Dissertations / Theses on the topic 'Pancreatic differentiation'
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1
Yuan, Songyang. "Differentiation and transdifferentiation of adult pancreatic cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ30425.pdf.
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Myatt, Emily-Jane. "Differentiation of pancreatic and hepatic cell types." Thesis, University of Bath, 2013. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.616573.
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The endoderm gives rise to many different cell types including those that will form the liver and pancreas. How cells differentiate during embryonic development is an important focus for the field of regenerative medicine. Understanding the normal development of liver and pancreatic cell types may allow us to develop strategies for the production of hepatocytes and pancreatic beta-cells for therapeutic purposes. One potential method of producing cells for therapeutic purposes is via transdifferentiation, or, the conversion of one cell type to another. In this thesis we aimed to establish a protocol for the transdifferentiation of liver ductal cells (termed cholangiocytes) to either hepatocyte or pancreatic lineages. We also aimed to investigate the signalling pathways important for normal differentiation of embryonic liver and pancreas. In order to address the potential of a cholangiocyte cell line (biliary epithelial cells or BECs) to transdifferentiate to other cell types, BECs were infected with a combination of candidate transcription factors known as ‘master switch’ genes that have previously been demonstrated to induce transdifferentiation to hepatic or pancreatic lineages. We demonstrated that overexpression of the hepatic transcription factors C/EBPα, C/EBPβ and HNF4 resulted in the up-regulation of the hepatocyte genes Albumin and Gs and de novo expression of Afp. In complementary experiments we also demonstrated that overexpression of the pancreatic transcription factors Pdx1, Ngn3, NeuroD and Pax4 resulted in de novo expression of insulin II in BECs. While these results were encouraging further work is necessary to enhance the maturation status of the nascent cells. We also addressed the role of the Notch signalling pathway in the differentiation of embryonic hepatic and pancreatic cells using ex vivo organ culture models of liver and pancreas development. We treated pancreata with N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S- phenylglycine t-Butyl Ester (DAPT) a gamma-secretase inhibitor. Treatment with DAPT inhibits the Notch signalling pathway. Following treatment with DAPT we observed reduced branching morphogenesis, loss of the acinar cell phenotype (amylase expression) and an enhancement in endocrine differentiation (insulin and glucagon expression). We propose that in the absence of Notch signalling the proendocrine gene Ngn3 is no longer repressed by the Notch target Hes1, allowing endocrine differentiation to take place. Finally we observed that β-cells in pancreata treated with DAPT are functionally more mature in terms of responsiveness to glucose stimulation. Overall these results have important implications for the development of potential therapies in the treatment of liver failure and diabetes.
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Decker, Kimberly Jean. "Gata6 regulates pancreatic branching morphogenesis and endocrine differentiation /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2007.
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Thesis (Ph.D. in Molecular Biology) -- University of Colorado Denver, 2007.Typescript. Includes bibliographical references (leaves 160-175). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;
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Kimura, Yoshito. "ARID1A Maintains Differentiation of Pancreatic Ductal Cells and Inhibits Development of Pancreatic Ductal Adenocarcinoma in Mice." Kyoto University, 2018. http://hdl.handle.net/2433/235986.
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Blyszczuk, Przemyslaw. "Differentiation of embryonic stem cells into pancreatic insulin-producing cells." [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=97560032X.
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Murad, Nadia Yousif. "Differentiation of human embryonic stem cells to the pancreatic lineage." Thesis, University of Sheffield, 2008. http://etheses.whiterose.ac.uk/6102/.
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Human embryonic stem (hES) cells have great therapeutic potential for the treatment of degenerative conditions such as Parkinson's disease, cardiac failure and type I diabetes. This potential is based on the ability of hES cells in vitro to self-renew and also differentiate to cells of all three germ layers; ectoderm, mesoderm and endoderm. Type I diabetes is due to an autoimmune disease destroying the insulin-secreting cells of the pancreas (β-cells) that regulate plasma glucose concentration. The pancreas develops from the endoderm lineage. 2. To find a cure for type I diabetes based on the use of hES, it is essential to understand the differentiation process of ES cells into the endodermal, β-cell lineage. The aim of this study was to investigate the generation of insulin-secreting cells using hES cells in vitro and to compare sue with those in the developing pancreas of the foetus.
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Shah, Nadia Nisa. "Human embryonic stem cells : prospects for pancreatic β-cell differentiation." Thesis, University of Manchester, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.495052.
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The focus of this thesis was to explore different strategies in trying to generate putative pancreatic β-cells using one of the initial Wisconsin H7 hES cell lines. Prior to this, human pancreas development was assessed during the first trimester of pregnancy in an attempt to determine the spatial and temporal expression of development and mature pancreatic β-cell markers during this period. Spontaneous differentiation of hES cells can be induced by the formation of embryoid bodies (EBs) in suspension culture. EBs began to express markers of pancreatic β-cell development and function at a molecular, protein and functional level upon differentiation over a 3-week period. The constitutive over-expression of the terminal β-cell marker PAX4 enhances this process, whereas karyotypic abnormalities induced in hES cells over prolonged culture can hinder differentiation potential towards pancreatic β-cells. Directed differentiation strategies which mimic mouse pancreas development have led to the elucidation of an in vitro protocol to generate putative definitive endoderm from hES cells through the application of Wnt3a and Activin A in the presence of low serum. Indirect co-culture of this H7 hES cell-derived putative definitive endoderm with mouse islets did not lead to the differentiation of fully functional pancreatic β-cells. The hES cell-derived putative definitive endoderm did however influence the aging mouse islets in a positive manner by allowing the maintenance of insulin secretagogue-induced functional responses which are usually lost in culture. This may prove useful in maintaining viability of human islets during culture to be used for transportation therapies.
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Uroić, Daniela Sonja. "Differentiation of embryonic stem cells towards pancreatic β-like cells." Thesis, University of Aberdeen, 2011. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=167694.
