Heterozygous mutations in the transcription factor, hepatocyte nuclear factor 1B (HNF1B), result in multisystem disease including diabetes due to beta-cell dysfunction and pancreatic hypoplasia. However, the mechanisms that underlie development of diabetes in HNF1B mutation carriers are still not fully understood due to lack of an appropriate model system. Human induced pluripotent stem cells (hiPSCs), which are capable of self-renewal and can differentiate into any cell type, provide an advantageous alternative to model human developmental diseases. The aim of this project was to develop a hiPSC based model system to determine the molecular mechanisms by which HNF1B mutations cause pancreatic hypoplasia and diabetes. HNF1B mutant hiPSC lines were produced using CRISPR-Cas9 genome editing. Isogenic HNF1B wild-type, homozygous and heterozygous mutant hiPSC lines were directed to differentiate along the pancreatic lineage and cells were phenotyped at each stage of the differentiation process to check for appropriate expression of lineage markers. The normal expression pattern of HNF1B in human pancreas development was analysed and showed up-regulation of HNF1B at the foregut stage, and during pancreas specification. Homozygous knockout of HNF1B resulted in failure of foregut and pancreatic progenitor development, while heterozygous knockout of HNF1B resulted in impairment of pancreatic progenitor and endocrine cell differentiation as well as impaired insulin secretion upon glucose stimulation. Cell proliferation analyses showed a significant decrease in the proliferation rate in HNF1B heterozygous and homozygous mutant cells compared with wild-type cells at the foregut stage while no change in the apoptosis rate could be detected. RNA-sequencing and ATAC-sequencing, were used to further define the molecular mechanisms controlled by HNF1B and the effect HNF1B on modulation of chromatin accessibility during pancreas development. These results provide further insights into the molecular mechanisms by which HNF1B regulates human pancreas development and function, revealing that HNF1B haploinsufficiency impairs the expansion and maintenance of pancreatic progenitor cells in vitro. In vivo, this would likely result in reduced beta cell numbers at birth and diabetes later in life in patients with HNF1B-associated disease. These mechanisms suggest that the capacity to produce pancreatic progenitor cells during embryonic life could determine individual susceptibility to diabetes.

Breast and pancreatic cancers are among the major causes of cancer mortality in our society. There has been a significant decline in mortality from breast cancer over the last two decades, while pancreatic cancer has an exceptionally poor prognosis. Although these malignancies have very different clinical outcomes they share the common feature that metastatic disease is almost uniformly fatal. The existence of cancer stem cells has been postulated as a major factor in tumour recurrence after traditional chemo- or radio-therapy. Addressing this important clinical question requires a deeper understanding of the biology of normal and cancer stem cells and the signalling pathways involved in their regulation. The identity of the pancreatic stem cell remains elusive. However, using a murine model of haematopoietic stem cell (HSC) transplantation I have demonstrated for the first time transdifferentiation of these bone marrow derived cells into mature pancreatic acinar cells, where they appear to contribute to cell turnover ultimately forming acini and lobules. These data show that HSC have surprising developmental plasticity and provide insight into a potential stem cell niche in the pancreas. The Hedgehog, Wnt and Notch signalling pathways play a critical role in early development and in the maintenance and self-renewal of stem cells. There is also increasing evidence that dysregulation of these pathways contributes to the development of many malignancies. There is relatively little information regarding their role in breast cancer development and progression. I used immunohistochemistry for key proteins in these pathways, sonic hedgehog, beta-catenin and Notch 1 in three substantial series of human breast lesions and determined that abnormal expression of these proteins is an early event in the development in breast cancer, and is associated with particular breast cancer subtypes, Shh and beta-catenin expression is associated predominantly with the basal-like phenotype and Notch 1 with the HER2 amplified phenotype. Overexpression of Shh in particular confers a worse clinical outcome in invasive ductal carcinoma. Furthermore, increased levels of Shh in a 3D culture model of non-transformed mammary epithelial cells resulted in disorganisation of acini and the development of an abnormal discohesive phenotype. Finally the role of Shh was investigated in a mammary epithelial transplantation model, where overexpression of Shh resulted in the development of hyperplasia of the mammary ductal epithelium. Together these data confirm that the Hedgehog, Wnt and Notch developmental pathways are dysregulated in breast cancer and represent viable targets for further investigation of potential novel therapies in breast cancer.

