Dissertations / Theses on the topic 'Human pancreatic islet'

Background: Pancreatic islet transplantation for type-1 diabetes has resulted in considerable success over the past decade. However, the worldwide shortage of pancreatic donors remains a major challenge. In an attempt to expand the donor pool, pancreases from obese organs donors (>30 kg/m²) are now routinely offered to islet transplantation in the UK. In addition, it has been suggested that pancreases from donors with early type-2 diabetes mellitus may also be suitable. However, for both these donor groups, although high islet yields (IEQ) may be obtained, it is unclear whether these islets function optimally. An additional approach to the donor shortage is to minimise the number of donors required per islet recipient. One strategy to achieve this is to use different pharmacological agents to enhance post-transplant islet function. Aims: The aims of this thesis were fourfold; 1) To determine whether islets isolated from high BMI donors function normally in vitro and in vivo; 2) To establish why islet yields are higher from obese donors; 3) To determine whether islets from donors with type-2 diabetes are suitable for islet transplantation; 4) To investigate whether glucagon-like peptide 1 receptor agonist therapy improves post-transplant islet graft function. Results: Stimulated insulin and glucagon secretion, and markers of mitochondrial function (intra-islet ATP content and mitochondrial copy number) were compared in islets isolated from obese (BMI>30kg/m²; n= 27) and non-obese (BMI<28kg/m²; n=25) donors. No differences in secretory function or intra-islet ATP were observed between the two groups. Pancreatic lipid and intra-islet triglyceride concentrations were higher in the obese group. In vivo clinical outcomes of patients transplanted in Oxford and a larger cohort (n=35) in Edmonton, Canada with islets from obese or non-obese donors showed no differences in post-transplant outcomes. Improved islet yields were shown to be a result of improved islet recovery of larger islets, in obese donors. Abnormal insulin and glucagon secretory responses were observed in islets from type-2 diabetic subjects (n=6) and islet amyloid content was significantly higher in diabetes. The glucagon-like peptide 1 receptor agonist, exenatide, administered for 20 weeks, significantly improved graft function in patients whom islet function was impaired. Conclusion: The high lipid environment of islets isolated from donors with high BMI appears not to be deleterious to their function either in vitro or when transplanted, supporting the use of islets from high BMI donors for clinical islet transplantation. However, islet dysfunction and pathological changes indicate that islets from type-2 diabetic donors are unsuitable. Therapeutic options such as exenatide, improve transplanted islet viability and function, and could have a significant role in the future of beta-cell replacement therapy for type-1 diabetes.

This thesis focuses on enteroviral effects on human pancreatic islets. Most knowledge of viral effects on host cells relies on studies of immortalized cell lines or animal models. The islets represent a fundamentally different and less well studied cellular host. Also, enterovirus has been implicated in the etiology of type 1 diabetes (T1D). We show that when enterovirus replicates in human islets it activates innate immunity genes and induces secretion of the chemokines MCP-1 and IP-10. An important difference in activation of innate immunity by replicating EV and synthetic dsRNA is suggested, since the chemokine secretion induced by EV infection but not by dsRNA is reduced by female sex hormone. We also demonstrate a direct antiviral effect of nicotinamide, and even though this substance failed to prevent T1D in a large-scale study, this finding could have implications for the treatment/prevention of virus- and/or immune-mediated disease. We also had access to human pancreata from two organ donors with recent onset T1D and several donors with T1D-related autoantibodies, which gave us the opportunity to study ongoing pathogenic processes at and before the onset of T1D. Despite this, we could neither confirm nor reject the hypothesis that EV is involved in T1D development. Several observations, such as ultrastructural remodeling of the beta cell, activation of innate immunity, and immunopositivity to EV capsid protein 1, supported an ongoing virus infection, but direct evidence is still lacking. An interesting finding in the donors with recent onset T1D was that the islets were positively stained for insulin, but did not secrete insulin in response to glucose-stimulation. A similar effect was observed in EV-infected islets in vitro; EV destroyed islet function and insulin gene expression, but the islets still stained positive for insulin. This may be indicative of that a functional block in addition to beta cell destruction is involved in T1D pathogenesis. In conclusion, these studies of EV in isolated human islets in vitro support that this virus can cause T1D in vivo, but future studies will have to show if and how frequently this happens.

Type 1 diabetes is an autoimmune disease resulting in the selective destruction of the insulin producing β-cells in the pancreas. Pro-inflammatory cytokines and the free radical nitric oxide (NO) have been implicated in mediating the destruction of β-cells, possibly through activation of the mitogen activated protein kinases (MAPKs) JNK, ERK and p38. In addition to MAPKs, cytokine signaling also results in activation of the transcription factor nuclear factor-kappaB (NF-κB). The upstream signaling events leading to MAPK and NF-κB activation in β-cells are not well known. The work presented in this thesis therefore aims at characterizing the regulation of MAPKs and NF-κB in human islets, with emphasis on the role of the MAPK activator MAP/ERK kinase kinase-1 (MEKK-1) in islet cell death. It was found that MEKK-1 was phosphorylated in response to the nitric oxide donor DETA/NONOate (DETA/NO), the β-cell toxin streptozotocin (STZ) and pro-inflammatory cytokines and that MEKK-1 downstream signaling in response to the same treatments involved activation of JNK but not ERK and p38. MEKK-1 was also found to be essential for cytokine-induced NF-κB activation. MEKK-1 downregulation protected human islet cells from DETA/NO-, STZ, and cytokine-induced cell death. Furthermore, overexpression of the NF-κB subunit c-Rel protected human islet cells from STZ and hydrogen peroxide-induced cell death indicating that NF-κB activity protects against cell death in human islets. In summary, these results support an essential role for MEKK-1 in the activation of JNK and NF-κB, with important consequences for human islet cell death and that strategies preventing human islets death by inhibition of the JNK pathway instead of NF-κB might be suitable.

Islet plasticity has proven to be an important platform for the engineering of alternative islet tissue for transplantation. In vitro studies have shown the ability of islets to transdifferentiate into duct-like epithelial structures (DLS) thought to possess progenitor cells capable of replenishing damaged tissue within the pancreas. The aim of this study was to investigate the natural history of human derived duct-like epithelial structures transplanted into nude mice.
Human islet derived duct-like structures from three cadaver pancreases were subcutaneously transplanted into 6-8 week old male HSD athymic nude-Foxn1 mice. Six mice were sacrificed at day 3, 7, 14 and 21 from each time period. DLS were also placed in matrigel for in-vitro control samples. DLS were processed for immunohistochemistry for endocrine markers, epithelial markers, cell death and proliferation markers, islet maturation markers and angiogenic factors.
Our results show that as DLS are transplanted, there is an increase in cell death and proliferation. This increase in cell death and proliferation causes an increase in PDX-1 expression as well as VEGF, an angiogenic factor. But over time, transplanted DLS do not show an increase in cell death and show a small decrease in cell proliferation from pre-transplanted DLS. At day 3 of engraftment, DLS show a significant expression of PDX-1. We see a small increase in endocrine tissue after 3 days of transplantation, then an increase in endocrine cell death, which returns the percentage of endocrine cells back to pre-transplantation levels at day 21. DLS were shown to express VEGF, and once transplanted into an initial hypoxic environment there is a substantial increase in expression, followed by a recruitment of microvessels. Although there is a dynamic change in expression of cell markers throughout engraftment, there is no significant change in DLS size, nuclei per DLS or cell morphology over time.
DLS have been shown to survive subcutaneous transplantation and possess an initial increase in cell proliferation leading to increases in PDX-1 and VEGF expression. Transplanted DLS have shown to possess significant angiogenic properties with the recruitment of microvessels into subcutaneous DLS grafts. Subcutaneous DLS transplantation could be used in combination with islet transplantation to alleviate current problems with islet transplantation such as islet cell death and insufficient blood supply.

Diabetes mellitus is a metabolic disorder characterized by a rise in blood sugar levels, estimated to currently affect more than 300 million people worldwide. Even though pancreatic islet β-cells play a central role in all major forms of the disease, their transcriptome is still not fully characterised. Furthermore, type 2 diabetes susceptibility is known to be affected by non-coding genomic variation, but the impact of these variants on gene regulation and their disease implications remain to be elucidated. In the first part of this project, I integrated the analysis of RNA sequencing datasets with maps of epigenomic features, resulting in the identification of 1,128 long non-coding RNA (lncRNA) genes in human pancreatic islets. Islet lncRNAs were enriched for tissue-specific expression, and a subset were shown to be regulated during endocrine differentiation or in response to changes in glucose. Furthermore, lncRNA orthologs in mouse islets were identified, which displayed similar regulatory properties to their human counterparts. Taken together, these analyses led to the identification of a novel class of genes active in human and mouse pancreatic islets, opening new avenues for the study of diabetes development and therapeutic strategies. In the second part of this project, I analysed the RNA-seq datasets and genotypes of 27 human islet samples, and developed a methodology to study the effect of non-coding genomic variation on gene regulation. This revealed hundreds of genes under allele-specific expression, consistently across many independent individuals. Some of these genes performed known roles in human islets, or resided within GWAS loci for type 2 diabetes and related traits. These findings could be used to identify functional cis-regulatory genetic variants impacting the human islet transcriptome, some of which could lead to the development of diabetes. Collectively, these projects uncovered novel components of the human islet transcriptome, and provided new methodologies to study the effects of genomic regulatory variation on gene transcription.

Type I diabetes mellitus (T1DM) is characterized by insulin deficiency due to β cells death caused by inflammation and immune reaction. Pro-inflammatory cytokines play a key role in T1DM pathogenesis by activating the pro-apoptotic pathway. Cytokine-activated NF-κB and STAT1 signalling also leads to oxidative stress and triggers the antigen presentation pathway. Stressful stimuli and self-defence responses combined cause mitochondrial dysfunction and endoplasmic reticulum (ER) stress, which progress to β cell functional impairments and death. Some molecular mechanisms involved in the progressive loss of functions are still unknown. In this contest, a study, which analyses proteomic changes of β cells upon cytokine prolonged exposure, can be useful. The global effects of pro-inflammatory cytokines, IL-1β and INF-γ, on protein expression, Nε-acetylation, and thermal stability was studied using different proteomic approaches and β cell models (rat INS-1E cells and human pancreatic islets). At first, the impact of cytokines on β cell insulin secretion, survival and apoptosis activation was examined to confirm functional impairment. Differential expression proteomics showed that cytokines dysregulated the expression of many proteins, which are components of pathways involved in T1DM pathogenesis. The study of protein lysine acetylome highlighted 83 and 242 differentially modified proteins in human islets and INS-1E cells, respectively. Most proteins are related to metabolic, mitochondrial dysfunction, inflammation, and insulin secretion pathways. In INS-1E cells, an analysis of Proteome Integral Solubility Alteration (PISA) was also performed. This technique allowed to identify 504 proteins, which thermal stability was modified by cell exposure to cytokines. Many of these proteins participate to protein synthesis, protein trafficking, and antigen presentation or are directly involved in mediating cytokine effects. Overall, this multi-level proteomic analysis unveiled new potential players of cytokine-induced β cells dysfunction.