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Embryonic stem (ES) cells were used as a model system to understand the signalling events in pancreas development. ES cells were differentiated through known precursor stages towards the tissue of interest in order to recapitulate development in vitro. Thus, protocols directing differentiation of mouse ES cells towards definitive endoderm and pancreatic β-cells were developed. A combination of activin A and bone morphogenic protein 4 resulted in a population of enriched cells expressing genetic markers of definitive endoderm. In vitro differentiation of ES cells into functional pancreatic β-cells has only been partially successful, as it results in cells that are not fully differentiated or functional. This might be due to a lack of cues emanating from surrounding cells present in the developing pancreas. Conditioned media from the mouse MIN6 β-cell line were used on the basis that differentiated β- cells might send out signals affecting the differentiation of the surrounding islet cells. Mouse ES cells were enriched in definitive endoderm and then treated with MIN6 conditioned medium. Gene expression of the β-cell markers Insulin1, Insulin2, and Glucose transporter 2 was significantly increased relative to the untreated control group after 10 days of treatment with conditioned medium. This result was specific for conditioned medium from MIN6 cells as conditioned medium from a kidney-, a neuronal-, and an exocrine pancreatic cell line had no effect. In order to characterise the secreted factor(s) the conditioned medium was subjected to protein precipitation. The pancreatic differentiation factor was present in a protein fraction, suggesting that the factor(s) was proteinaceous. The protein in question was neither proinsulin nor insulin. This knowledge will support the efficient generation of insulin-secreting cells for diabetes therapy.
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Chong, Tsz-yat Ian, and 莊子逸. "Inducing the progressive differentiation of hESCs into pancreatic progenitor cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/196433.
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Diabetes is a chronic disorder of the pancreas, where a decline in the insulin-producing β-cell population disrupts metabolic homeostasis. Pancreatic transplantation has shown to be effective in circumventing the problem of β-cell insufficiency. However, availability of donor islets remains an obstacle. Although progressive differentiation of embryonic stem cells (ESCs) to pancreatic β-cells is a solution, current protocols are wrought with inefficiencies. It is obvious that to realize ESC differentiation for therapy many steps need to be optimized, and this study describes improvement of Pdx1+pancreatic progenitor derivation, a critical determinant of pancreatic fate.
The compounds melatonin and sPDZD2 have been suggested to act through the
Protein Kinase A (PKA) pathway to exert transcriptional effects, and in particular sPDZD2 stimulates the expression of pancreatic genes in INS-1E rat pancreatic cells. This led to the hypothesis that the PKA-targeting characteristics of said molecules could be exploited for pancreatic specification through post-translational activation ofPdx1. hESCs were first induced to form definitive endoderm before treatment with melatonin and sPDZD2. Pdx1 expression induced by these molecules was then compared with levels triggered by known pancreatic progenitor inducer Indolactam V (ILV). A secondary objective of this study was to assess the endoderm induction potential of small molecules in hESCs, which claim to be potentially useful in differentiation.
In this research, I show that small molecules are noticeably more challenging to use in the hESC context. Between the TGF-β pathwayactivatorsIDE-1 and 2, the latter is more potent at inducing endoderm formation, though it does not surpass the capabilities of Stauprimide, a molecule originally thought to only serve a priming purpose in mESCs.IDE-2 and Stauprimide consistently perform better than Activin A, the near universal factor for endoderm induction. Possible synergy between IDE-2 and Stauprimide was explored, but their combination appears detrimental to Sox17expression. Subsequent pancreatic differentiation was also inefficient, and my results affirm the immaturity of chemically-induced endoderm by contrasting with mainstream means of endoderm induction; levels of endoderm marker expression between the two methods are millions of folds apart. This work exposes the risks of using small molecules, and they necessitate proper characterization before being adopted for differentiation.
Most favorably, both sPDZD2 and melatonin were able to trigger Pdx1 expression in STEMDiffTm derived definitive endoderm; 10 and 30folds respectively, comparable to the known Pdx1 inducer ILV (25 folds). I also reveal concentration-mediated differentiation and proliferative purposes of ILV and sPDZD2, which are highly reminiscent of the signaling mechanisms involved during pancreatic development. Preliminary quantification of Pdx1+ cells suggest that high concentrations of ILV and sPDZD2 favor self-renewal of Pdx1+ progenitors, whilst lower doses elevate Pdx1 expression. Demonstration of Pdx1 at both gene and protein expression levels was encouraging, but it remains uncertain if melatonin and sPDZD2 manipulate PKA signaling to exert Pdx1 promoting effects. My work supports the use of melatonin as a candidate for pancreatic differentiation, and suggests involvement of sPDZD2 in deriving and expanding progenitors during pancreatic organogenesis.published_or_final_version
Biochemistry
Master
Master of Philosophy
10
Gsour, Amna. "Differentiation of human cell line towards a pancreatic endocrine lineage." Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/differentiation-of-human-cell-line-towards-a-pancreatic-endocrine-lineage(0c2c21fe-724d-449f-804c-02741c89828c).html.