The development of different organs and tissues along the gastrointestinal tract, including the pancreas, depends on signalling between the endoderm and the adjacent mesenchyme. The Nkx gene Bapx1 is involved in spatial control of organ-positioning in the spleno-pancreatic region, and deficiency in this gene results in unacceptable proximity of the splenic mesenchyme to the pancreas. This permits agitating signals from the splenic mesenchyme to induce an in vivo (and in vitro) transformation of pancreatic epithelium to a cystic structure with gut like features. Also, wild type splenic mesenchyme is competent to induce a similar transformation. These findings illustrate the importance for strict control of organ positioning during spleno-pancreatic development. Several growth factors and receptors involved in pancreatic development are activated by protease processing. Some of these growth factors have been implicated as substrates for members of the A Disintegrin And Metalloprotease (ADAM) family. The ADAMs 9, 10, and 17 are expressed during pancreatic development and in the adult pancreas, suggesting a possible role for these ADAMs in pancreatic development and function. Animal model systems are widely used to investigate gene function during development and disease. However, spatial, molecular, and quantitative phenotype screening in animals is a time consuming effort. Optical Projection Tomography is a 3-dimensional imaging technique that, in combination with improvements in sample preparation and computer processing, can be used to visualize and quantify characteristics of intact adult mouse organs such as the total β-cell content in the pancreas.

No description available.

A pancreatic stem cell could provide the tissue necessary for widespread β-cell transplantation therapy for diabetes. It is disputed whether pancreatic stem cells or β-cell replication are responsible for maintenance and regeneration of endocrine cells. Evidence presented here shows that pancreatic stem cells express insulin and produce multiple endocrine, exocrine and neural cells in vitro and in vivo. The human pancreas also contains stem cells that produce functional β-cells capable of reducing blood sugar levels in a diabetic mouse. Initial studies of pancreatic stem cells grown clonally in vitro indicated that they produced large numbers of neurons, suggesting they may be derived from the neural crest. Evidence shows that there are at least two distinct developmental origins for stem cells in the pancreas; one from the pancreatic lineage that produces endocrine and exocrine cells and one from the neural crest lineage that produces neurons and Schwann cells. Furthermore, pancreatic stem cells require the developmental transcription factor, Pax6, for endocrine cell formation suggesting they are using expected differentiation pathways. There is an interesting evolutionary connection between pancreatic β-cells and neurons which was applied to the derivation of pancreatic stem cells from human embryonic stem cells by using a clonal neural stem cell assay. These pancreatic stem cells express pancreatic and neural markers, self-renew and differentiate into insulin-expressing cells. The overexpression of SOX17 in these cells increases stem cell formation and self-renewal but inhibits differentiation. Overall I will show that there is a genuine stem cell in the adult mammalian pancreas capable of producing functional β-cells, that this stem cell is derived from the pancreatic developmental lineage but the pancreas also contains stem cells from the neural crest lineage, and that the neural stem cell assays that have identified these adult stem cells can be applied to the derivation of a pancreatic stem cell from hESCs.

A pancreatic stem cell could provide the tissue necessary for widespread β-cell transplantation therapy for diabetes. It is disputed whether pancreatic stem cells or β-cell replication are responsible for maintenance and regeneration of endocrine cells. Evidence presented here shows that pancreatic stem cells express insulin and produce multiple endocrine, exocrine and neural cells in vitro and in vivo. The human pancreas also contains stem cells that produce functional β-cells capable of reducing blood sugar levels in a diabetic mouse. Initial studies of pancreatic stem cells grown clonally in vitro indicated that they produced large numbers of neurons, suggesting they may be derived from the neural crest. Evidence shows that there are at least two distinct developmental origins for stem cells in the pancreas; one from the pancreatic lineage that produces endocrine and exocrine cells and one from the neural crest lineage that produces neurons and Schwann cells. Furthermore, pancreatic stem cells require the developmental transcription factor, Pax6, for endocrine cell formation suggesting they are using expected differentiation pathways. There is an interesting evolutionary connection between pancreatic β-cells and neurons which was applied to the derivation of pancreatic stem cells from human embryonic stem cells by using a clonal neural stem cell assay. These pancreatic stem cells express pancreatic and neural markers, self-renew and differentiate into insulin-expressing cells. The overexpression of SOX17 in these cells increases stem cell formation and self-renewal but inhibits differentiation. Overall I will show that there is a genuine stem cell in the adult mammalian pancreas capable of producing functional β-cells, that this stem cell is derived from the pancreatic developmental lineage but the pancreas also contains stem cells from the neural crest lineage, and that the neural stem cell assays that have identified these adult stem cells can be applied to the derivation of a pancreatic stem cell from hESCs.