Dans les conditions normales, les organismes maintiennent une masse cellulaire endocrine stable tout au long de leur vie. En cas d’obésité, la masse de cellules b pancréatiques est capable de maintenir des taux de glucose plasmatique en augmentant la sécrétion en insuline. L’incapacité de ces cellules à fournir de l’insuline entraîne alors l’apparition d’une hyperglycémie et d’un diabète de type II. Cliniquement, la majorité des individus obèses ne développent pas de diabète car les îlots pallient à cette résistance à l’insuline. La preuve de l’adaptation de la masse d’îlots humains à l’obésité, in vivo, n’a pas été clairement décrite et, de plus, peu d’informations existent sur les mécanismes et les types cellulaires impliqués. Actuellement, la mise en évidence de l’augmentation de la masse des cellules b chez les humains obèses repose uniquement sur des études histologiques.But : Au cours de cette thèse, nous avons cherché, dans un premier temps (partie 1), à évaluer la morphologie des îlots pancréatiques et la distribution des cellules a et b chez des donneurs en état de mort cérébrale normaux, en surpoids et obèses. Dans un second temps (partie 2), nous avons étudié l’adaptation au cours du temps des îlots humains à un environnement obésogène. Nous avons montré ainsi que les îlots humains non diabétiques s’adaptent in vivo à l’obésité en modifiant la masse de cellules b, leur fonction et leur expression génique. En suite (partie 3), on a identifié le mécanisme de transdifférenciation des cellules alpha et beta en utilisant la méthode de lineage tracing. Finalement (partie 4), on a déterminé la différence sur l’expression de gène des ilots humains greffé chez les souris sous régime control ou régime riche en graisse en utilisant les puces d’ARN (Illumina).Methodes et Resultats: Des coupes de pancréas humains inclues en paraffines ont été analysées. Les donneurs obèses étaient caractérisés par une augmentation de la masse endocrine totale, de la taille des îlots, de la graisse intra-pancréatique et du ratio b:a dans les îlots, mais avec une diminution du ratio a:b dans les îlots. Au cours de l’étude longitudinale, des souris Rag2-/- non diabétiques ont été greffées sous la capsule rénale avec des îlots humains issus de donneurs en état de mort cérébrale (donneurs non diabétiques ou donneurs avec un dysfonctionnement métabolique déclaré). Les animaux ont été nourris pendant 2 semaines avec soit un régime contrôle soit un régime riche en graisse (high fat diet HFD). Un suivi du poids, du taux des triglycérides, de la glycémie et du C-peptide a été mis en place. Après sacrifice des souris, les greffons et les pancréas endogènes ont été analysés pour le volume endocrine, la distribution des cellules b et a et les mécanismes de régénération des cellules pancréatiques. Après 12 semaines sous régime gras, les souris montraient toutes les caractéristiques typiques de l’obésité, à savoir, une augmentation du poids, un doublement de la graisse abdominale, des triglycérides, de la glycémie et une sensibilité à l’insuline réduite. De plus, l’apparition sur ces animaux d’un doublement rapide de la quantité de C-peptide humain dans le sérum murin nous indique la mise en place d’une compensation fonctionnelle. Une analyse histologique des greffons a permis de mettre en évidence une adaptation de la masse endocrine des îlots avec une augmentation des cellules b. D’autres analyses ont identifié la prolifération et la néogénèse comme les mécanismes responsables de ce doublement de la masse endocrine humaine.Discussion: Ce nouveau modèle animal permet d’étudier, in vivo sur une longue période, l’adaptation des îlots humains à un environnement obésogène murin. Il peut être utilisé comme un outil dans le décryptage des voies de signalisation impliquées dans l’expansion des cellules b humaines et permettre également l’identification des facteurs prédisposant ces cellules à subir une décompensation
Under normal healthy conditions, organisms maintain a dynamic endocrinecell mass throughout life. Pancreatic beta cell mass are able to maintain plasma glucose levels increasing insulin secretion in conditions as obesity.Beta cell inability to compensate in insulin demand provokes hyperglycemia and Type 2 Diabetes. Clinically, most obese individuals do not develop diabetes because islets compensate for insulin resistance. Direct evidence that human islet mass adapts longitudinally to obesity in vivo was lacking and, moreover, little information was available on the mechanismsand cell type(s) involved.Current evidence for increased beta cell mass in obese humans (vs lean) is based entirely on postmortem histology.Aim: In this thesis, firstly (Part 1) we performed a descriptive cross sectional study by evaluating the pancreatic islet morphology and alpha and beta cell distribution from our archived human pancreatic sections of obese and normal subjects. Secondly, (Part 2) we explored the longitudinal adaptation of human islets to an obesogenic environment and showed direct evidence that non-diabetic human islets adapt bothendocrine and beta cell mass, function and gene expression to obesity in vivo. Thirdly (Part 3) we performed lineage tracing to determine which cell type alpha or beta give rise to the increase islet mass in obesity. Finally (Part 4) in this diet induced obesity model we developed, we looked at the differential gene expression with Illumina gene chips in a kinetic study on human islets which were laser capture microdissected at 6, 8 and 10 weeks on control or high fat diet.Methods: Archived human pancreatic sections were immunostained for endocrine, beta, alpha, fat. In the obese/immunodeficient mouse model, non-diabetic Rag2–/– mice were transplanted under kidney capsule with human islets from human brain-deceased donors (non-diabetics donors and donors with overt metabolic dysfunction). Animals were fed for 12 weeks with a control or high-fat diet (HFD), and followed for weight, serum triacylglycerol, fasting blood glucose and human C-peptide. After the mice were killed, human grafts and the endogenous pancreas were analyzed for endocrine volume, distribution of beta and alpha cells, and mechanisms of regeneration.Results: The cross-sectional study, performed on archived human paraffin embedded sections of normal weight, overweight, or obese subjects showed that obese donors were characterized by an increased total endocrine mass, bigger individual islet size, increased intrapancreatic fat, increased β to  cell ratio and decreased :β cell ratio in islets. In the longitudinal study, concomitant with the increased weight gain, doubling of abdominal fat, increased serum triacylglycerol and reduced insulin sensitivity in 12 week HFD animals we reported that human islet grafts showed functional compensation, measured as a more than doubling of fasting human C-peptide in mouse serum, and histological adaptation of islet endocrine mass including increased beta cells. Further analysis of the human grafts revealed proliferation and neogenesis as the responsible mechanisms for the doubling of the human endocrine mass.Discussion: This novel model allows, for the first time, longitudinal studies of human islet adaptation to an obese murine environment and may be instrumental in deciphering pathways involved in human beta cell expansion, as well as in helping to identify factors predisposing human beta cells to undergo decompensation

Pancreatic islets are composed of endocrine cells that secrete hormones essential for blood-glucose homeostasis. Prior research has revealed that the gene expression and functionality of human islet cells is heterogenous. However, it is not currently understood how the heterogeneity correlates to normal islet cell function and dysfunction in diabetes pathogenesis. Subsequently, an international collaborative project has been initiated to elucidate what constitutes islet cell heterogeneity from a transcriptional, proteomic, and functional perspective in both health and disease. In this study, a highly multiplex tissue image assay was developed to allow for the study of the localization and distribution of proteins previously identified to correlate with functional activity and heterogeneity in islet cells using the CO-Detection by indEXing (CODEX) platform. In total, 22 proteins were studied simultaneously of which 10 were specifically expressed in islet cells. These included generic pancreatic markers such as C-peptide (C-pep) marking insulin-secreting β-cells, glucagon (GCG) and somatostatin (SST), but also less well-characterized proteins such as Shisa like 2B (FAM159B) and Neural proliferation, differentiation and control 1 (NPDC1). The multiplex tissue imaging allowed for single-cell analysis of protein expression in human islet cells showing that most islet specific proteins were heterogeneously expressed. The observations made in this study serves as a validation to that the human islet microenvironment is highly complex due to islet cell heterogeneity. Additionally, the study demonstrated that multiplex tissue imaging has the potential to reveal novel cell types and interactions.
Langerhanska öar består av endokrina celler som utsöndrar hormoner nödvändiga för reglering av blodsockernivåerna. Tidigare forskning har visat att genuttrycket och funktionaliteten är heterogen i de celler som utgör mänskliga Langerhanska öar. Dock är förståelsen för hur heterogeniteten korrelerar till normal cellfunktion och dysfunktion i diabetespatogenes fortfarande ofullständig. Följaktligen har ett internationellt samarbete inletts i syfte att  undersöka  vad som utgör heterogenitet  i  Langerhanska öar ur ett transkriptionellt, proteomiskt och funktionellt perspektiv i såväl friska som sjuka individer. I denna studie utvecklades en metod för multiplex mikroskopisk avbildning av vävnad för att möjliggöra undersökningen av hur proteiner som tidigare korrelerats med endokrin cellspecifik aktivitet och heterogenitet i Langerhanska öar var lokaliserade med hjälp av plattformen för Co-Detection by indEXing (CODEX). Totalt undersöktes 22 proteiner samtidigt varav 10 var specifikt uttryckta i celler som utgör Langerhanska öar. Bland dessa proteiner fanns generella markörer för vanligt förekommande celltyper i Langerhanska öar såsom C-peptid (C-pep) som markör för insulinsekreterande β-celler, glukagon (GCG) och somatostatin (SST) såväl som proteiner med färre kända funktioner såsom Shisa like B (FAM159B) och Neural proliferation, differentiation and control 1 (NPDC1). Med hjälp av multiplex mikroskopisk avbildning av vävnad kunde uttrycket av proteiner specifikt uttryckta i celler som utgör Langerhanska öar analyseras för enskilda celler i vävnaden. Denna analys visade att de flesta proteiner specifikt uttryckta i celler som Langerhanska öar består av var heterogent uttrycka. Resultaten från denna studie validerar att mikromiljön i Langerhanska öar är mycket komplex på grund av cellernas heterogenitet. Vidare visade denna studie att multiplex mikroskopisk avbildning av vävnad har potentialen att identifiera nya celltyper och interaktioner.