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Islet transplantations have been successful in restoring glucose homeostasis in patients with diabetes; however, the limited number of donor organs limits the success of this treatment. The lineage reprograming of different cell sources to beta cells potentially provides an unlimited supply of insulin-producing cells for regenerative therapy for patients with diabetes. The aim of this study was to investigate the ability to transdifferentiate two cell lines into an endocrine lineage. Insulin production in pancreatic beta cells can be increased using a small molecule, 3,5-disubstituted isoxazole, N-cyclopropyl-t-(thiophen-2-yl)isoxazole-3-carboxamide (isoxazole) but its effect on other cell types has not been reported. Here, we investigated the lineage reprogramming of PANC-1 pancreatic ductal cells to insulin producing cells by isoxazole treatment. Gene expression was performed using RT-PCR and qPCR for approximately 30 genes critical to beta cell development and function. In addition, quantitative proteomic profiling was performed using LC-MS by monitoring protein abundance in isoxazole-treated PANC-1 cells compared to time-matched controls. Isoxazole treatment stimulated PANC-1 cells to aggregate into islet-like clusters and gene expression analysis revealed induction of important developmental beta cell markers including NGN3, NEUROD1 and INSULIN. In addition, beta cell surface markers were also upregulated such as CD200, GPR50, TROP-2, GLUT2 and SLC30A8. Using LC-MS a catalogue of approximately 2400 identified proteins was generated; 257 proteins were differentially expressed in isoxazole-treated cells compared to DMSO-vehicle controls at p < 0.05. Amongst the proteins upregulated were molecules that regulate metabolic processes and cytoskeletal reorganisation. The expression of the majority of these proteins has not been previously reported or studied in the context of beta cell differentiation. Functional analysis of the relative protein changes was determined using Ingenuity Pathway Analysis, IPA, and gene ontology, GO, software, which revealed the regulation of several cellular canonical pathways including metabolic pathways, cell adhesion, remodelling of epithelial adherens junctions and actin cytoskeleton signalling. The effects of isoxazole were further studied in the A549 lung cancer cell line. Similar effects were observed, such as the induction of pro-endocrine markers NGN3 and NEUROD1 and endocrine-specific hormones INS and GCG. These results indicate that isoxazole has the capacity to transdifferentiate pancreatic and non-pancreatic cell origins into an endocrine lineage. This study reveals the powerful induction capacity of isoxazole in inducing cellular reprogramming events.
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Halvorsen, Tanya L. "Growth regulation and differentiation in the human pancreatic beta cell /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2001. http://wwwlib.umi.com/cr/ucsd/fullcit?p3000408.
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Yeo, Wendy Wai Yeng. "Differentiation of skeletal muscle-derived stem cells into beta pancreatic lineage." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONTS091.
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Le diabète de type 1 (DT1) est caractérisé par des niveaux élevés de glucose en raison de la destruction des cellules ß pancréatiques sécrétrices d'insuline. Cependant, les thérapies actuelles de remplacement des cellules bêta du pancréas impliquant la transplantation d'îlots pancréatiques sont techniquement difficiles et limitées par la disponibilité de don d'organes. Bien que les cellules souches embryonnaires et les cellules souches pluripotentes induites soient intensément étudiées, aucune de ces deux sources de cellules souches ne peut être utilisée directement sans le risque de développement de tumeurs. Les cellules souches dérivées du muscle squelettique (MDSC) sont une source de cellules alternative intéressante car elles sont multi-potentes et peuvent donc se différencier vers plusieurs lignages cellulaires tels que des cellules cardiaques à battement autonome “pacemaker-like” et des cellules neuronales. Par conséquent, nous avons émis l'hypothèse qu'elles pourraient se différencier en lignées de type pancréatique. Les objectifs de cette étude étaient donc d'étudier le potentiel des MDSC (1) à se différencier in vitro en cellules beta pancréatiques exprimant l'insuline et (2) à se différentier in vivo dans le pancréas et ainsi réduire l'hyperglycémie chez la souris modèle d'un diabète de type 1. Dans cette étude, les MDSC de muscle de souris ont été isolées via une série de passages des cellules les moins adhérentes en culture. Les cellules souches ainsi isolées peuvent adhérer sur une couche de cellules de types fibroblastes ou sur une matrice extra-cellulaire de type laminine pour ensuite se différentier in vitro ou bien être utilisées comme cellules souches MDSC non-adhérentes et non différentiées pour les études in vivo. In vitro, les MDSC peuvent se différencier spontanément en agrégats de cellules formant des îlots et exprimant des marqueurs de cellules bêta identifiés par immunofluorescence et analyse “PCR transcription inverse”. Ceci a été confirmé par immuno-analyse montrant l'expression des protéines nécessaires à la fonction des cellules ß, comme Nkx6.1, MafA et Glut2. Les MDSC différenciées en aggrégats cellulaires de type îlots pancréatiques montrent une sécrétion d'insuline en réponse au glucose in vitro. Cependant, dans des modèles murins de DT1 induit par la streptozotocine, l'injection intra-péritonéale des MDSC n'a pas permis de rétablir chez les souris diabétiques une normoglycémie du glucose sanguin en dépit d'un engreffement des MDSC dans les tissus pancréatiques. Ces données montrent que les MDSC peuvent constituer une source de cellules souches alternative intéressante pour le traitement du diabèteType 1 Diabetes (T1D) is characterized by high and poorly controlled glucose levels due to the destruction of insulin-secreting pancreatic ß-cells. However, current ß-cell replacement therapies, involving pancreas and pancreatic islet transplantation are technically demanding and limited by donor availability. While embryonic stem cells and induced pluripotent stem cells are intensely investigated, neither can be used due to safety issues. Skeletal muscle-derived stem cells (MDSC) are an attractive alternative cell source as they have the potential to undergo multilineage differentiation into beating pacemaker-like cells and neuronal cells. Hence, it is hypothesised that they can differentiate into pancreatic lineages. This led to the goals of this study, which were (1) to investigate the potential of MDSC to differentiate into mature insulin expressing cells in vitro and (2) to reduce hyperglycemia in mouse model type 1 diabetes. In this study, MDSC were isolated from mouse via a serial pre-plating based on the adhesive characteristics of cultured cells, in which the cells of interest adhered to plates at a later time for in vitro differentiation, while the non-adherence undifferentiated MDSC were used for in vivo study. The MDSC were found to spontaneously differentiate into islet-like aggregates and expressed ß-cell markers in vitro, as determined by immunofluorescence and reverse transcription PCR analyses. This was further confirmed by immunoblotting analysis showing expression of proteins required for ß-cell function, such as Nkx6.1, MafA and Glut2. The differentiation of MDSC into islet-like clusters demonstrated glucose responsiveness in vitro. In streptozotocin-induced T1D mouse models, intraperitoneal injection of the undifferentiated MDSC did not restore the blood glucose levels of the diabetic mice to normoglycemia despite successful engraftment of MDSC into the pancreatic tissues. Taken together, these data show that MDSC may serve as an alternative source of stem cells for the treatment of diabetes
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Wright, Elli Alexander. "The differentiation of human embryonic stem cells towards a pancreatic endoderm lineage." Thesis, University of Manchester, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.509868.