El objeto de estudio de esta tesis ha sido dilucidar los diferentes mecanismos a partir de los cuales las serina proteasa activadoras del plasminógeno, uPA y tPA, llevan a cabo sus efectos en la progresión tumoral del cáncer de colon y de páncreas. Los resultados descritos muestran de forma detallada las vías de transducción de señal utilizadas por estos PAs. Se muestra por un lado como uPA, proteolíticamente activo, estimula el efecto de scatter en las células de cáncer de colon HT29-M6, y por otro como tPA mediante la unión a anexina II y la transactivación de EGFR estimula la invasión y la proliferación en células de cáncer de páncreas. / The object of study of this thesis was to elucidate the different mechanisms from which serine proteases plasminogen activators, uPA and tPA, carry out their effects in tumor progression of colon cancer and pancreatic cancer.The results described show in detail the signal transduction pathways used by these APs. It shows the one hand as uPA, proteolytically active, enhance the effect of scatter in colon cancer cells HT29-M6, and by another as tPA by binding to annexin II and EGFR transactivation stimulated invasion and proliferation in pancreatic cancer cells.

La diabetes de tipo 1 resulta de la destrucción autoinmune de las células ß pancreáticas, que conduce a una falta en la producción de insulina y la consiguiente hiperglucemia. La terapia sustitutiva con inyecciones subcutáneas de insulina permite a los pacientes llevar un vida activa, sin embargo esta terapia es imperfecta y no evita la aparición de graves complicaciones secundarias. El transplante de páncreas o islotes pancreáticos se ha realizado con éxito en algunos pacientes, sin embargo la escasez de donantes impide que esta terapia se pueda aplicar a todos los individuos diabéticos. Por ello, una gran cantidad de esfuerzos se han centrado en la diferenciación de células madre, embrionarias o adultas, en células ß. Las células de la médula ósea (BMC) poseen propiedades de célula madre adulta y además son fáciles de obtener, por ello se han propuesto como una fuente alternativa para la formación de nuevas células ß. El factor de crecimiento a la insulina de tipo I (IGF-I) participa en la regeneración muscular e incrementa la atracción y diferenciación de BMC en el músculo dañado. Además, la expresión de IGF-I específicamente en células ß de ratones diabéticos es capaz de regenerar la masa de células ß. Así, el primer objetivo de este trabajo fue estudiar la capacidad de la expresión de IGF-I en las células ß para atraer y diferenciar las BMC en nuevas células ß, tanto en ratones sanos como en ratones diabéticos. Con esta finalidad se transplantó la médula ósea de ratones transgénicos que expresaban la proteína verde fluorescente (GFP) constitutivamente, en los ratones transgénicos para IGF-I. Los resultados obtenidos demostraron que ni la sobreexpresión de IGF-I en células ß, ni la inducción de diabetes mediante estreptozotocina fueron causa suficiente para atraer y diferenciar las BMC en células ß pancreáticas in vivo. Estos datos sugerían que la regeneración del páncreas endocrino observada en los ratones transgénicos para IGF-I no era mediada por las BMC, indicando que la replicación de células ß preexistentes o bien la diferenciación a partir de precursores no hematopoyéticos son los mecanismos que actuarían en la regeneración de las células ß mediada por IGF-I.La diabetes se ha intentando curar mediante estrategias de terapia génica, sin embargo hasta el momento no se ha conseguido ninguna terapia efectiva. La recuperación completa del paciente diabético de tipo 1 requeriría la regeneración de las células ß. Una aproximación para conseguir este objetivo es la manipulación genética del páncreas endocrino in vivo, con la finalidad de expresar factores que induzcan replicación o neogénesis de las células ß y además contrarrestar la respuesta inmune. Sin embargo, el riesgo de inducir pancreatitis al manipular el páncreas es elevado, y por tanto se han realizado escasos intentos de modificar genéticamente este órgano hasta la fecha. Por ello, nuevas aproximaciones de transferencia génica in vivo son necesarias para avanzar en el desarrollo de nuevas aproximaciones de terapia génica para la diabetes. En este trabajo hemos estudiado la eficiencia de diferentes vectores virales y diferentes vías de administración para transducir el páncreas in vivo, tanto en ratones como en perros. En primer lugar, observamos que las células ß pancreáticas fueron transducidas eficientemente por adenovirus inyectados vía sistémica en ratones a los cuales se les había cerrado la circulación hepática. Este resultado obtenido con vectores adenovirales de primera generación también se obtuvo cuando usamos vectores adenovirales de última generación, también llamados gutless. Además de vectores adenovirales, también se estudio la capacidad de transducir el páncreas de los vectores adenoasociados de serotipo 8 (AAV8). Así, se demostró que la vía de administración de los vectores AAV8 por el conducto pancreático era más efectiva que la administración de estos vectores por vía endovenosa o intraperitoneal.El