO), has been shown to have cardioprotective and neuroprotective functions but with no adverse effects like that of sialylated rhuEPO. Heretofore, the anti-apoptotic effect of asialo-rhuEPO on pancreatic beta-cells has not been reported. In the current study, we investigated the cytoprotective properties of plant-produced asialo-rhuEPO (asialo-rhuEPOP) against staurosporine-induced cell death in the pancreatic beta-cell line RIN-m5F. Our results showed that 60 IU/ml asialo-rhuEPOP provided 41% cytoprotection while 60 IU/ml rhuEPO yielded no effect. Western blotting results showed that asialo-rhuEPOP treatment inhibited both MST1 and caspase-3 activation with the retention of PDX1 and insulin levels close to untreated control cells. Our study provides the first evidence indicating that asialo-rhuEPOP-mediated protection involves the reduction of MST1 activation, which is considered a key mediator of apoptotic signaling in beta-cells. Considering the many advantages its plant-based expression, asialo-rhuEPOP could be potentially developed as a novel and inexpensive agent to treat or prevent diabetes after further performing studies in cell-based and animal models of diabetes.

KNOCKOUT OF MIR‐223 INDUCES BETA‐CELL DYSFUNCTION THROUGH ACTIVATION OF THE FOXO1 SIGNALING PATHWAY

BackgroundMicroRNAs (miRNAs) are small, endogenous non‐coding RNAs, well‐known to regulate wide ranges of biological processes and tissue metabolism homeostasis. Recently, miR‐223 has been reported to coordinate systemic cholesterol under basal conditions and ameliorate adipose tissue‐associated insulin resistance in mice fed with high‐fat diet. However, the functional role of miR‐223 in the diabetic pancreas remains less studied.Method and ResultsDiabetes was induced in C57BL6 mice (5‐week old, male) by either daily intraperitoneal injection of streptozotocin (STZ, 50μg/g body weight) for 5 days (type 1 diabetes, or T1D), or 60% high‐fat diet (HFD) feeding for 24 weeks (type 2 diabetes, or T2D). Subsequently, pancreases were collected from such diabetic mice to measure miR‐223 levels using RT‐PCR. We observed that the levels of miR‐223 were increased by 8‐fold in the T1D pancreas, and by 2‐fold in T2D pancreas, compared to respective controls. Similarly, in vitro studies using Min6 β‐cell line revealed a 3‐fold increase of miR‐223 levels upon high glucose treatment. These results indicate that miR‐223 may involve in the development of diabetes. Of important, global miR‐223 knockout (KO) mice displayed insulin resistance, as evidenced by higher levels of plasma glucose and lower levels of insulin than wild‐type (WT) mice under both basal and diabetic conditions (n=5–11, p<0.05)). Further investigation showed that the diabetic phenotype of miR‐223‐KO mice may be attributed to diminished β‐cell proliferation and increased β‐cell apoptosis. Using luciferase reporter assay, we mechanistically identified that miR‐223 could directly target the 3′‐UTR of Foxo1, a critical transcriptional factor that regulates β‐cell function and survival. Using Western‐blotting, we validated that the protein levels of Foxo1 were increased by 3‐fold in the pancreas of miR‐223‐KO mice, compared to WTs, leading to subsequent decreased levels of Pdx1 and Glut2, two key regulators of β‐cell proliferation and function.ConclusionOur study reveals that loss of miR‐223 triggers β‐cell dysfunction by post‐transcriptional activation of the Foxo1 signaling pathway, resulting in the development of diabetes.Support or Funding InformationNIH R01 GM‐112930

Foxa2 and Pdx1 cooperatively regulate postnatal maturation of pancreatic β-cells

ObjectiveThe transcription factors (TF) Foxa2 and Pdx1 are key regulators of beta-cell (β-cell) development and function. Mutations of these TFs or their respective cis-regulatory consensus binding sites have been linked to maturity diabetes of the young (MODY), pancreas agenesis, or diabetes susceptibility in human. Although Foxa2 has been shown to directly regulate Pdx1 expression during mouse embryonic development, the impact of this gene regulatory interaction on postnatal β-cell maturation remains obscure. MethodsIn order to easily monitor the expression domains of Foxa2 and Pdx1 and analyze their functional interconnection, we generated a novel double knock-in homozygous (FVFPBFDHom) fluorescent reporter mouse model by crossing the previously described Foxa2-Venus fusion (FVF) with the newly generated Pdx1-BFP (blue fluorescent protein) fusion (PBF) mice. ResultsAlthough adult PBF homozygous animals exhibited a reduction in expression levels of Pdx1, they are normoglycemic. On the contrary, despite normal pancreas and endocrine development, the FVFPBFDHom reporter male animals developed hyperglycemia at weaning age and displayed a reduction in Pdx1 levels in islets, which coincided with alterations in β-cell number and islet architecture. The failure to establish mature β-cells resulted in loss of β-cell identity and trans-differentiation towards other endocrine cell fates. Further analysis suggested that Foxa2 and Pdx1 genetically and functionally cooperate to regulate maturation of adult β-cells. ConclusionsOur data show that the maturation of pancreatic β-cells requires the cooperative function of Foxa2 and Pdx1. Understanding the postnatal gene regulatory network of β-cell maturation will help to decipher pathomechanisms of diabetes and identify triggers to regenerate dedifferentiated β-cell mass.