O Diabetes mellitus (DM) do tipo 1 é uma doença causada pela destruição, por mecanismo auto-imune, das células beta das ilhotas pancreáticas, produtoras de insulina. O tratamento convencional da doença é realizado por meio de injeções diárias de insulina exógena. O transplante de ilhotas pancreáticas inclui-se, atualmente, como uma das alternativas terapêuticas à insulinoterapia. Entretanto, para atingir a insulino-independência, é necessário transplantar um grande número de ilhotas por paciente. O conhecimento do mecanismo de proliferação das células beta pode possibilitar a realização do transplante a partir da expansão celular ex vivo. A glicose é um dos principais indutores da proliferação de células beta. Neste trabalho, foi estabelecida e executada a tecnologia de isolamento e purificação de ilhotas pancreáticas humanas, visando sua estimulação com glicose. Para identificar genes regulados por glicose nestas ilhotas, foi utilizada a técnica de hibridização subtrativa SSH, associada ao rastreamento da biblioteca através de macroarranjos de DNA. Num primeiro rastreamento, foram identificados dois fragmentos gênicos induzidos pela glicose. Um destes apresentou homologia com uma proteína hipotética humana de função desconhecida e o segundo com o receptor de polipetídeo pancreático. Este trabalho permitiu a identificação de novos genes regulados pela glicose em ilhotas pancreáticas humanas, os quais podem estar relacionados à proliferação celular deste tecido.
Type 1 Diabetes mellitus (T1DM) is caused by autoimmune destruction of the insulin-producing pancreatic islet b-cells. Treatment is generally approached by daily subcutaneous injections of exogenous insulin. Nowadays, pancreatic islet transplantation is considered as an effective alternative treatment to insulin therapy. However, in order to reach insulin-independence, a large number of islets is required for each patient. Knowledge of the mechanisms regulating islet b-cell proliferation may allow ex-vivo b-cell expansion prior to transplant. Glucose is considered one of the main inducers of islet b-cells proliferation. We established and executed the technology of human islet isolation and purification. The islets were then stimulated in culture with glucose. In order to identify glucose-regulated genes in cultured human islets, we utilized the suppression subtractive hybridization (SSH) method, followed by cDNA library screening by DNA macroarrays. Preliminary screening allowed us to isolate two cDNAs displaying glucose regulation, one of which is similar to a human hypothetical protein of unknown function and the other shows similarity to the pancreatic polypeptide receptor. This work allowed identification of glucose-regulated genes in human pancreatic islets, which may be related to cell proliferation in this tissue.

Islet transplantation is a relatively new treatment for Type I diabetes which offers great potential for normoglycaemia and ultimate avoidance of life- threatening complications(1). However, the treatment has only been partially successful and poor graft survival rates clinically have contrasted with reversal of diabetes by islet transplantation in rodent models. One major reason for graft loss is rejection of the islets and to study this an in vitro model for human allogeneic islet transplantation was developed. The aim of this study was to develop the mixed lymphocyte islet coculture (MLIC) and so investigate the in vitro human allogeneic lymphoproliferative response to untreated human islets. The possibility of developing a short duration MLIC as a pretransplant model for clinical use was initially investigated, and then the parameters and conditions of the MLIC response determined. Titration and kinetic studies of the MLIC (islet immunogenicity) and the MLAC (acinar tissue immunogenicity) showed that ten human islets or acinar tissue pieces (average 150mum diameter) cocultured with 1 x 10.;5 HLA mismatched responder PBLs for a duration of ninedays, gave an optimal response. Splenocyte, islet and acinar cell stimulator populations from the same donor source showed that the MLR was greater than the MLIC and the response of both were greater than the MLAC. It was shown that soluble products of untreated acinar cells inhibited lymphocyte proliferation in the MLR and may have led to a reduced MLAC response compared with that in the MLIC. Immunocytochemical investigations showed that upregulation of MHC class II antigen expression on acinar cells and induction on islets by cytokine treatment, did not enhance the stimulatory capacity in this model. The development of the MLIC as a model for in vitro islet immunogenicity showed that human islets can stimulate an allogeneic response which is not enhanced by the presence of freshly isolated acinar tissue.

The objective was to measure the effects of varying time points of pellet-induced hypoxic¹ exposure on human pancreatic islets through viability and function as compared to normoxic conditions. Hypoxic conditions were induced by storing the islets in pelletized conditions by which the islets lacked proper access to both oxygen and media. The islets were cultured for varying time points and then allowed to recover. The effects were measured upon immediate normalization of conditions as well as 24 or 48 hours later. When the islets were subjected for 12 hours, they showed an inability to recover through lack of both viability and function. The islets exposed to hypoxic conditions for only 4 hours were unable to recover any function, though no significant detriment to their viability was detectable. This would suggest that even as little as four hours of hypoxic exposure causes a naturally irreversible molecular change that damages cell function, even if no detectable lack of viability is present.

Diabetes mellitus is a heterogeneous group of metabolic diseases characterized by impaired blood glucose homeostasis that affects more than 415 million people worldwide and is a leading cause of mortality. The most prevalent form of diabetes is Type 2 Diabetes (T2D) that accounts for 90% of diabetes cases. An interplay of environmental and genetic risk factors contributes to etiology of T2D via a progressive loss of pancreatic beta cell function coupled with insulin resistance. Genome Wide Association Studies (GWAS) identified more than 400 independent genetic loci associated with T2D risk, although the molecular mechanisms underlying these genetic signals remain poorly understood. A comprehensive understanding of gene regulation in human pancreatic islets and identifying the role of T2D risk variants on different components of gene regulation will enlighten our insights into T2D etiology. In this work, we performed an in-depth characterization of human pancreatic islets transcriptional regulatory elements, attaining a greater granularity at transcriptional enhancers. We further identified glucose responsive enhancers which regulate glucose-dependent gene expression programs via three-dimensional chromatin interactions. This allowed us to gain insights into human islet transcriptional gene regulation and how glucose, a primary physiological stimulant of pancreatic islets, modulates human islet genome function. We also generated comprehensive transcriptome annotations in human islets using short- and long-read sequencing data along with accurate maps of transcriptional start sites. This revealed islet-specific promoters, transcript isoforms and novel coding sequences. This underscored the importance of generating transcript models in disease relevant tissue to progress in the understanding of gene regulation. Finally, these parallel efforts allowed us to create pioneer maps of genetic effects on human alternative splicing that revealed for the first time the noteworthy contribution of human islet mRNA splicing to T2D pathophysiology. These results have thus the potential to blossom in the discovery of novel T2D drug targets.

Type-2 diabetes (T2D), a multi-factorial disease characterised by chronic hyperglycaemia, is caused by a complex interaction between genetic and environmental factors. Genetic and phenotypic characterisations of diabetic patients suggest that a combination of β-cell failure, culminating in defective insulin secretion, as well as impairment of glucagon secretion in α-cells is central to the aetiology of the disease. Mouse models represent a valuable tool in such investigations. With the advent of large-scale genetic tools, a myriad of novel susceptibility loci associated with T2D have been identified. For many of these genes, it is unclear how genetic variation is linked to increased disease susceptibility. Our first study elucidates the implication of a transcription factor, SOX4, which is believed to underlie a T2D susceptibility locus (CDKAL1) in human. Using an N-ethyl-N-nitrosourea (ENU) mouse model, we explored β-cell function in mice carrying a point mutation in Sox4 (Sox4mt mice). This mouse strain displayed a significant reduction in glucose-stimulated insulin secretion (GSIS) that was associated with a 2-fold increase in wild-type Sox4 expression. The exocytotic events in mutant β-cells, as measured by single-vesicle (on-cell) capacitance measurements, suggested the presence of a persistent fusion pore. Subsequent failure of fusion pore expansion beyond the initial 1–2 nm results in an incomplete insulin release due to steric hindrance (insulin diameter: 3–4 nm). The proportion of full fusion events diminished in favour of kiss-and-run events in mutant β-cells. Stxbp6, which encodes amisyn, was shown to be the target gene of Sox4. Increased expression of amisyn, a protein previously shown to be involved in the stabilisation of the fusion pore in chromaffin cells, was observed in islets isolated from Sox4mt mice. The possible involvement of amisyn is further suggested by the finding that overexpression of amisyn mimicked the effect of the Sox4 mutation and resulted in reduced insulin secretion. Knockdown of amisyn expression restored the secretory defect in Sox4mt-overexpressing cells. Importantly, the effect of the Sox4 mutation was recapitulated by the overexpression of Sox4. Similar effects were obtained in the human β-cell line EndoC-βH2. We also observed a negative correlation between SOX4 expression and GSIS in a large collection of human islet preparations. There was also a positive correlation between SOX4 expression and STXBP6 (amisyn) expression and a tendency towards increased SOX4 expression in islets from organ donors with T2D. The second part of the thesis focuses on the role of the Krebs cycle enzyme fumarate hydratase (FH) in insulin release. Ablation of the Fh1 gene (which is initially implicated as a tumour suppressor in hereditary leiomyomatosis and renal cell cancer) in pancreatic β-cells led to a complete loss of GSIS, as determined by ex vivo pancreatic perfusion studies, although this was not associated with any detectable abnormalities in [Ca²⁺]_i homeostasis, ATP production or glucose oxidation. The phenotype was rescued by the introduction of the human orthologue FH into the cytosol alone or in both the cytoplasm and mitochondria of Fh1 knockout (Fh1KO) mice, confirming the role of Fh1 in insulin secretion. Moreover, the addition of exogenous glutamate, previously implicated as a 2^nd messenger between glucose metabolism and insulin secretion, corrected the insulin secretory defect in Fh1^-/- β-cells. We hypothesise that the loss of GSIS in Fh1KO mice results from enhanced anaplerosis, which is necessary to replenish Krebs cycle reactants. Consequently, this is followed by the depletion of the intracellular amino acid pool (including glutamate). Thus, our study demonstrates that the pancreatic Fh1KO mouse is a novel model of severe hyperglycaemia that harbours dysregulated metabolic features at the interface between both cancer and diabetes. The final study investigates the effect of ageing, a risk factor for T2D, on glucose-stimulated insulin and glucagon release. However, GSIS increased rather than decreased with ageing in both human and mouse islets (6 and 20 mmol/l glucose). Notably, ageing was not associated with reduced insulin content. Normal calcium homeostasis was observed in old (24-month-old) mice, demonstrating that the glucose sensing machinery was intact. In human islets, the inhibitory effect of glucose on glucagon secretion deteriorated with age. In the oldest group (>60 years of age), the inhibitory effect was completely abolished with 20 mmol/l glucose, while 6 mmol/l glucose only achieved less than 20% inhibition (which was not statistically significant). Our study reports the exciting possibility that hypersecretion of glucagon represents a link between senescence and increased diabetes risk.