páncreas del perro presenta una estructura lobular y una vascularización similar al humano, por tanto constituye un buen modelo para ensayar estrategias de transferencia génica a páncreas. En este trabajo se estudió la capacidad de los vectores adenovirales para transferir genes a páncreas in vivo en animales sometidos a un clamp circulatorio de los vasos pancreáticos. Adenovirus con el gen marcador de la ß-galactosidasa se inyectaron en la vena pancreaticoduodenal y el clamp se mantuvo durante 10 minutos. Usando esta técnica se consiguió transducir células acinares, ductales y también islotes pancreáticos sin evidencias de daño pancreático. Esta técnica también se ensayó con éxito en un perro diabético.Por consiguiente, la metodología descrita en este trabajo puede ser usada para transducir el páncreas in vivo, ya sea en ratones o en perros, con la finalidad de estudiar la biología de las células ß o bien para desarrollar nuevas aproximaciones terapéuticas para la diabetes mellitus y otras enfermedades pancreáticas. / Type 1 diabetes is characterized by progressive destruction of pancreatic ?-cells, resulting in insulin deficiency and hyperglycemia. Insulin replacement therapy allows diabetic patients to lead active lives, but this therapy is imperfect and does not prevent development of severe secondary complications. Transplantation of pancreatic tissue or islets has been performed successfully in a limited numbers of patients. However, the shortage of donors is a primary obstacle that prevents this treatment from becoming more widespread. Therefore, many efforts have been focused on differentiating embryonic or adult stem cells into ß-cells. Bone marrow cells (BMCs) are an important source of easily procurable adult stem cells and have been proposed as an alternative source of ß-cells. Insulin-like growth factor-I (IGF-I) participates in skeletal muscle regeneration and enhances the recruitment of BMCs at the sites of muscle injury. In addition, IGF-I expression in ß-cells of diabetic transgenic mice regenerates pancreatic ß-cell mass. Therefore one of the objectives of this study was to investigate whether IGF-I expression in ß-cells could increase BMC recruitment and differentiation into ß-cells under steady-state conditions or after STZ treatment. To this end, BMCs from ß-actin/GFP transgenic donor mice were transplanted into IGF-I transgenic mice. Our experiments have demonstrated that IGF-I overexpression or STZ-induced pancreatic damage were not sufficient to recruit and differentiate GFP-labelled BMCs into ß-cells in vivo, indicating that these cells did not contribute to the endocrine pancreas regeneration observed in IGF-I transgenic mice. These data suggest that replication of pre-existing ß-cells and/or differentiation from non-BMC precursors is the most likely mechanism for IGF-I-mediated regeneration.Diabetes mellitus has long been targeted, as yet unsuccessfully, as being curable with gene therapy. Recovery from type 1 diabetes requires ß-cell regeneration. One approach to do so is by genetically engineering the endocrine pancreas in vivo to express factors that induce ß-cell replication and neogenesis and counteract the immune response. However, the pancreas is difficult to manipulate and pancreatitis is a serious concern, which has made effective gene transfer to this organ elusive. Thus, new approaches for gene delivery to the pancreas in vivo are required. In this study we have examined different viral vectors and routes of administration in rodents and also in large animals, to determine the most efficient method to deliver exogenous genes to the pancreas. First, we observed that pancreatic ß-cells were efficiently transduced to express ß-galactosidase after systemic injection of adenoviral vectors in mice with clamped hepatic circulation. This was true both for first generation as well as for helper-dependent adenoviral vectors. In addition to adenoviruses, we have compared the ability of AAV vectors to transduce the pancreas in vivo after intravascular, intraperitoneal or intraductal delivery, being the last the most efficient route of administration. Like the human pancreas, the canine pancreas is compact, with similar vascularization and lobular structure. It is therefore a suitable model in which to assess gene transfer strategies. Here we examined the ability of adenoviral vectors to transfer genes into the pancreas of dogs in which pancreatic circulation has been clamped. Adenoviruses carrying the ß-galactosidase (ß-gal) gene were injected into the pancreatic-duodenal vein and the clamp was released 10 min later. These dogs showed ß-gal-positive cells throughout the pancreas, with no evidence of pancreatic damage. ß-gal was expressed mainly in acinar cells, but also in ducts and islets. ß-gal expression in the exocrine pancreas of a diabetic dog was also found to be similar to that observed in healthy dogs. Thus, the methodology described herein may be used to transfer genes of interest to murine and canine pancreas in vivo, both for the study of islet biology and to develop new gene therapy approaches for diabetes mellitus and other pancreatic disorders.