Hepcidin links gluco-toxicity to pancreatic beta cell dysfunction by inhibiting Pdx-1 expression.

ObjectiveGluco-toxicity is a term used to convey the detrimental effect of hyperglycemia on β-cell function through impaired insulin synthesis. Although it is known that the expression and activity of several key insulin transcription regulators is inhibited, other molecular mechanisms that mediate gluco-toxicity are poorly defined. Our objective was to explore the role of hepcidin in β-cell gluco-toxicity.DesignWe first confirmed that high glucose levels inhibited hepcidin expression in the mouse insulinoma cell line, MIN6. The downregulation of hepcidin decreased Pdx-1 expression, which reduced insulin synthesis.MethodsMIN6 cells were exposed to high glucose concentrations (33.3 mmol/L). Glucose-stimulated insulin secretion (GSIS) and serum hepcidin levels were measured by ELISA. The mRNA levels of insulin1, insulin2, Pdx-1 and hepcidin were measured by real-time polymerase chain reaction. Western blot analysis was used to detect the changes in PDX-1 expression. Transient overexpression with hepcidin was used to reverse the downregulation of Pdx-1 and insulin synthesis induced by gluco-toxicity.ResultsExposure of MIN6 cells to high glucose significantly decreased GSIS and inhibited insulin synthesis as well as Pdx-1 transcriptional activity and expression at both the mRNA and protein levels. High glucose also decreased hepcidin expression and secretion. Hepcidin overexpression in MIN6 cells partially reversed the gluco-toxicity-induced downregulation of Pdx-1 and insulin expression and improved GSIS. The restoration of insulin synthesis by transfection of a hepcidin overexpression plasmid confirmed the role of hepcidin in mediating the gluco-toxic inhibition of insulin synthesis.ConclusionsOur observations suggest that hepcidin is associated with gluco-toxicity-reduced pancreatic β-cell insulin synthesis in type 2 diabetes by inhibiting Pdx-1 expression.

Preserving expression of Pdx1 improves β-cell failure in diabetic mice

Pdx1, a β-cell-specific transcription factor, has been shown to play a crucial role in maintaining β-cell function through transactivation of β-cell-related genes. In addition, it has been reported that the expression levels of Pdx1 are compromised under diabetic conditions in human and rodent models. We therefore aimed to clarify the possible beneficial role of Pdx1 against β-cell failure and generated the transgenic mouse that expressed Pdx1 conditionally and specifically in β cells (βPdx1) and crossed these mice with Ins2Akita diabetic mice. Whereas Pdx1 mRNA levels were reduced in Ins2Akita mice compared with their non-diabetic littermates, the mRNA levels of Pdx1 were significantly recovered in the islets of βPdx1; Ins2Akita mice. The βPdx1; Ins2Akita mice exhibited significantly improved glucose tolerance, compared with control Ins2Akita littermates, accompanied by increased insulin secretion after glucose loading. Furthermore, histological examination demonstrated that βPdx1; Ins2Akita mice had improved localization of SLC2A2 (GLUT2), and quantitative RT-PCR showed the recovered expression of Mafa and Gck mRNAs in the islets of βPdx1; Ins2Akita mice. These findings suggest that the sustained expression of Pdx1 improves β-cell failure in Ins2Akita mice, at least partially through the preserving expression of β-cell-specific genes as well as improved localization of GLUT2.

Abstract B03: Pancreatic cancer stem cells: can it be restrained?