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Chen, Jim-Ray. "Hepatocytic differentiation of normal but not neoplastic cultured rat pancreatic duct cells." Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=28996.
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This thesis represents an effort to examine certain aspects of the differentiation potential of normal and neoplastic (spontaneously- and chemically-transformed) cultured rat pancreatic ductal cells under the influence of two microenvironments.The technique of in vivo implantation of cells to subcutaneous and intraperitoneal sites is used as it not only reveals the intrinsic potential of the implanted cells but also reflects the effects of the microenvironment on phenotypic expression.
The results of these studies indicate that when implanted in vivo normal propagable cultured cells derived from the duct epithelium of adult rat pancreas develop phenotypic features of a hepatocyte, and that the extent of this phenotypic expression is influenced by the microenvironment in which these cells are implanted. When localized subcutaneously, the cells displayed partial differentiation toward hepatocytes but retained some of their ductal phenotype. In contrast, when the same cells were implanted intraperitoneally, they expressed the full phenotypic properties of mature hepatocytes. Both spontaneously- and chemically-transformed pancreatic ductal cell lines did not display phenotypic differentiation along the hepatocytic lineage after in vivo implantation. It is concluded that (1) pancreatic ductal cells can be the progenitor cell for pancreatic hepatocytes; (2) Neoplastic transformation of these cell lines results in partial or total loss of hepatoctyic differentiation.
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Shepherd, Jessica. "Malignant transformation and differentiation of adult rat pancreatic duct cells in vitro." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=61214.
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The development of animal models for pancreatic cancer has magnified the controversy regarding the cell of origin of the tumor in animals and man. In rats, most pancreatic tumors have an acinar histology. By contrast, tumors in hamsters have a ductular morphology, resembling the predominant type of human exocrine pancreatic cancer. While some workers advocate a ductular origin for hamster tumors, others insist that the progenitor is an acinar cell.With the use of normal adult rat pancreatic ductular cell lines, this work shows that four representative pancreatic carcinogens all display dose-related genotoxicity to rat pancreatic ductular cells in vitro. Further, repeated exposure to sublethal doses induced anchorage-independent growth. Transformed cells, injected subcutaneously into isogeneic newborn rats, produced malignant tumors with ductular morphology. A novel achievement of this work was the production of ductular adenocarcinomas using streptozotocin, previously reported as only inducing islet cell tumors.
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Delaspre, Fabien. "Stepwise differentiation of pancreatic acinar cells from mES cells by manipulating signalling pathway." Doctoral thesis, Universitat Pompeu Fabra, 2011. http://hdl.handle.net/10803/38364.
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Tot i que es coneix l’involucrament de les cèl·lules pancreàtiques
acinars en patologies exocrines (pancreatitis i càncer de pàncrees),
la manca de models normals basats en cèl·lules ha limitat l’estudi
de les alteracions que succeeixen en el programa de diferenciació
pancreàtica. Hem demostrat prèviament que les cèl·lules mare
embrionàries murines, que són pluripotents, poden adquirir un
fenotip acinar in vitro. Això es va aconseguir, en part, amb una
combinació de senyals que provenien del cultiu de pàncrees fetals
que no era, però, específic del llinatge pancreàtic. L’objectiu
d’aquest treball ha estat el de desenvolupar un protocol selectiu
pel llinatge acinar basat en l’activació seqüencial de vies de
senyalització que recapitulin el desenvolupament pancreàtic in
vivo, a través de la formació definitiva de l’endoderm,
l’especificació pancreàtica i acinar i l’expansió/diferenciació de
progenitors acinars. El tractament de cossos embrionaris amb
Activina A va promoure l’expressió de gens d’endoderm com està
prèviament descrit. El tractament subsegüent amb àcid Retinoic,
FGF10 i Ciclopamina, un inhibidor de la via de Hedgehog, va
resutar en la inducció dels marcadors de progenitors pancreàtics
Pdx1, Ptf1a i Cpa1 però també d’aquells expressats en el llinatge
pancreàtic, que van ser reduïts amb la inhibició de BMPs. Les
cèl·lules van ser a continuació cultivades en Matrigel utilitzant un
sistema de cultiu en 3D en presència de fol·listatina,
dexametasona i KGF comportant una inducció significativa dels
nivells de mRNA i proteïna de marcadors acinars i una
disminució de l’expressió dels de marcadors acinars. A més, es va
veure que Amyl es secretava en el medi. Aquestes dades indiquen
que l’activació selectiva del programa de diferenciació acinar en
cèl·lules mare embrionàries es pot dur a terme mitjançant una
inducció esgraonada de vies de senyalització involucrades en el
desenvolupament pancreàtic exocrí proporcionant una eina
potencial per estudiar la diferenciació pancreàtica i malalties
relacionades amb el pàncrees.Despite known involvement of pancreatic acinar cells in exocrine pathologies (pancreatitis and pancreatic cancer), the lack of normal cell-based models has limited the study of the alterations that occur in the acinar differentiation program. We have previously shown that mESC (murine embryonic stem cells), which are pluripotent, can acquire an acinar phenotype in vitro. This was achieved, in part, by a combination of signals provided by the culture of foetal pancreases which was, however, no specific for the acinar lineage. The aim of this work was to develop a protocol selective for the acinar lineage based on the sequential activation of signaling pathways that recapitulate pancreatic development in vivo, through the definitive endoderm formation, the pancreatic and acinar specification and the expansion/differentiation of acinar progenitors. Treatment of embryoid bodies with Activin A enhanced the expression of endodermal genes as previously described. Subsequent treatment with Retinoic acid, FGF10 and Cyclopamine, an inhibitor of the Hedgehog pathway, resulted in the enhancement of pancreatic progenitor markers Pdx1, Ptf1a and Cpa1 but also of those expressed in the hepatic lineage, which were reduced by BMPs inhibition. Cells were further cultured in Matrigel using a 3D culture system in the presence of follistatin, dexamethasone, and KGF leading to a significant enhancement of the mRNA and protein levels of acinar markers while decreasing the expression of endocrine ones. Moreover, active Amyl was released into the medium. These data indicate that the selective activation of the acinar differentiation program in ES cells can be achieved by stepwise induction of signaling pathways involved in pancreatic exocrine development providing a potential tool for studying pancreatic differentiation and pancreas-related diseases.