Sammanfattning Inledning: Den årliga incidensen för pankreascancer är 9 per 100 000 invånare. En tidig diagnos ger förbättrad 5-årsöverlevnad men botar fortfarande få patienter. De senaste åren har den dåliga prognosen förbättras tack vare utvecklingen inom bilddiagnostiken. Författaren i denna litteraturstudie har valt att jämföra multidetektor datortomografi (MDCT) och magnetisk resonans (MR) med kontrastmedel för diagnostik av pankreastumör vid misstänkt pankreascancer. Syfte: Syftet med denna litteraturstudie är att undersöka vilken radiologisk undersökningsmetod som är att föredra vid diagnostisering av pankreascancer, MDCT eller MR. Frågeställning: Är det MDCT eller MR som är bäst vid diagnostisering av pankreascancer? Metod: En litteraturstudie baserad på 12 antal artiklar som är funna i databasen PubMed. Resultat: MDCT och MR har likvärdig diagnostisk säkerhet att upptäcka pankreastumör samt påvisa kärlinväxt och förutse operabilitet. Båda metoderna har en jämförbar hög noggrannhet för karakterisering av förändringens aggressivitet. Slutsats: Såväl MDCT och MR är likvärdiga två mycket bra radiologiska metoder för att diagnostisera pankreascancer. MDCT har fortfarande en större tillgänglighet är MR men dess nackdel är man utsätter patienterna för joniserande strålning. MR är därför en metod att föredra och med stor sannolikhet kommer dess tillgänglighet att öka kraftigt i framtiden.

<p>Insulin secretion is a fundamental process of pancreatic beta-cells required for the maintenance of glucose homeostasis. Fuel-stimulated insulin secretion occurs in proportion to the rate of metabolism of fuel substrates, yet the signals generated by metabolism of these secretagogues are incompletely understood. The increased burden placed on the beta-cell in conditions of obesity and insulin resistance often leads to dysregulation of stimulous-secretion coupling. Therefore, better understanding of the metabolic events required for insulin release is likely to be helpful in development of more effective treatments for diabetes.</p><p>Previous work in our lab revealed a critical role for the pyruvate-isocitrate cycling pathway in glucose-stimulated insulin secretion. It has been our hypothesis that this series of reactions plays a unique role in the beta-cell, and may be responsible for the generation of second-messenger signals critical for insulin secretion in response to increased fuel metabolism. One of the intermediates in the pyruvate/isocitrate cycle is cytosolic 2-oxoglutarate (2OG). In an effort to better understand the components of the pyruvate/isocitrate cycle and the signals that it generates, we initially focused our studies on the transporter protein responsible for the return of 2OG to the mitochondria, the 2-oxoglutarate carrier (OGC).</p><p>OGC was overexpressed in rat insulinoma 832/13 beta-cells and suppressed in both 832/13 cells and islets, and effects on metabolism and insulin secretion were measured. While overexpression of the OGC failed to alter insulin secretion, its siRNA-mediated suppression resulted in decreased insulin secretion in response to glucose, glutamine + BCH, and dimethyl-2-oxoglutarate. Suppression of OGC did not affect core pathways of fuel metabolism such as glucose usage, glucose oxidation or ATP production during glucose-stimulated insulin secretion (GSIS) or glutamine oxidation or ATP production during amino acid-stimulated insulin secretion (AASIS). Similar to previous findings, glucose-induced NADPH production was determined to be decreased in response to OGC suppression, whereas NADPH production during AASIS in untreated cells was already much lower than for GSIS, and suppression of OGC failed to decrease NADPH further.</p><p>As an additional approach to studying the role of 2OG metabolism in insulin secretion, we also investigated the mitochondrial enzyme glutamate dehydrogenase (Glud1). Overexpression of wild-type Glud1 failed to alter insulin secretion in 832/13 cells or in islets; however, suppression of Glud1 decreased both GSIS and AASIS, but did not affect dimethyl-2OG-stimulated insulin secretion. The reduction in AASIS was most likely the result of reduced glutamine oxidation. In contrast, during GSIS, NADPH production was decrease by Glud1 suppression, similar to our observation with the OGC.</p><p>In summary, these data expand our understanding of the metabolic pathways necessary for insulin secretion, and support the idea of a common metabolic pathway required for fuel-stimulated insulin release, including flux through the OGC, Glud1, and ICDc. However, while these data support the hypothesis that NADPH production is necessary for robust GSIS, it plays a less-prominent role during AASIS, and most likely works in concert with additional coupling-factors and signals.</p> / Dissertation