Abstract Over the past decades world-wide, research towards not only understanding the biology, but also attempting to decrease the mortality rate in pancreatic carcinoma has become the forefront in cancers of solid tumor. Recent evidences suggest that pancreatic carcinomas harbour a distinct subpopulation of putative cancer stem cells defined by their self-renewal capacity, differentiation ability, tumorigenicity and ability to drive towards metastasis. Therefore, there is not only a need to identify this population, and also design newer approaches to target these chemo-resistant cell populations, especially in pancreatic cancer which till date shows 5-year survival rate to be below 5%. Our study identified the potential of CD44hiCD24lo MiaPaCa2/PanC1 cells to possess lower capability to generate spheroids whereas CD44hiCD24hi MiaPaCa2/PanC1 showed higher spheroid forming capacity. Spheroids obtained from both cell-lines showed similar metastatic cancer gene expression pattern. However, a distinctive CXCR4 expression levels were observed between CD44hiCD24lo PanC1-spheroids and CD44hiCD24hi PanC1-spheroids. It was also observed that EMT, CXCR4 and Muc1 were highly expressed in MiaPaCa2-spheroids. Hence, suggesting only a sub-population of tumor cells behave as “cancer initiating cells/stem-like cells” which are associated with cell-cell adhesion and cell matrix interaction. In order to determine the role of cancer stem-like cells (spheroids/CSCs) to migrate and invade to metastasize particularly in pancreatic carcinoma, experiments were designed to evaluate their self-renewal capability and epithelial to mesenchymal transition (EMT) and their association with developmental genes. An exponential spheroid formation was observed along with stem cell transcriptional genes (Oct4 and Nanog). Interestingly, MiaPaCa2-spheroids exhibited higher expression levels of Sox-2, MMP-9 and Vimentin in comparison to PanC1-spheroids suggesting that MiaPaCa2-spheroids may be more aggressive or has higher invasive potential. It appears that pancreatic CSCs undergoing EMT probably express higher levels of Sox-9, Ngn-3, Pdx-1 and c-Myc genes which attributes to its embryonic stemness. The results from the study clearly project the role of CSCs in pancreatic carcinoma and thus further studies to delineate the mechanism which might lead to suppression of pancreatic CSCs is crucial. In this direction, a pilot study has been initiated to explore the potential of Mesenchymal Stem Cells to target the pancreatic CSCs. Citation Format: Neha Chopra, Sangeeta Choudhury, Saima Wajid, Kriti Jain, Rizwana Mirza.{Authors}. Pancreatic cancer stem cells: can it be restrained? [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B03.

Abstract B19: Kras mutation imparts neoplastic potential on duct cells but not acinar cells in a mouse model of obstructive chronic pancreatitis

Abstract Understanding the progression of human pancreatic cancer is difficult due to the late stage of diagnosis of this deadly disease. We must therefore rely upon robust model systems to understand how pancreatic cancer arises, how its acute and chronic phases of progression are regulated, and how best to identify and treat it, hopefully still in the early stage. Models of pancreatic ductal adenocarcinoma (PDAC) that accurately reflect human disease are still being developed. To mimic human disease, PDAC must arise from mutations occurring in adult animals and with etiologies relevant to humans. We developed a new mouse model that incorporates obstructive chronic pancreatitis with tissue-specific, adult-onset expression of mutant Kras. We found that KrasG12D expression in duct cells but not in acinar cells led to progression of metaplastic ducts to eventual dysplasia and cancer. In early-stage disease, Kras mutation in acinar cells led to increased acinar-to-ductal metaplasia but did not downregulate p53, thus leading to reduced cell survival. Ducts naturally express much lower p53 levels, however, and there was increased survival of KrasG12D-mediated, duct-derived metaplastic cells. Furthermore, acinar cells upregulated Pdx1 during acinar-to-ductal metaplasia while duct cells did not do so during their metaplastic transition. Without chronic pancreatitis, Kras mutation in acinar cells causes abundant Pancreatic Intraepithelial Neoplasm (PanIN)-like lesions. However, overexpression of Pdx1 in acinar cells in this context permitted acinar-to-ductal metaplasia but prevented PanIN-like lesion formation, suggesting that Pdx1 can repress neoplastic progression. In summary, in the setting of obstructive chronic pancreatitis, ducts are the principal source of cancer development via reduced Pdx1 and p53 levels and increased cell survival. Citation Format: Fong C. Pan, Jessica N. Kim, Chanjuan Shi, Mary K. Washington, Maike Sander, Maureen Gannon, Robert D. Beauchamp, Christopher V. Wright, Anna L. Means.{Authors}. Kras mutation imparts neoplastic potential on duct cells but not acinar cells in a mouse model of obstructive chronic pancreatitis. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B19.

The combined effect of PDX1, epidermal growth factor and poly-L-ornithine on human amnion epithelial cells' differentiation.