Introduction : L'utilisation des îlots humains de Langerhans est la référence pour la recherche, tant physiologique que pour le développement de nouvelles molécules thérapeutiques pour le traitement du diabète de type 2. La demande d'îlots de Langerhans humains pour des projets de recherche est en constante augmentation, cependant, la disponibilité est limitée et les différentes préparations d'îlots de Langerhans révèlent une grande variabilité entre elles.Objectifs : L'objectif principal de cette thèse était de proposer une alternative aux îlots de Langerhans humains natifs qui permettrait d’obtenir des îlots pancréatiques homogènes et en quantité abondante pour les projets de recherche.Pour ce faire, nous avions deux objectifs principaux : 1) la production de pseudo-îlots de diamètre contrôlé à partir de pancréas humain, et l'évaluation de leur fonction in vitro et in vivo par rapport à leurs équivalents îlots natifs ; 2) l'optimisation de la production de cellules endocrines pancréatiques à partir de différentes lignées de cellules souches pluripotentes et l'évaluation des effets de la progérine sur la différenciation et la maturation des cellules produites. Les cellules souches pluripotentes utilisées provenaient de donneurs sains (H1, WiCell) et de patients atteints de Progeria (HGPS, iStem).Matériel et méthodes : Les pseudo-îlots ont été formés dans un milieu d'îlots clinique (CMRL 1066 albumine humaine, insuline) pendant 7 jours en utilisant les Sphericalplate 5D (Kulgelmeiers) et comparés aux îlots natifs J1 (jour 1) et J7 (jour 7) du même donneur.La différenciation des cellules souches pluripotentes (cellules iPS DF19.9, H1 et iPS HGPS) a été optimisée par différents protocoles : le protocole Rezania, le SD Kit (StemCell Technologies) et le protocole Nostro. L'expression des gènes de maturation in vitro entre différentes lignées cellulaires a été évaluée par qPCR. L'expression des protéines a été évaluée par immunofluorescence et par cytométrie en flux (plateforme EGID).Pour la maturation in vivo, après la transplantation sous la capsule rénale de souris immunodéficientes, des mesures de glycémie et de c-peptide humain ont été effectuées, ainsi que des tests métaboliques comme l'ipGTT.12Résultats : Les pseudo-îlots (n=4) générés ont sécrété significativement moins d'insuline in vitro que les îlots natifs à J1 mais sans différence significative avec les îlots natifs à J7. Dans les deux groupes à J7, on a observé une diminution significative de l'insuline intracellulaire comparativement aux îlots natifs à J1. In vivo, les îlots natifs à J1 sécrètent significativement plus de c-peptide humain que les îlots natifs à J7, alors que la différence n'est pas significative entre les îlots natifs à J1 et les pseudo-îlots à J7. De plus, l'analyse morphométrique des greffons a révélé que les pseudo-îlots ont tendance à avoir plus de cellules glucagon-positives que les deux autres groupes.L'optimisation de la différenciation des cellules souches pluripotentes a permis d'obtenir plus de 95% d'endoderme pour les cellules H1 et 80% pour les cellules iPS HGPS. Pour les deux lignées, nous avons généré 95 % de progéniteurs pancréatiques. La comparaison des gènes de maturation a révélé que la progérine conduisait à une légère augmentation de la maturation cellulaire dans le groupe iPS HGPS par rapport aux cellules H1. Des marqueurs liés à l'âge (53BP1, IGF1r et yH2AX) ont été validés dans un pancréas provenant d'un donneur âgé et un insulinome. Cependant, aucune différence de la fonctionnalité in vivo n’a été observée. Six mois post transplantation, nous avons identifié yH2AX dans des cellules endocrines et non endocrine des greffons H1 alors que dans les greffons HGPS, nous l’avons observé dans une plus vaste proportion de cellules présentant différentes formes de noyaux [...]
Introduction: The use of human islets of Langerhans is the gold standard for research, both for physiological research and for the development of new therapeutic molecules for the treatment of type 2 diabetes. The demand of human islets for research projects is constantly growing however, the availability is limited and different islet preparations show significant variability between human pancreata.Objectives: The main objective of this thesis was to propose an alternative to native human islets that can provide homogeneous and abundant pancreatic islets for research. To do this, we had two main objectives: 1) the production of controlled diameter pseudo-islets from human pancreata, and the evaluation of their function in vitro and in vivo compared to their native islet counterparts; 2) the optimization of the production of pancreatic endocrine cells from different pluripotent stem cell lines and evaluation of the impact of progerin on the differentiation and maturation of the cells produced. Pluripotent stem cells from healthy donors (H1, WiCell) and from patients affected with accelerated aging disease Progeria (HGPS, iStem).Material and Methods: The pseudo-islets were formed in clinical islet medium (CMRL 1066 human albumin, insulin) 7 days using the 5D Sphericalplate (Kulgelmeiers) and compared to the native islets D1 (day 1) and D7 (day 7) from the same donor.The differentiation of pluripotent stem cells (iPS DF19.9, H1 and iPS HGPS cells) was optimized using different protocols: the Rezania protocol, the SD Kit (StemCell Technologies) and the Nostro protocol. For in vitro maturation gene expression among different cell lines was evaluated by qPCR. Protein expression was assessed by immunofluorescence technique and Flow cytometry analysis (EGID).For in vivo maturation, after transplantation under the kidney capsule of immunodeficient mice, blood glucose and human c-peptide measurements were assessed as well as metabolic test such as IPGTT were performed.Results: The pseudo-islets (n=4) generated in clinical islet medium secreted significantly less insulin in vitro than the native islets at D1 but with no significant difference from the native islets at D7. In both groups at D7, a significant decrease in intracellular insulin was observed compared10to native islets at D1. In vivo, the native islets at D1 secrete significantly more human c-peptide than the native islets at D7, while the difference is not significant between the native islets at D1 and the pseudo-islets at D7. In addition, morphometric analysis of the grafts revealed that the pseudo-islets tend to have more glucagon positive cells than the other two groups.Optimization of the differentiation of pluripotent stem cells allowed us to obtain more than 95% endoderm for H1 cells and 80% for iPS HGPS cells. For both lines, we generated 95% of pancreatic progenitor cells. The comparison of maturation genes revealed that progerin lead to a slight increase of cell maturation in the iPS HGPS group compared to H1 cells. However, no differences in in vivo function was observed. Age-related markers (53BP1, IGF1r, p16 and yH2AX) which validated in a pancreas from an elderly donor and an insulinoma. We identified yH2AX after 6 months transplantation of H1-grafts in endocrine and non-endocrine cells, while the expression in iPS HGPS-grafts appeared in the majority of cells, which had various shape of nucleiConclusion: This work provided positive results in terms of functional pseudo-islets and stem cells derived pancreatic endocrine cells. However, they remain preliminary and further studies must be conducted to provide realistic alternatives to native human islets for research

Human pancreatic islet transplantation presents an attractive method for type I diabetes cellular therapy. However, there remain several limitations that should be addressed in order to increase the efficacy of the transplantation procedure; the most important of which are addressing the shortage of available pancreatic tissue and the viability of post-isolation islets. These obstacles are overcome through methods directed at long-term in vitro preservation, culture, and expansion of post-isolation islets in order to arrive at functionally viable islet populations for transplantation purposes. This thesis presents a novel system that promotes in vitro human pancreatic islet preservation and culture in a controlled microenvironment that is monitored noninvasively. The development of this system is accomplished in four consecutive stages: i) optimization of a two-dimensional surface-modified extracellular matrix (ECM) substrate, ii) fabrication of a geometrically controlled and interconnected scaffold suitable for islet culture, iii) three-dimensional in vitro culture incorporating the optimized ECM components and fabricated scaffold, and iv) incorporating the three-dimensional microenvironments within a multi-welled perfusion bioreactor, coupled with dielectric measurement electrodes, for the long-term in vitro culture of human pancreatic islets. Several novelties attributed to the developed system are also presented. The two-dimensional studies revealed fibronectin to enhance glucose-dependent insulin functionality and morphology, while collagen I/IV contribute to adhesion. The microfabricated Poly(lactic-co-glycolic acid) (PLGA) scaffolds were constructed to give suitable pore structures and full interconnectivity, in a reproducible geometrically controlled manner. Furthermore, the incorporation of the optimized ECM components into the scaffolds was accomplished through islet embedding in an ECM laden gel that is seeded within the microfabricated scaffold pore structures. The three-dimensional microenvironment encouraged long-term human islet culture, giving high insulin release indices of ~1.8, while increasing islet gene expression. Finally, a fabricated multi-well perfusion bioreactor, equipped with an electrical impedance dielectric spectroscopy monitoring system, was employed in the controlled three-dimensional culture of the isolated islets. This system was successful in the long-term monitoring of human pancreatic islet differentiation and redifferentiation in a controlled three-dimensional microenvironment, yielding a population that is characteristically, morphologically, and functionally analogous to freshly isolated islets.
La transplantation humaine d'îlots pancréatiques se présente comme une méthode intéressante pour le traitement du diabète de type I au niveau cellulaire. Cependant, plusieurs problèmes persistants limitent l'efficacité de la procédure de transplantation; les principaux sont le manque de tissu pancréatique disponible et la viabilité des îlots après leur isolement. Ces obstacles sont surmontés par des méthodes de préservation, de culture, et d'expansion in vitro à long terme d'îlots isolés afin de parvenir à des populations d'îlots fonctionnellement viables pour des fins de transplantation. Cette thèse présente un nouveau système qui favorise la préservation et la culture in vitro d'îlots pancréatiques humains dans un microenvironnement contrôlé et surveillé de manière non invasive. Le développement de ce système est réalisé en quatre étapes successives : i) l'optimisation d'un substrat 2D de matrice extracellulaire modifié en surface, ii) la fabrication d'un échafaudage interconnecté à géométrie contrôlée, élément approprié à la culture des îlots, iii) la culture in vitro 3D intégrant le substrat de la matrice optimisé et l'échafaudage fabriqué, et iv) l'intégration des microenvironnements en trois dimensions au sein d'un bioréacteur à perfusion multi-sources, couplé avec des électrodes de mesure diélectrique pour la culture in vitro à long terme des îlots pancréatiques humains. Plusieurs nouveautés attribuées au système développé sont également présentées. Les études 2D révèlent que la fibronectine améliore la fonctionnalité et la morphologie de l'insuline dépendant du glucose, tandis que les collagènes I/IV contribuent à l'adhésion. Les échafaudages en PLGA (acide poly-lactique-co-glycolique) micro-fabriqués ont été élaborés de façon à fournir des structures poreuses et une inter-connectivité complète de manière reproductible et géométriquement contrôlée. De plus, l'incorporation de composants optimisés de matrices extracellulaires dans les échafaudages a été accomplie grâce à un enrobage des îlots dans un gel de matrices extracellulaires semé dans les structures poreuses d'échafaudages micro-fabriqués. Le microenvironnement 3D a favorisé la culture à long terme d'îlots humains, donnant des indices élevés de libération d'insuline d'approximativement 1.8, tout en augmentant l'expression des gènes des îlots. Enfin, un bioréacteur à perfusion multi-sources, équipé d'un système de surveillance d'impédance électrique par spectroscopie diélectrique, est utilisé pour la culture 3D contrôlée des îlots isolés. Ce système permet de surveiller à long terme la différenciation et la re-différenciation des îlots pancréatiques humains dans un microenvironnement 3D contrôlé, donnant une population dont les caractéristiques morphologiques et fonctionnelles en particulier équivalent à celles d'îlots fraîchement isolés.