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Massumi, Mohammad. "Directed Differentiation of ES cells by pancreatic transcription factors p48, RBPJL and Mist1." Doctoral thesis, Universitat Pompeu Fabra, 2009. http://hdl.handle.net/10803/7231.
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A pesar de la abundancia de estudios realizados sobre el papel de las células acinares en las patologías exocrinas del páncreas (i.e. pancreatitis y cáncer), el estudio de las modificaciones producidas durante la diferenciación acinar en dichas patologías, se ha visto limitado por la escasez de modelos celulares no tumorales. Resultados previos de nuestro laboratorio, muestran que las células mES (células madre embrionarias de ratón )- pluripotentes y con la capacidad para generar tipos celulares especializados- pueden desarrollar un fenotipo acinar in vitro. Los objetivos de esta tesis han sido aumentar el contenido de enzimas digestivos así como las propiedades funcionales de las células generadas. Para ello se sobreexpresaron de forma estable p48, RBPJL y Mist1en células madre por transducción lentiviral de estos genes. Obtuvimos, gracias a una estrategia de infección en múltiples etapas, líneas celulares transgénicas mES que expresaban de forma constitutiva RBPJL y/o Mist1. La superimposición de la expresión de p48 por infección lentiviral en células en proceso de diferenciación dio lugar a una fuerte expresión de enzimas digestivos, con un patrón de regulación similar al que acontece in vivo durante el desarrollo pancreático. En esta inducción, tanto p48 como RPBJL son indispensables. Por otro lado, hemos mostrado un aumento elevado en la producción de varios componentes de la maquinaria secretota dependiente de Mist1. Además, hay que hacer notar ,que las células p48/RBPJL/Mist1 exhiben una regulada-secreción en respuesta a los secretagogos acinares y una mejor actividad de que la línea celular acinar 266-6. La expresión combinada de genes clave implicados en el desarrollo pancreático en células ES es un prometedor abordaje que nos llevará a una comprensión de los sutiles procesos del desarrollo exocrino pancreático.Despite known involvement of acinar cells in pancreatic exocrine pathologies (i.e pancreatitis and pancreatic cancer), the lack of normal cell-based models has limited the study of the alterations that occur in the acinar differentiation program. Our previous data showed that mES (murine embryonic stem) cells, which are pluripotent and have the ability to generate specialized cell types, can acquire an acinar phenotype in vitro. The aim of this work was to increase the digestive enzyme content of the generated cells as well as their functional properties based on stable overexpression of p48, RBPJL and Mist1 by lentiviral gene transduction. Thus, we engineered transgenic mES cell lines constitutively expressing RBPJL and/or Mist1 using a multi-step infection strategy. The superimposition of p48 expression by lentiviral infection of differentiating cells resulted in a strong expression of digestive enzymes, with a pattern of regulation similar to what occurs in vivo during pancreatic development. In this induction, both p48 and RPBJL are indispensable. On the other hand, we showed a high increase in the production of several components of the secretory machinery which was dependent of Mist1. Importantly, p48/RBPJL/Mist1 cells exhibited a regulated-secretory in response to acinar secretagogues and a better secretion activity than the 266-6 acinar cell line. Combined expression of key genes involved in pancreatic development in ES cells may be a promising approach to better understand subtle steps of pancreatic exocrine development.
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Lehnert, Lasse. "Morphological and molecular characterization of human pancreatic adenocarcinoma cells undergoing duct-like differentiation." [S.l.] : [s.n.], 2000. http://e-diss.uni-kiel.de/diss/d385.pdf.
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Svensson, Per. "Functional analysis of Ipf1/Pdx1, MFng and Id during pancreatic growth and differentiation." Doctoral thesis, Umeå universitet, Umeå centrum för molekylär medicin (UCMM), 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1671.
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The pancreas is an endodermally derived organ consisting of three major cell lineages. The endocrine cells, organised into the Islets of Langerhans, regulate blood glucose homeostasis by producing and secreting hormones such as glucagon and insulin into the bloodstream. The major part of the pancreas consists however of acinar cells that produce digestive enzymes that are transported via a highly branched ductal system to the duodenum where they function in breakdown of food. Early in pancreas development a dorsal and ventral evagination of the foregut epithelium appear, resulting in the formation of the dorsal and ventral pancreatic bud. These pancreatic buds subsequently grow, branch and differentiate to form the mature pancreas via a process controlled by intrinsic factors, such as transcription factors, and extracellular signals. Insulin promoter factor 1 (Ipf1), also known as Pdx1 (for Pancreatic duodenal homeobox gene 1), is required for pancreas development. Although the evagination of pancreatic buds still occurs in Ipf1/Pdx1 mutant mice, the subsequent proliferation, branching and differentiation is impaired, resulting in complete pancreatic agenesis. Gene array profiling identified several candidate Ipf1/Pdx1 target genes, including FgfR2IIIb, ErbB3, Ptf1a/p48, Pax6 and Nkx6.1, in pancreatic progenitor cells. Together these genes provide a mechanistic explanation for the pancreatic growth arrest observed in Ipf1/Pdx1 deficient mice. In addition, Spondin1, which has not previously been described in the pancreas, was identified to be regulated by Ipf1/Pdx1. The spatial and temporal expression pattern of Spondin1 defines Spondin1 as a marker for early pancreatic progenitor cells. The Notch signalling pathway controls cell type specification and differentiation during pancreas development. The Fringe family of proteins have previously been shown to regulate Notch signalling by altering the interaction between Notch receptors and their ligands, hence affecting the cellular response. Manic Fringe (MFng) is transiently expressed in pancreatic pro-endocrine cells between E9.5 and E14.5. The expression of MFng is regulated by Ngn3, which may suggest a role for MFng in pro-endocrine cell maturation. The lack of a pancreatic phenotype in transgenic mice overexpressing MFng in the pancreatic epithelium and in MFng null mutant mice, however, provide evidence that MFng is dispensable for the specification, differentiation and function of the adult pancreas. Inhibitors of DNA binding (Id) proteins are generally known as inhibitors of differentiation, a feature they mainly perform by forming non-functional heterodimers with bHLH proteins, thereby inhibiting downstream targets of the bHLH proteins. Id proteins also promote cell proliferation by interacting with the cell cycle machinery. In the developing pancreas Id2 and Id3 are co-expressed in an overlapping manner during the period of massive proliferation and expansion of the pancreatic epithelium, suggestive of a role for the Id proteins during these processes. In addition, Id4 expression is also detected in the embryonic pancreas, albeit at lower levels. Gain- and loss- of- function analyses suggest however that specification, differentiation and function of the adult pancreas are largely independent of Id function.