BackgroundIt has been suggested that the ectopic expression of PDX1, a dominant pancreatic transcription factor, plays a critical role in the developmental programming of the pancreas even from cells of unrelated tissues such as keratinocytes and amniotic fluid stem cells. In this study we have chosen to drive pancreatic development in human amnion epithelial cells by inducing endogenous PDX1 expression. Further, we have investigated the role of Epidermal Growth Factor (EGF) and Poly-L-Ornithine (PLO) on this differentiation process.ResultsHuman amnion epithelial cells expressed high levels of endogenous PDX1 upon transduction with an adenoviral vector expressing murine Pdx1. Other markers of various stages of pancreatic differentiation such as NKX6.1, SOX17, RFX6, FOXA2, CFTR, NEUROD1, PAX4 and PPY were also expressed upon Pdx1 transduction.Although initial expression of pancreatic progenitor markers was higher in culture conditions lacking EGF, for a sustained and increased expression EGF was required. Culture on PLO further increased the positive impact of EGF.ConclusionPancreatic marker expression subsequent to mPdx1 transduction suggests that this approach may facilitate the in vitro differentiation of hAECs into cells of the endocrine pancreas. This result may have important implications in diabetes therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0108-y) contains supplementary material, which is available to authorized users.

Exendin-4 enhances expression of Neurod1 and Glut2 in insulin-producing cells derived from mouse embryonic stem cells.

IntroductionStem cells involved cell replacement therapies for type 1 diabetes mellitus is promising, yet time-consuming and inefficient. Exendin-4 is a glucagon-like peptide-1 (GLP-1) receptor agonist which has been reported to possess anti-apoptotic effects, thereby increasing β-cell mass and improving β-cell function. The present study aimed to investigate whether exendin-4 would enhance the differentiation of embryonic stem cells into insulin-secreting cells and improve the pancreatic differentiation strategy.Material and methodsR1 embryonic stem cells were treated with different concentrations of exendin-4 and divided into three groups. In the high dosage group (group H), exendin-4 was added at the dosage of 10 nmol/l. In the low dosage group (group L), exendin-4 was added at the dosage of 0.1 nmol/l. Group C was a control. Expression of genes related to the β-cell phenotype and immunofluorescence staining of insulin and C-peptide were detected.ResultsCompared with groups L and C, group H had the highest mRNA expression levels of Isl1, Pdx1, Ngn3, and Insulin1 (p < 0.05). Neurod1 and Glut2 only emerged at the final stage of differentiation in group H. Immunofluorescence analysis revealed that exendin-4 upregulated the protein expression of insulin and C-peptide.ConclusionsExendin-4 remarkably facilitated Neurod1 and Glut2 gene transcription, and was able to induce differentiation of embryonic stem cells into endocrine and insulin-producing cells.

Dysregulated miRNA Associated with Transcription Factors of Insulin Gene Expression in Chronic Pancreatitis

Background/Aim: MicroRNAs with regulatory functions in gene expression are implicated in different diseases. The present study investigated differentially expressed miRNAs that possibly influence transcription factors involved in insulin gene expression in Chronic Pancreatitis (CP) employing bioinformatics approaches. Methods: Pancreatic tissues were collected from CP patients undergoing partial pancreatectomy (n = 16) and controls (n = 15) undergoing resections for non-pancreatic malignancies. MiRNA profiles obtained using microarrays were validated by qRT-PCR. Target search involving miRWalk and TarBase as well as functional annotation employing KEGG (Kyoto encyclopedia of genes and genomes) and DAVID (Database for Annotation) databases were performed. Ingenuity pathway analysis (IPA) was used to construct networks relating miRNAs to their target genes. mRNA and proteins related to insulin gene transcription factors and hormones were evaluated by qRT-PCR and western blotting followed by confirmation upon immunofluorescent staining. Results: Microarray data revealed 10 up-regulated and 15 down-regulated miRNAs in CP as compared to controls (Log2 FC > 2). Bioinformatic analysis showed 8399 target genes and KEGG pathway analysis suggested a role for the dysregulated miRNAs in modulating cytokine signaling, fibrosis, JAK-STAT signaling and insulin synthesis. IPA analysis suggested a simplified network attributing dysregulated miRNAs to NFκB-dependent cytokine signaling. Further, associations could be noted between miRNA 200b with Maf A, 138-1 with Neuro D and 27b with FoxO1. Decreases in mRNA levels of Pdx1, Neuro D and increases of Maf A and FoxO1 transcription factors could be noted (P Conclusion: Our results identified dysregulation of miRNAs 138-1, 27b and 200b which were found to be associated with insulin gene transcription factors Neuro D, FoxO1 and Maf A respectively.

Engineering tissue substrates for generation of islet-like organoids from human pluripotent stem cell differentiation

Engineering tissue substrates for generation of islet-like organoids from human pluripotent stem cell differentiation