The process of β-cell destruction that leads to type 1 diabetes (T1D) is incompletely understood and it is believed to be a result of both genetic and environmental factors. Enterovirus (EV) infections of the β-cells have been proposed to be involved, however, the effects of EV infections on human β-cells have been little investigated. This thesis summarises studies of three different Coxsackie B4 virus strains that have previously been shown to infect human islets. The effects of infections with these EV were studied in vitro in human islets and in a rat insulin-producing cell line. In addition, a pilot study was performed on isolated human islets to investigate the ability to treat such infections with an antiviral compound.

It was found that one of the virus strains replicated in human β-cells without affecting their main function for at least seven days, which in vivo may increase a virus’s ability to persist in islets.

Nitric oxide was induced by synthetic dsRNA, poly(IC), but not by viral dsRNA in rat insulinoma cells in the presence of IFN-γ, suggesting that this mediator is not induced by EV infection in β-cells and that poly(IC) does not mimic an EV infection in this respect.

All three virus strains were able to induce production of the T-cell chemoattractant interferon-γ-inducible protein 10 (IP-10) during infection of human islets, suggesting that an EV infection of the islets might trigger insulitis in vivo.

Antiviral treatment was feasible in human islets, but one strain was resistant to the antiviral compound used in this study.

To conclude, a potential mechanism is suggested for the involvement of EV infections in T1D. If EV infections induce IP-10 production in human islet cells in vivo, they might recruit immune cells to the islets. Together with viral persistence and/or virus-induced β-cell damage, this might trigger further immune-mediated β-cell destruction in vivo.

De l'isolement des îlots pancréatiques à leur implantation, l'inflammation est omniprésente au cours de la transplantation d'îlots pancréatiques. Le maintien d'une inflammation contrôlée est essentiel pour préserver la survie et la fonctionnalité du greffon à court et long terme. L'objectif de ce travail de thèse est d'identifier précisément les mécanismes inflammatoires à l'origine de la perte précoce des îlots et de déterminer des cibles thérapeutiques pour limiter ces réactions inflammatoires.Nous avons ainsi démontré que les conditions de culture induisent des réactions à l'origine du développement d'un phénotype pro-inflammatoire et pro-oxydant propre à l'îlot. Cette induction se caractérise par une élévation de la sécrétion de cytokines, de chimiokines pro-inflammatoires, une activation des voies de l'inflammation Toll-like récepteurs (TLRs)-dépendantes et une génération d'espèces réactives de l'oxygène (ROS). Toutefois, ce processus peut être prévenu par l'activation de l'Hème oxygénase-1 (HO-1), une enzyme anti-oxydante et anti-inflammatoire.Par l'étude des réactions inflammatoires sur un modèle animal de transplantation mimant les conditions de transplantation humaine, nous avons démontré qu'un changement des médiateurs plasmatiques de l'inflammation et du protéome hépatique s'opère 12 heures après transplantation. De plus, ces résultats sont associés à une infiltration des îlots par les cellules immunitaires qui s'organise 12 heures après transplantation. Nous avons également établi le rôle anti-inflammatoire de la rapamycine (une drogue immunomodulatrice) sur les îlots et les macrophages in vitro. Nous avons ainsi démontré que l'usage de la rapamycine avec la mise en place d'un pré-traitement des îlots et du receveur avant la greffe serait envisageable. Ces travaux ont permis de caractériser les mécanismes inflammatoires mis en oeuvre immédiatement avant et après transplantation. Ainsi, ces données offrent de nouvelles pistes thérapeutiques susceptibles de prévenir et/ou limiter l'inflammation au cours de la transplantation d'îlots pancréatiques.

Le diabète de type 2 est une pathologie chronique complexe associant une altération de sécrétion de l'insuline par le pancréas et une résistance à l'insuline au niveau des tissus périphériques, notamment au niveau du foie et du muscle squelettique. Son origine est multifactorielle, alliant des anomalies génétiques et environnementales, en particulier nutritionnelles. Un des mécanismes par lesquels les facteurs nutritionnels (comme les glucides et les lipides en excès) contribuent au développement du diabète et à son aggravation est la glucolipotoxicité. En effet, l'élévation de la glycémie et des lipides plasmatiques, ainsi que l'accumulation ectopique de lipides dans les tissus, participent au développement de l'insulinorésistance hépatique et musculaire et aux dysfonctions des cellules bêta, en partie via l'induction d'un stress métabolique, impliquant notamment le stress oxydant, le stress du réticulum endoplasmique (RE) et la perturbation de l'homéostasie calcique. Nous avons étudié les effets de la glucotoxicité et de la lipotoxicité dans les cellules bêta pancréatiques et les mécanismes impliqués. Nous nous sommes aussi intéressés à des traitements potentiellement protecteurs de la fonction bêta-pancréatique. Nous avons fait l'hypothèse que les effets bénéfiques de l'inhibition du système rénine-angiotensine sur l'incidence du diabète de type 2 chez l'homme étaient médiés par une action directe sur les cellules bêta. Nos résultats montrent que le glucose chronique à une dose élevée entraine une réduction de la sécrétion d'insuline des cellules bêta des îlots de Langerhans humains par une action conjointe sur le stress du RE, le stress oxydant et l'homéostasie calcique. L'inhibition du SRA a permis de restaurer cette fonction grâce notamment à une action inhibitrice sur la voie Phospholipase C-IP3-Calcium
This study addressed the hypothesis that inhibiting the soluble epoxide hydrolase (sEH)-mediated degradation of epoxy-fatty acids, notably epoxyeicosatrienoic acids, has an additional impact against cardiovascular damage in type 2 diabetes, beyond its previously demonstrated beneficial effect on glucose homeostasis. The cardiovascular and metabolic effects of the sEH inhibitor t- AUCB (10 mg/l in drinking water) were compared to those of the sulfonylurea glibenclamide (80 mg/l), both administered for 8 weeks in FVB mice subjected to a high-fat diet (HFD, 60% fat) for 16 weeks. Mice on control chow diet (10% fat) and non-treated HFD mice served as controls. Glibenclamide and t-AUCB similarly prevented the increased fasting glycemia in HFD mice but only t-AUCB improved glucose tolerance and decreased gluconeogenesis, without modifying weight gain. Moreover, t-AUCB reduced adipose tissue inflammation, plasma free fatty acids and LDL cholesterol, and prevented hepatic steatosis. Furthermore, only the sEH inhibitor improved endothelium-dependent relaxations to acetylcholine, assessed by myography in isolated coronary arteries. This improvement was related to a restoration of epoxyeicosatrienoic acid and nitric oxide pathways, as shown by the increased inhibitory effects of the NO-synthase and cytochrome P450 epoxygenase inhibitors, L-NA and MSPPOH, on these relaxations. Moreover, t-AUCB decreased cardiac hypertrophy, fibrosis and inflammation, and improved diastolic function, as demonstrated by the increased E/A ratio (echocardiography) and decreased slope of the enddiastolic pressure-volume relation (invasive hemodynamics). These results demonstrate that she inhibition improves coronary endothelial function and prevents cardiac remodeling and diastolic dysfunction in obese type 2 diabetic mice

Diabetes mellitus tipo 1 resulta da produção insuficiente ou da ausência de insulina, decorrente da destruição de células β, por mecanismo auto-imune. O tratamento deste tipo de diabetes consiste na administração subcutânea de insulina exógena. Recentemente, foi demonstrado que o transplante de ilhotas pancreáticas é capaz de tornar o portador de diabetes tipo 1 independente de insulina exógena. Apesar do sucesso alcançado, a necessidade permanente de imunossupressão é uma das principais barreiras para que o transplante de ilhotas possa ser realizado em número maior de pacientes. Assim, o desenvolvimento de novas metodologias que evitem a rejeição do enxerto, como o macro e o microencapsulamento de ilhotas, continua sendo crucial para o estabelecimento definitivo do transplante de ilhotas como opção terapêutica no tratamento de diabetes tipo 1. Neste trabalho, foi padronizado um modelo animal para avaliar, in vivo, a funcionalidade das ilhotas pancreáticas humanas isoladas e purificadas na Unidade de Ilhotas Pancreáticas Humanas do IQUSP. Ratos NIH nude foram tornados diabéticos através de injeção de estreptozotocina para o implante de ilhotas pancreáticas humanas nuas e microencapsuladas. As ilhotas foram microencapsuladas em Biodritina, um novo heteropolissacarídeo patenteado e cedido ao nosso laboratório, tendo sido possível padronizar a produção de microcápsulas uniformes e homogêneas, com tamanho médio entre 400µm e 600 µm. A reversão do diabetes ocorreu em 24% dos ratos nude transplantados com ilhotas pancreáticas humanas nuas. Por outro lado, não observamos reversão do diabetes quando ilhotas encapsuladas foram implantadas, apesar do teste de atividade funcional realizado in vitro ter demonstrado que elas continuam a secretar insulina e a responder ao estímulo com glicose após o encapsulamento. Para elucidar este efeito, cápsulas vazias foram implantadas em ratos nude e em ratos imunocompetentes, os quais desenvolveram processo inflamatório acompanhado de processo fibrótico no local do implante. Estudo imuno-histoquímico está sendo realizado para esclarecer a natureza e a intensidade destes processos.
Type 1 diabetes mellitus results from insufficient or absence of insulin production, as a consequence of destruction of pancreatic β cells, by an auto-imune mechanism. Treatment for this type of diabetes consists of subcutaneous administration of exogenous insulin. Recently, it has been demonstrated that pancreatic islet cell transplantation is capable of rendering type I diabetic patients independent of exogenous insulin. However, in spite of the success achieved, permanent immunosuppression is still required, being the main barrier to expand this treatment to a large number of patients. Therefore, development of new technologies, such as islet macro and microencapsulation to avoid rejection of the tissue implanted, is still crucial for definitive establishment of islet transplantation as a therapeutic alternative for type I diabetes. In the present work, an animal model was established for in vivo evaluation of the functional ability of human pancreatic islets, which were isolated and purified at the Human Pancreatic Islet Unit of the University of São Paulo Chemistry Institute. Diabetes was induced in NIH nude rats through streptozotocin injection followed by implantation of naked or microencapsulated human pancreatic islets. Biodritin, a new and patented heteropolyssaccaride was used to microencapsulate the islets. The production of uniform and homogeneous microcapsules with diameters in the range of 400µm e 600 µm was successfully established. Reversion of diabetes occurred in 24% of the nude rats transplanted with human pancreatic islets. On the other hand, no reversion of diabetes was observed when encapsulated islets were implanted, although their functional activity in vitro indicated that they secreted insulin and responded to glucose stimulation upon encapsulation. In order to elucidate this effect, empty capsules were implanted in nude rat and in immunocompetent rats, both of which developed an inflammatory process accompanied by a fibrotic process in the site of the implant. Immunohistochemical studies are underway to address the nature and the intensity of these inflammatory processes.