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Wang, Nan. "In vitro differentiation of mouse embryonic stem cells into pancreatic insulin-producing cells." Thesis, University of Nottingham, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.508245.
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Weng, Chen. "SINGLE-CELL TRANSCRIPTOMICS OF HUMAN PANCREATIC ISLETS IN DIABETES AND ΒETA CELL DIFFERENTIATION." Case Western Reserve University School of Graduate Studies / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=case1612882103714773.
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Kondo, Yasushi. "Identification of a small molecule that facilitates the differentiation of human iPSCs/ESCs and mouse embryonic pancreatic explants into pancreatic endocrine cells." Kyoto University, 2018. http://hdl.handle.net/2433/230976.
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Cho, Hsin-Hua. "Differentiation of human embryonic stem cells into pancreatic progenitors using chemically defined culture systems." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608076.
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Zhao, Min. "Molecular studies on the differentiation and proliferation of human pancreatic beta cells and exocrine cells." Thesis, King's College London (University of London), 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.405642.
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Piccand, Julie. "Regulation of pancreatic and intestinal endocrine cell differentiation and function : roles of Pak3 and Rfx6." Thesis, Strasbourg, 2012. http://www.theses.fr/2012STRAJ057/document.
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Les hormones sécrétées par les cellules endocrines pancréatiques et intestinales participent à la régulation de l’homéostasie énergétique. Leur différenciation repose sur des programmes génétiques similaires contrôlés par le facteur de transcription Ngn3. Peu de choses sont connues sur les gènes activés par Ngn3 et leurs implications dans les mécanismes contrôlant la spécification et la maturation des cellules endocrines. Par conséquent, le transcriptome des progéniteurs endocrines a été déterminé dans l’équipe. Parmi les gènes fortement enrichis dans le lignage endocrine, j’ai caractérisé l’expression et la fonction de la kinase Pak3 et j’ai continué l’étude de la fonction pancréatique et intestinale du facteur de transcription Rfx6. J’ai montré que Pak3 est exprimé dans le lignage endocrine pendant le développement et chez l’adulte. Avec des expériences de perte de fonction, j’ai montré que ce gène inhibe la prolifération des progéniteurs endocrines et des cellules bêta durant l’embryogénèse. De plus, une étude métabolique a montré que les souris mutantes pour Pak3 sont intolérantes au glucose. En parallèle, en utilisant une souris conditionnelle pour Rfx6, j’ai montré que Rfx6 est nécessaire en aval de Ngn3 pour la différenciation des cellules endocrines pancréatiques et intestinales. Finalement, des expériences dans les souris adultes suggèrent que Rfx6 est nécessaire pour maintenir les cellules bêta, renouveler les cellules entéroendocrines et absorber les lipides dans l’intestin. En conclusion, ces études révèlent deux nouveaux gènes clés dans la régulation de la différenciation des cellules endocrines et de l’homéostasie énergétique dans le pancréas et l’intestinPancreatic and intestinal endocrine cells, and their secreted hormones, contribute to the regulation of energy homeostasis. Their differentiation relies on similar genetic programs controlled by the proendocrine transcription factor Ngn3. However, our knowledge of the endocrinogenic programs implemented by Ngn3 is still fragmentary. Therefore, the transcriptome of endocrine progenitors has been determined in the lab. Among the genes which showed a strong enrichment in the endocrine lineage, I studied the expression and function of Pak3, a serine/threonine kinase and further pursued the dissection of the function of the transcription factor Rfx6 in the pancreas and the intestine. I showed that Pak3 is expressed throughout pancreas development and maintained in adult islets. Using ex vivo loss of function experiments and in vivo characterisation of the Pak3-deficient mice, I identified Pak3 as an inhibitor of islet progenitors and beta-cell proliferation in the embryonic mouse pancreas. Furthermore, we performed metabolic studies which revealed that Pak3-deficient micehave an impaired glucose homeostasis, especially under challenging high fat diet. In parallel, using a conditional knockout mouse for Rfx6, we showed that Rfx6 is necessary downstream of Ngn3 for endocrine cell differentiation in the pancreas as well as in the intestine. Finally, additional experiments in adult mice suggest that Rfx6 is necessary to maintain pancreatic beta-cells, enteroendocrine cell turnover and intestinal lipid absorption. In conclusion, these studies revealed two novel key players in the regulation of endocrine cell differentiation and energy homeostasis in the pancreas and the intestine
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Santos, Cravo Ana Maria. "The role of epithelial cell de-differentiation in the context of improved chemotherapy applied to pancreatic cancer." Thesis, University of Bath, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.642055.