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Amylin is a 37-amino acid peptide usually cosecreted with insulin from pancreatic islet β-cells. It is implicated in the regulation of normal glucose metabolism and thought to induce pathological features of non-insulin-dependent diabetes mellitus (NIDDM). In particular, human amylin (hA) deposits as islet amyloid, and is associated with the loss of insulin-producing islet β-cells in NIDDM. The biochemical mechanism of hA-evoked death in cultured RINm5F pancreatic islet β-cells has been investigated in this thesis. Synthetic hA but not rat amylin (rA) aggregated in aqueous solution, formed fibrils, and evoked β-cell death in a time- and concentration-dependent manner. The cell death exhibited apoptotic features, including inter-nucleosomal DNA fragmentation, mitochondrial dysfunction, delayed membrane lysis, aurintricarboxylic acid suppression and cell membrane blebbling. Cytotoxicity of hA was inhibited by Congo red (an amyloid-binding dye), 8-37hA fragment (fibril-forming but non-toxic), 1-40βA or 25-35βA (Alzheimer-associated peptide), but neither by sorbitol (inhibitory to hA fibril formation), rA nor its 8-37rA peptide (non-fibril-forming and non-toxic). Preformed large amyloid deposits of hA were less potent in causing β-cell death than small aggregates. These data suggest that hA induces β-cell apoptosis via small aggregates through a possible membrane receptor pathway. Inhibitors of protein and mRNA synthesis did not inhibit hA-evoked apoptosis, but rather enhanced or directly triggered β-cell death during prolonged exposure. Likewise, Ca2+ modulators, which alter intracellular free Ca2+ concentration ([Ca2+]i), failed to prevent hA cytotoxicity and were ultimately cytotoxic themselves. Fura-2 loading and 45Ca2+ uptake studies indicated that hA did not mobilise intracellular Ca2+ during its toxicity. These results indicate a protein synthesis- and Ca2+-independent process of hA toxicity RINm5F islet β-cells. The studies reported in this thesis have established a new in vitro model of hA-evoked apoptosis using cultured RINm5F pancreatic islet β-cells. A new model of NIDDM pathogenesis is presented and discussed.

Additionally, cyclosporin A, an inhibitor of the mitochondrial permeability transition (MPT) pore, failed to prevent hIAPP-induced DeltaPsim collapse, cytochrome c and AIF release and caspase-3 activation, indicating that the MPT pore was not involved in hIAPP-induced apoptosis. On the other hand, potential crosstalk between the extrinsic and intrinsic apoptotic pathways was demonstrated by cleavage of Bid by caspase-8 in the apoptotic process triggered by hIAPP.
It is widely accepted that human islet amyloid polypeptide (hIAPP) aggregation plays an important role in the loss of insulin-producing pancreatic beta cells. Insulin secretion impairment and cell apoptosis can be due to mitochondrial dysfunction in pancreatic beta cells. hIAPP-induced cytotoxicity is mediated by the generation of reactive oxygen species (ROS). Phycocyanin (PC) is a natural compound from blue-green algae that is widely used as food supplement. Currently, little information is available about the effect of hIAPP on mitochondrial function of beta cells and protection of PC against hIAPP-induced cytotoxicity. In this thesis, I hypothesize that hIAPP may impair beta cell function with the involvement of mitochrondrial dysfunction, and this effects could be attenuated by PC. Therefore, the aim of this study was to investigate the role of mitochondria in hIAPP-induced apoptosis, the in vitro protective effects of PC and explore the underlying mechanisms.
It was found that hIAPP induced apoptosis in INS-1E cells with the disruption of mitochondrial function, as evidenced by ATP depletion, mitochondrial mass reduction, mitochondrial fragmentation and loss of mitochondrial membrane potential (DeltaPsim). Further molecular analysis showed that hIAPP induced changes in the expression of Bcl-2 family members, release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria into cytosol, activation of caspases and cleavage of poly (ADP-ribose) polymerase. Interestingly, the hIAPP-induced mitochondrial dysfunction in INS1-E cells was effectively restored by co-treatment with PC.
Our results showed that hIAPP inhibited the INS-1E cell growth in a dose-dependent manner. However, cytotoxicity of hIAPP was significantly attenuated by co-incubation of the cells with PC. hIAPP induced DNA fragmentation and chromatin condensation, which were key characteristics of cell apoptosis. These changes were inhibited by PC as examined by TUNEL assay and DAPI staining. Moreover, PC significantly prevented the hIAPP-induced overproduction of intracellular ROS and malonaldehyde (MDA), as well as changes of activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzymes. Furthermore, hIAPP triggered the activation of mitogen-activated protein kinases (MAPKs) such as c-Jun N-terminal kinase (JNK) and p38 kinase, and these effects were effectively suppressed by PC.
Taken together, I have demonstrated for the first time the involvement of mitochondrial dysfunction in hIAPP-induced INS-1E cell apoptosis, which was attenuated by PC through attenuating oxidative stress, modulating JNK and p38 pathways and reducing mitochondrial dysfunction.
Li, Xiaoling.
Adviser: Juliana Chung Ngor Chan.
Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: .
Thesis (Ph.D.)--Chinese University of Hong Kong, 2010.
Includes bibliographical references (leaves 150-159).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

碩士
高雄醫學大學
醫學研究所
96
Type 1 diabetes is an autoimmune destruction of pancreatic islet cell disease. It is an important development goal to find new alternative source of the islet cells to replace the damaged cells. Human embryonic stem (hES) cells are derived from the isolated inner cell mass of blastocysts, and they possess pluripotent potential and self-renewal capacity. The Embryoid Bodies (EBs) from hES cells were first formed by the suspension culture method, and the EBs were then treated with series of stimulus-induction to produce the pancreatic islet-like cells in vitro. The pancreatic islet-like cells were shown to express pancreatic specific markers of insulin, glucagons and somatostatin, and so on. The expression profiles of mRNAs and microRNAs from hES cells, EBs and pancreatic islet-like cells were further analyzed. MicroRNAs are endogenous small noncoding RNAs, and they negatively regulate the expression of protein-coding mRNAs. In this study, pancreatic islet-like cell were found to exhibit high expression of miR-186, 199a and 339, which possibly down-regulated the expression of undifferentiated markers such as LIN28. These islet-like cells were also found to have low expression of miR-19b, 20b, 221, 222, 92, 375, 200c, 302d, 367 and 372, which possibly resulted in the high expression of CNTN4, PIK3R1 and C11orf 9. Therefore, microRNAs are likely to play important regulatory roles in the cell differentiation and early embryonic development.

Another growth factor candidate is a recently recognized bioactive peptide, islet-neogenesis associated protein (INGAP). A master pancreatic transcription factor, pancreatic duodenal homeobox-1 (Pdx-1), was overexpressed in PSCs by the adenovirus-mediated transfer method in the present study. With the infection of adenovirus expressing Pdx-1, several beta-cell developmental genes, including Isl-1, Beta2, Nkx2.2, Nkx6.1 and the endogenous Pdx-1 were found to be upregulated temporally in our PSCs-derived ICCs. Meanwhile, previous study has shown that Pdx-1/INGAP-positive cells represent a new stem cell subpopulation during early stage of pancreatic development. We thus explore whether any functional integration of Pdx-1 and INGAP in the growth and functional maturation of PSCs. In order to achieve this proposition, the effects of over-expressing PSCs with the Pdx-1 adenovirus in conjunction with the treatment of INGAP were then investigated. Interestingly, differentiation of the PSC-derived ICCs was not further enhanced by the synergistic treatment of Pdx-1 and INGAP when compared to those ICCs infected with adenovirus expressing Pdx-1 alone, as revealed by the endogenous Pdx-1 and insulin gene expression and their C-peptide content. These data might provide some clues to the intricate interaction between Pdx-1 and INGAP in regulating the ICC and/or the pancreatic endocrine differentiation. (Abstract shortened by UMI.)
Due to the scarcity of fetal pancreas for generating functional insulin-secreting cell clusters for sufficient islet transplantation, we targeted for searching pancreatic stem/progenitor cells. Putative PSCs can be aggregated and differentiated into islet-like cell clusters (ICCs) when exposed to serum-free medium containing various conventional growth factors, including HGF, GLP-1, betacellulin and nicotinamide.
Fetal pancreatic tissue consisting of immature progenitor cells serves as a potential source of stem cells as they possess a higher replicative capacity and longevity than their adult counterparts.
Two novel candidates and a key pancreatic transcription factor on the PSC/ICC proliferation and differentiation were investigated in the present study. One of them is a ubiquitously expressed multi-PDZ-domain protein, PDZ-domain-containing 2 (PDZD2), which was previously found to express in the mouse beta cells and exhibit mitogenic effects in beta cell line. Results showed that PDZD2 was detected in high levels in both human fetal pancreas and in PSCs. Results indicate the potential involvement of PDZD2 in regulating PSCs proliferation and differentiation and pancreatic development.
Suen Po Man, Ada.
"July 2007."
Adviser: P.S. Leung.
Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0051.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (p. 194-214).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
School code: 1307.

Ma, Man Ting.
"July 2010."
Thesis (M.Phil.)--Chinese University of Hong Kong, 2010.
Includes bibliographical references (leaves 186-207).
Abstracts in English and Chinese.
Abstract --- p.I
List of Publications --- p.VI
Acknowledgements --- p.VIII
Table of Contents --- p.X
List of Figures --- p.XV
List of Tables --- p.XVIII
List of Abbreviations --- p.XIX
Chapter CHAPTER1 --- INTRODUCTION
Chapter 1.1 --- The Pancreas --- p.2
Chapter 1.1.1 --- Structure of pancreas --- p.2
Chapter 1.1.2 --- Structure and function of exocrine pancreas --- p.6
Chapter 1.1.3 --- Structure and function of endocrine pancreas --- p.9
Chapter 1.1.3.1 --- Pancreatic islet and islet cells --- p.9
Chapter 1.1.3.2 --- Glucose-stimulated insulin secretion from islets --- p.12
Chapter 1.2 --- Type 1 Diabetes Mellitus (T1DM) --- p.14
Chapter 1.2.1 --- Pathophysiology of Diabetes Mellitus --- p.14
Chapter 1.2.2 --- Autoimmunity in T1DM --- p.17
Chapter 1.2.3 --- Management ofTlDM --- p.20
Chapter 1.2.3.1 --- Insulin replacement --- p.20
Chapter 1.2.3.2 --- Pancreas and islet transplantation --- p.21
Chapter 1.2.3.3 --- Stem-cell-based transplantation --- p.22
Chapter 1.3 --- The Adaptive Immune System --- p.26
Chapter 1.3.1 --- T-lymphocytes --- p.26
Chapter 1.3.2 --- B-lymphocytes --- p.29
Chapter 1.3.3 --- Major histocompatibility complex (MHC) --- p.30
Chapter 1.3.3.1 --- Classification of MHC molecules --- p.30
Chapter 1.3.3.2 --- Structure of MHC class I and II molecules --- p.32
Chapter 1.3.3.3 --- Function and regulation of MHC molecules --- p.34
Chapter 1.3.4 --- HLA-G and its immuno-modulatory properties --- p.36
Chapter 1.4 --- Transplantation Rejection --- p.40
Chapter 1.4.1 --- Mechanisms involved in transplantation rejection --- p.40
Chapter 1.4.2 --- Immunobiology of rejection --- p.41
Chapter 1.4.2.1 --- Direct allorecognition pathway --- p.42
Chapter 1.4.2.2 --- Indirect allorecognition pathway --- p.43
Chapter 1.4.2.3 --- Semi-direct allorecognition pathway --- p.43
Chapter 1.4.3 --- Xenotransplantation --- p.46
Chapter 1.5 --- Cytokines and Immunity --- p.48
Chapter 1.5.1 --- Interferons --- p.48
Chapter 1.5.1.1 --- Interferon-γ and its immune regulation --- p.49
Chapter 1.5.1.2 --- Effect and kinetics of interferon-γ on MHC molecules expression --- p.53
Chapter 1.5.1.3 --- Regulation of interferon-γ production --- p.56
Chapter 1.5.2 --- Interlukins --- p.58
Chapter 1.5.2.1 --- IL-10 and its immune regulation --- p.58
Chapter 1.5.2.2 --- IL-10 and HLA-G --- p.59
Chapter 1.6 --- Stem Cells and their Immunogenicity --- p.62
Chapter 1.6.1 --- Embroynic stem cells --- p.62
Chapter 1.6.2 --- Mesenchymal stem cells --- p.64
Chapter 1.6.3 --- Neural stem cells --- p.68
Chapter 1.6.4 --- Fetal stem cells --- p.69
Chapter 1.6.5 --- Potential immuno-study in human fetal pancreatic stem cells --- p.70
Chapter 1.7 --- Aims and Objectives of study --- p.72
Chapter CHAPTER2 --- MATERIALS AND METHODS
Chapter 2.1 --- Isolation of Pancreatic Progenitors (PPCs) from Human Fetal Pancreas and Induction of Islet-like Cell Cluster (ICCs) Differentiation --- p.75
Chapter 2.1.1 --- Tissue procurement --- p.75
Chapter 2.1.2 --- Tissue processing and PPCs culture --- p.75
Chapter 2.1.3 --- In vitro differentiation of PPCs into ICCs --- p.78
Chapter 2.1.4 --- Interferon-γ and IL-10 treatment --- p.80
Chapter 2.2 --- Cell culture of human placental Choriocarcinoma JEG-3 Cell Line --- p.81
Chapter 2.3 --- RNA Expression Detection --- p.82
Chapter 2.3.1 --- RNA isolation --- p.82
Chapter 2.3.2 --- Reverse transcriptase (RT) --- p.83
Chapter 2.3.3 --- Design of primers for Polymerase Chain Reaction (PCR) and Real-time PCR --- p.84
Chapter 2.3.4 --- PCR --- p.86
Chapter 2.3.5 --- Real-time PCR analysis --- p.88
Chapter 2.3.6 --- Calculation using the comparative CT method --- p.90
Chapter 2.4 --- Flow Cytometry --- p.91
Chapter 2.5 --- Western Blotting Analysis --- p.93
Chapter 2.5.1 --- Protein extraction and quantification --- p.93
Chapter 2.5.2 --- Western blotting --- p.93
Chapter 2.6 --- Mixed Lymphocyte Reaction (MLR) --- p.95
Chapter 2.6.1 --- Isolation of peripheral blood mononuclear cells (PBMCs) --- p.95
Chapter 2.6.2 --- PPC-PBMCs MLR --- p.98
Chapter 2.6.3 --- ICC-PBMCs MLR --- p.98
Chapter 2.6.4 --- Proliferation assay --- p.99
Chapter 2.7 --- ICC Transplantation --- p.101
Chapter 2.7.1 --- Streptozotocin-induced diabetic animals for transplantation --- p.101
Chapter 2.7.2 --- Procedures of ICCs transplantation --- p.102
Chapter 2.8 --- Histological Analysis of ICC Graft --- p.105
Chapter 2.8.1 --- H&E staining --- p.105
Chapter 2.8.2 --- DAB staining --- p.106
Chapter 2.8.3 --- Immunofluorescence staining --- p.107
Chapter 2.9 --- Enzyme-linked Immunosorbent Assay (ELISA) --- p.109
Chapter 2.10 --- Statistical Data Analysis --- p.110
Chapter CHAPTER3 --- RESULTS
Chapter 3.1 --- Immuno-characterization of PPCs and ICCs --- p.112
Chapter 3.2 --- Effect of cytokines on immune-properties of PPCs and ICCs --- p.115
Chapter 3.2.1 --- Effect of lFN-γ on MHC-I expression in PPCs --- p.115
Chapter 3.2.2 --- Effect of lFN-γ and IL-10 on HLA-G expression in PPCs and ICCs --- p.119
Chapter 3.2.3 --- Effect of IFN-γ on B7H4 expression in PPCs --- p.123
Chapter 3.3 --- Comparison of immune-properties of PPCs and ICCs from 1st and 2nd trimester --- p.125
Chapter 3.3.1 --- Differential expression of MHC molecules in PPCs --- p.125
Chapter 3.3.2 --- Different immune-related gene expression in PPCs and ICCs --- p.128
Chapter 3.3.3 --- Comparison of IFN-γ activated MHC molecules expression in PPCs/ICCs --- p.134
Chapter 3.3.4 --- Comparison of other IFN-γ activated genes expression in PPCs --- p.139
Chapter 3.4 --- Mixed lymphocyte reaction of PPCs from 1st and 2nd trimester --- p.143
Chapter 3.4.1 --- Effect of PPCs on proliferation of PBMC --- p.143
Chapter 3.4.2 --- Effect of ICCs on proliferation of PBMC --- p.145
Chapter 3.4.3 --- Effect of PPCs on cytokine production in PBMC --- p.149
Chapter 3.5 --- Xenotransplantation of ICCs into diabetic mouse model --- p.152
Chapter 3.5.1 --- Blood glucose level of diabetic mice after transplantation --- p.152
Chapter 3.5.2 --- Histological evaluation of transplanted ICCs grafts --- p.154
Chapter 3.5.3 --- Infiltration of CD45 into transplanted grafts of 1st and 2nd trimester --- p.158
Chapter CHAPTER4 --- DISCUSSION
Chapter 4.1 --- Expression of selected immuno-regulated genes in PPCs and ICCs --- p.163
Chapter 4.2 --- Effect of IFN-g and IL-10 on expression of immuno-regulated genes in PPCs and ICCs --- p.166
Chapter 4.3 --- In vitro studies on immunogenicity of PPCs and ICCs from first and second trimester --- p.171
Chapter 4.3.1 --- Immune-related genes expression --- p.171
Chapter 4.3.2 --- IFN-γ activated gene expression --- p.173
Chapter 4.3.3 --- Mixed lymphocyte reaction --- p.175
Chapter 4.3.4 --- Cytokine production of PBMC in MLR --- p.179
Chapter 4.4 --- In vivo Xenotransplantation of ICCs into diabetic mouse model --- p.181
Chapter 4.5 --- Conclusion --- p.187
Chapter 4.6 --- Further studies --- p.188
Chapter CHAPTER5 --- BIBLIOGRAPHY
Bibliography by Alphabetical Order --- p.189