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In cancer, epithelial cell de-differentiation is a feature of rapidly dividing cells under non-controlled growth and it often reflects a change in the gene expression pattern; however, the relationship between proliferation and alterations in cellular differentiation has not yet been identified. This work examined how changes in the characteristics of cells that discriminate their differentiated and proliferative states can be used to improve on current pancreatic cancer chemotherapeutic strategies. PepT1, a high substrate-capacity and low-affinity transporter system, has been suggested as an attractive drug delivery target for pancreatic cancer. Through a combination of immunological assays, PepT1 normally restricted to the apical surfaces in polarised intestinal epithelial cells, was shown to distribute at the cell membrane of non-polarised cancerous ductal cells. Anti-inflammatory or anti-cancer agents, like ibuprofen or gemcitabine, were conjugated to selected amino acids to enhance their uptake via PepT1. Studies with these conjugates demonstrated enhanced uptake into pancreatic cancer cells, AsPc-1 and HPAFII. Subsequent studies investigated how cell polarity that is typically disrupted in cancer can be modulated to affect the balance of epithelial differentiation versus proliferation. Pharmacological attenuation of YAP (c-Yes associated protein) using a β adrenergic agonist, dobutamine, increased functional tight junction (TJ) structures and diminished proliferation rates of two pancreatic cancer cells, AsPc-1 and HPAFII. Dobutamine also primed an apoptotic cell response. When given in combination with gemcitabine, dobutamine further reduced cell proliferation. Overall, these studies have provided support for using PepT1 as a method to target pancreatic cancer cells for the delivery of anti-cancer agents. Additionally, dobutamine was identified as a potential pharmacological agent to suppress the proliferation of pancreatic cancer cells by altering increasing cell programming that drives epithelial cell differentiation.
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Jung, Matthias Verfasser], Gerald [Akademischer Betreuer] Moritz, Bernd [Akademischer Betreuer] [Fischer, and Heike [Akademischer Betreuer] Walles. "The establishment of non‐viral reprogramming methods and pancreatic differentiation in organotypic models for the production of patient‐specific pancreatic cells / Matthias Jung. Betreuer: Gerald Moritz ; Bernd Fischer ; Heike Walles." Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2014. http://d-nb.info/1068504536/34.
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Daoud, Jamal. "Development of a three-dimensional microenvironment and dielectric monitoring system for long-term «in vitro» culture and differentiation of human pancreatic islets." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=104749.
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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.
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Kuen, Janina [Verfasser], Manfred [Gutachter] Lutz, and Alois [Gutachter] Palmetshofer. "Influence of 3D tumor cell/fibroblast co-culture on monocyte differentiation and tumor progression in pancreatic cancer / Janina Kuen ; Gutachter: Manfred Lutz, Alois Palmetshofer." Würzburg : Universität Würzburg, 2017. http://d-nb.info/1149510390/34.
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McBain, Stuart C. "Development of an inducible expression system to regulate protein expression in pancreatic beta cells : studies of IRS-2 and wolframin in relation to beta cell growth and differentiation." Thesis, Keele University, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.288431.
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Vaissié, Alix. "Alternatives to “native human islets” for research in vitro and in vivo : pseudo-islets and pancreatic endocrine cells from pluripotent stem cells – the role of progerin in differentiation and maturation." Thesis, Lille 2, 2019. http://www.theses.fr/2019LIL2S035.
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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
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Rovira, Clusellas Meritxell. "Estrategias para la diferenciación in vitro de células ES de ratón a células acinares pancreáticas." Doctoral thesis, Universitat Pompeu Fabra, 2007. http://hdl.handle.net/10803/7104.
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Las patologías más importantes del páncreas exocrino, como la pancreatitis crónica (PC) o el cáncer de páncreas, representan un gran problema de salud pública en Europa. En la PC, el tejido acinar es substituido por complejos ductales. Además, es difícil mantener el fenotipo diferenciado de las células acinares en cultivo ya que sufren una transdiferenciación acinar-ductal.Las células madre embrionarias (ES) de ratón han sido utilizadas en la última década para generar in vitro células completamente diferenciadas de varios linajes celulares. No obstante, la capacidad de las células ES a diferenciarse a tipos celulares de origen endodérmico es muy limitada. El objetivo principal de este proyecto ha consistido en desarrollar estrategias para diferenciar células ES de ratón a células pancreáticas acinares con una elevada eficiencia mediante 1) la optimización de las condiciones de cultivo con tal de activar vías de señalización implicadas en el desarrollo/diferenciación pancreáticas; 2) la sobreexpresión de factores transcripcionales maestros utilizando vectores virales con el fin de recapitular específicamente un programa de diferenciación acinar; 3) la selección genética de las células comprometidas al linaje acinar con el objetivo de purificar las células acinares diferenciadas.
Mediante la integración de estos abordajes, hemos conseguido aislar células que comparten características fenotípicas con células acinares inmaduras según la expresión de marcadores de diferenciación y la respuesta funcional a secretagogos.
Exocrine pancreatic diseases such as chronic pancreatitis (PC) or pancreatic cancer are major health issues in Europe. In CP, the acinar tissue is substituted by ductal complexes. In addition, it is difficult to maintain the differentiated phenotype of the acinar cells in culture as within few days an acinar-ductal transdifferentiation takes place.
In the last decade, mouse embryonic stem cells (mES) have been used to generate differentiated cells of a variety of cellular lineages in vitro. However, the ability of ES cells to differentiate into endodermal lineages is limited. The main objective of this project has focused on the development of strategies to differentiate mES to pancreatic acinar cells with high efficiency by means of: 1) Optimization of cell culture conditions to activate signalling pathways involved in pancreatic differentiation/development; 2) the overexpression of master transcription factors involved in pancreas development using viral vectors in order to recapitulate specific acinar differentiation program; 3) the genetic selection of cells committed to the acinar linage in order to purify the differentiated cells.