Ng, Ka Yan.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2011.
Includes bibliographical references (leaves 179-204).
Abstracts in English and Chinese.
Abstract --- p.I
摘要 --- p.IV
Publications --- p.VII
Acknowledgements --- p.VIII
Table of contents --- p.IX
List of figures --- p.XV
List of tables --- p.XVII
List of abbreviations --- p.XVIII
Chapter Chapter 1 --- General Introduction
Chapter 1.1 --- The Pancreas --- p.2
Chapter 1.1.1 --- Anatomy of Pancreas --- p.2
Chapter 1.1.2 --- The Exocrine Pancreas --- p.4
Chapter 1.1.3 --- The Endocrine Pancreas --- p.5
Chapter 1.1.3.1 --- Structure of Islets --- p.5
Chapter 1.1.3.2 --- "Functions of α-, β-, y-, ð-, Σ-and PP-cells in Islets" --- p.7
Chapter 1.1.4 --- Overview of Pancreas Development --- p.9
Chapter 1.1.4.1 --- Organ Morphology --- p.10
Chapter 1.1.4.2 --- Cyto-differentiation --- p.12
Chapter 1.1.4.3 --- Control by Transcriptional Factors --- p.14
Chapter 1.1.5 --- Postnatal Pancreas Development and Regeneration --- p.18
Chapter 1.1.5.1 --- Proliferation of Pre-existing β-cells --- p.19
Chapter 1.1.5.2 --- Neogenesis from Precursor Cells --- p.20
Chapter 1.1.5.3 --- Transdifferentiation of other Cells --- p.20
Chapter 1.2 --- Diabetes Mellitus --- p.22
Chapter 1.2.1 --- Pathophysiology of Diabetes Mellitus and Current Treatments --- p.24
Chapter 1.2.1.1 --- Type I Diabetes Mellitus --- p.24
Chapter 1.2.1.2 --- Type II Diabetes Mellitus --- p.25
Chapter 1.2.1.3 --- Gestational Diabetes --- p.27
Chapter 1.2.1.4 --- Secondary Diabetes --- p.28
Chapter 1.3 --- Stem Cell therapy --- p.29
Chapter 1.3.1 --- Stem Cell --- p.29
Chapter 1.3.1.1 --- Mesenchymal Stem Sell --- p.31
Chapter 1.3.1.2 --- Embryonic Stem Cell --- p.35
Chapter 1.3.1.3 --- Induced Pluripotent Stem Cell --- p.36
Chapter 1.3.2 --- Islets Engineering --- p.37
Chapter 1.3.2.1 --- Genetic Modification --- p.37
Chapter 1.3.2.2 --- Directed Differentiation --- p.38
Chapter 1.3.2.3 --- Microenvironment --- p.38
Chapter 1.3.2.4 --- In vivo Regeneration --- p.39
Chapter 1.3.2.5 --- Cell Fusions --- p.40
Chapter 1.3.2.6 --- Combinatory Treatments --- p.40
Chapter 1.4 --- The Vitamin A & Vitamin D System --- p.42
Chapter 1.4.1 --- The Vitamin A --- p.42
Chapter 1.4.2 --- Vitamin A Metabolism --- p.44
Chapter 1.4.3 --- Roles of vitamin A in Pancreatic Development --- p.46
Chapter 1.4.4 --- The Vitamin D --- p.48
Chapter 1.4.5 --- Vitamin D Metabolism --- p.49
Chapter 1.4.6 --- Metabolic Functions of Vitamin D in Islets --- p.51
Chapter 1.4.7 --- Cod Liver Oil --- p.53
Chapter 1.4.8 --- Interactions between Vitamin A and Vitamin D --- p.53
Chapter 1.5 --- The Relations of Liver and Pancreas Development --- p.55
Chapter 1.5.1 --- Endoderm Induction for Hepatic and Pancreatic Differentiation of ESCs --- p.55
Chapter 1.5.2 --- Bipotential Precursor Population within Embryonic Endoderm --- p.56
Chapter 1.5.3 --- Pancreatic Islets Promote Mature Liver Hepatocytes Proliferation --- p.57
Chapter 1.5.4 --- Transdifferentiation --- p.57
Chapter 1.5.5 --- Transplantation in Liver Niche Promotes Maturation of Insulin-Producing Cells --- p.60
Chapter 1.5.6 --- Neuronal Relay from the Liver to Pancreatic --- p.61
Chapter 1.5.7 --- Development of Islets in the Nile Tilapia --- p.62
Chapter 1.6 --- The Insulin-like Growth Factor-I (IGF1) --- p.64
Chapter 1.6.1 --- IGF1 System --- p.64
Chapter 1.6.2 --- IGF 1 Regulation --- p.65
Chapter 1.6.3 --- Roles of IGF 1 in Pancreatic Development and Regeneration --- p.68
Chapter 1.7 --- Aims and Objectives of Study --- p.70
Chapter Chapter 2 --- General Materials and Methods
Chapter 2.1 --- Pancreatic progenitor cells (PPCs) and liver stromal cells (LSCs) isolation and cell culture --- p.72
Chapter 2.1.1 --- Tissue procurement --- p.72
Chapter 2.1.2 --- PPC and LSC culture --- p.72
Chapter 2.1.3 --- "Treatments of vitamin A, vitamin D and IGF 1" --- p.76
Chapter 2.1.4 --- "Cell culture of Caco-2, HepG2 and DU-145" --- p.76
Chapter 2.2 --- Induction of Islet-like Cell Clusters (ICCs) Differentiation --- p.77
Chapter 2.2.1 --- In vitro Directed Differentiation --- p.77
Chapter 2.2.2 --- In vitro LSC Microenvironment --- p.77
Chapter 2.3 --- RNA Expression Detection --- p.79
Chapter 2.3.1 --- RNA isolation --- p.79
Chapter 2.3.2 --- Reverse Transcription --- p.79
Chapter 2.3.3 --- Polymerase Chain Reaction (PCR) --- p.80
Chapter 2.3.4 --- Realtime PCR --- p.81
Chapter 2.4 --- Immunocytochemistry --- p.83
Chapter 2.5 --- Western Blotting --- p.85
Chapter 2.5.1 --- Protein extraction and quantification --- p.85
Chapter 2.5.2 --- Western Blotting --- p.85
Chapter 2.6 --- Enzyme-linked Immunosorbent Assay (ELISA) --- p.87
Chapter 2.6.1 --- Detection of cell viability --- p.87
Chapter 2.6.2 --- Detection of cell proliferation --- p.87
Chapter 2.6.3 --- Measurement of Cell death --- p.88
Chapter 2.6.4 --- Measurement of IGF 1 level in condition medium --- p.89
Chapter 2.6.5 --- Measurement of glucose induced insulin secretion --- p.90
Chapter 2.7 --- Regeneration model --- p.92
Chapter 2.7.1 --- Regeneration model in neonatal-STZ rat --- p.92
Chapter 2.7.2 --- Change in IGF1 expression in pancreas and liver --- p.92
Chapter 2.8 --- Statistical Data Analysis --- p.93
Chapter Chapter 3 --- Vitamin D and vitamin A receptor expression and the proliferative effects of ligand activation of these receptors on the development of pancreatic progenitor cells derived from human fetal pancreas. (Stem Cell Rev. 2011;7:53-63)
Chapter 3.1 --- Abstract --- p.95
Chapter 3.2 --- Introduction --- p.97
Chapter 3.3 --- Materials and Methods --- p.101
Chapter 3.3.1 --- Fetal Tissue Procurement --- p.101
Chapter 3.3.2 --- Culture of Pancreatic Progenitor Cells --- p.101
Chapter 3.3.3 --- RNA Expression Analysis by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.102
Chapter 3.3.4 --- Western Blot Analysis --- p.103
Chapter 3.3.5 --- Immunocytochemstry --- p.105
Chapter 3.3.6 --- PPC Proliferation Assays --- p.106
Chapter 3.3.7 --- PPC Cell Death Assays --- p.107
Chapter 3.3.8 --- Statistical Data Analysis --- p.108
Chapter 3.4 --- Results --- p.110
Chapter 3.4.1 --- "Expression and Localization of RAR, VDR and RXR, CYP26 and CYP24 in PPCs" --- p.110
Chapter 3.4.2 --- Incubation of PPC with atRA Enhances PPC Viability due to Increased Proliferation and Anti-apoptosis --- p.111
Chapter 3.4.3 --- Incubation of PPCs with Calcitriol Enhances Viability due to Increased Proliferation --- p.111
Chapter 3.4.4 --- Both atRA and Calcitriol Induce Up-regulation of both the RAR and the VDR but not the RXR --- p.112
Chapter 3.4.5 --- Combination Treatment with atRA and Calcitriol on Cell Viability and NGN3 Expression --- p.112
Chapter 3.5 --- Discussion --- p.114
Chapter Chapter 4 --- Human fetal liver stromal cell co-culture enhances the growth and differentiation of pancreatic progenitor cells into islet-like cell clusters (In submission to Gastroenterology)
Chapter 4.1 --- Abstract --- p.128
Chapter 4.2 --- Introduction --- p.129
Chapter 4.3 --- Materials and Methods --- p.133
Chapter 4.3.1 --- Use of human and animal tissues --- p.133
Chapter 4.3.2 --- "Cell preparation, characterizations and Differentiation" --- p.133
Chapter 4.3.3 --- Examination of PPC growth and ICC differentiation and functions with LSC co-culture --- p.133
Chapter 4.3.3 --- Identification of growth factors and investigation of their effects --- p.134
Chapter 4.3.4 --- Statistical Analysis --- p.135
Chapter 4.4 --- Results --- p.136
Chapter 4.4.1 --- "Isolation, Culture and Characterizations of LSCs" --- p.136
Chapter 4.4.2 --- Establishment of LSC co-culture system --- p.136
Chapter 4.4.3 --- LSC co-culture enhances PPC-derived ICC differentiation --- p.137
Chapter 4.4.4 --- Differential expression of mRNA for cytokines and growth factors between 1st and 2nd trimester LSCs --- p.138
Chapter 4.4.5 --- Characterization of IGF 1 receptors in PPCs and the effects of exogenous IGF1 on PPC growth and ICC differentiation --- p.139
Chapter 4.4.6 --- Neutralizing antibodies against IGF1R inhibit ICC differentiation --- p.140
Chapter 4.5 --- Discussion --- p.142
Chapter 4.6 --- Supplementary Materials and Methods --- p.147
Chapter 4.6.1 --- Cell Preparation and culture --- p.147
Chapter 4.6.2 --- In Vitro ICC differentiation --- p.148
Chapter 4.6.3 --- RNA expression analysis --- p.149
Chapter 4.6.4 --- Immunocytochemistry --- p.149
Chapter 4.6.5 --- PPC viability and cell count assays --- p.150
Chapter 4.6.6 --- IGF1 and insulin ELISA --- p.151
Chapter 4.6.7 --- Western blotting analysis --- p.152
Chapter 4.6.8 --- Neonatal streptozotocin regeneration model --- p.153
Chapter Chapter 5 --- General Discussion and Future Studies
Chapter 5.1 --- General Discussion --- p.165
Chapter 5.1.1 --- Proliferative effects and enhance expression of NGN3 by vitamin A and vitamin D on PPC --- p.166
Chapter 5.1.2 --- Induction of PPC derived ICCs by LSCs --- p.169
Chapter 5.1.3 --- Potential effects of liver stroma derived IGF1 on PPC derived ICCs differentiation --- p.172
Chapter 5.1.4 --- Significance of islet engineering in the management of diabetes --- p.174
Chapter 5.1.5 --- Conclusions --- p.176
Chapter 5.2 --- Future Studies --- p.177
Chapter Chapter 6 --- Reference
Reference --- p.180

Chronic hyperlipidemia (lipotoxicity) and hyperglycemia (glucotoxicity) have recently been shown to induce Endoplasmic Reticulum (ER) stress, which may contribute to pancreatic beta-cell dysfunction in type 2 diabetes. This thesis examined the involvement of ER stress in beta-cell lipotoxicity and glucotoxicity. Although chronic treatment with saturated free fatty acids (FFA) in vitro induced ER stress, altering ER stress by increasing or knocking-down GRP78 chaperone expression had no effect on apoptosis induction. Conversely, overexpression of ER chaperones rescued the reduction in proinsulin protein levels caused by chronic exposure to high glucose, although it had no effect on the decreased insulin mRNA levels and proinsulin translation rate. Thus, ER stress is likely not the main mechanism involved in saturated FFA-induced beta-cell apoptosis in vitro, but it may contribute to glucotoxic effects on proinsulin levels. These findings have increased our understanding of the link between ER stress and beta-cell dysfunction in type 2 diabetes.