The integration of these different strategies allowed us to isolate cells that share phenotypic features with immature acinar cells according to the expression of differentiation markers and the functional response to acinar secretegogues.
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Campos, Maria Luisa Morais Sarmento de. "ICAT: a novel Ptf 1A/P48 partner that modulates acinar expression." Doctoral thesis, Universitat Pompeu Fabra, 2010. http://hdl.handle.net/10803/7233.
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Ptf1a/p48 is a pancreas specific bHLH transcription factor that is required at early stages of embryonic development for pancreas formation and, during adulthood, for the proper exocrine pancreatic function. P48 also exerts an antiproliferative effect, which may exert a tumor suppressor activity. In this study, based on a yeast two-hybrid approach, we have identified new p48 partners that modulate the activity of p48. Among the newly identified putative interactors we found p/CAF, which is a coactivator that potentiates its transcriptional activity, and ICAT, an inhibitor of the β-catenin/TCF signaling pathway. ICAT binds to p48 and is coexpressed with it in the pancreas during development and postnatally. Using different cellular models, ICAT overexpression in acinar tumor cells resulted in changes of the pancreatic specific gene expression pattern. Furthermore, high levels of ICAT inhibited the interaction between p48 and p/CAF. While this hetero-oligomeric complex is required for the acinar gene expression, ICAT itself is shown to be present in a reconstituted PTF1 complex in vivo. Importantly, altered ICAT expression is demonstrated in several histological types of pancreatic tumors, possibly contributing to their differentiation phenotype and neoplastic properties.Ptf1a/p48 es un factor de transcripción bHLH específico del páncreas necesario durante los estadios tempranos del desarrollo embrionario para la formación del mismo, y para el correcto funcionamiento del páncreas exocrino en el adulto. P48 desempeña también una función antiproliferativa, la cual puede resultar en una actividad de supresión tumoral. En el presente estudio, basado en una estrategia de cribado de doble-híbrido en levadura, han sido identificadas nuevas proteínas que interaccionan y que modulan la actividad específica de p48. Entre las posibles proteínas que interaccionan y han sido identificadas de novo se encuentra p/CAF, un co-activador que potencia la actividad transcripcional de p48, y ICAT, un inhibidor de la vía de señalización de la β-catenina. Se ha demostrado que ICAT se une a p48 y ambos son co-expresados en el páncreas durante el desarrollo y en el adulto. Utilizando diferentes modelos celulares, la sobreexpresión de ICAT en células tumorales acinares resultó en un cambio en el patrón de expresión de genes específicos del páncreas. Al mismo tiempo, se observó que niveles elevados de ICAT inhiben la interacción entre p48 y su co-activador p/CAF. Mientras que este complejo hetero-oligomérico es necesario para la expresión de los genes acinares, se demostró que ICAT está presente en un complejo PTF1 reconstituido in vivo. Finalmente, se observaron alteraciones en la expresión de ICAT en varios tipos histológicos de tumores pancreáticos, que posiblemente contribuyen a su fenotipo de diferenciación y propiedades neoplásicas.
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Schroeder, Insa S., Sabine Sulzbacher, Tobias Nolden, Jörg Fuchs, Judith Czarnota, Ronny Meisterfeld, Heinz Himmelbauer, and Anna M. Wobus. "Induction and Selection of Sox17-Expressing Endoderm Cells Generated from Murine Embryonic Stem Cells." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-135990.
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Embryonic stem (ES) cells offer a valuable source for generating insulin-producing cells. However, current differentiation protocols often result in heterogeneous cell populations of various developmental stages. Here we show the activin A-induced differentiation of mouse ES cells carrying a homologous dsRed-IRES-puromycin knock-in within the Sox17 locus into the endoderm lineage. Sox17-expressing cells were selected by fluorescence-assisted cell sorting (FACS) and characterized at the transcript and protein level. Treatment of ES cells with high concentrations of activin A for 10 days resulted in up to 19% Sox17-positive cells selected by FACS. Isolated Sox17-positive cells were characterized by defini- tive endoderm-specific Sox17/Cxcr4/Foxa2 transcripts, but lacked pluripotency-associated Oct4 mRNA and protein. The Sox17-expressing cells showed downregulation of extraembryonic endoderm (Sox7, Afp, Sdf1)-, mesoderm (Foxf1, Meox1)- and ectoderm (Pax6, NeuroD6)-specific transcripts. The presence of Hnf4α, Hes1 and Pdx1 mRNA demonstrated the expression of primitive gut/foregut cell-specific markers. Ngn3, Nkx6.1 and Nkx2.2 transcripts in Sox17-positive cells were determined as properties of pancreatic endocrine progenitors. Immunocytochemistry of activin A-induced Sox17-positive embryoid bodies revealed coexpression of Cxcr4 and Foxa2. Moreover, the histochemical demonstration of E-cadherin-, Cxcr4-, Sox9-, Hnf1β- and Ngn3-positive epithelial-like structures underlined the potential of Sox17-positive cells to further differentiate into the pancreatic lineage. By reducing the heterogeneity of the ES cell progeny, Sox17-expressing cells are a suitable model to evaluate the effects of growth and differentiation factors and of culture conditions to delineate the differentiation process for the generation of pancreatic cells in vitroDieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich
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Schroeder, Insa S., Sabine Sulzbacher, Tobias Nolden, Jörg Fuchs, Judith Czarnota, Ronny Meisterfeld, Heinz Himmelbauer, and Anna M. Wobus. "Induction and Selection of Sox17-Expressing Endoderm Cells Generated from Murine Embryonic Stem Cells." Karger, 2012. https://tud.qucosa.de/id/qucosa%3A27696.
Full textAbstract:
Embryonic stem (ES) cells offer a valuable source for generating insulin-producing cells. However, current differentiation protocols often result in heterogeneous cell populations of various developmental stages. Here we show the activin A-induced differentiation of mouse ES cells carrying a homologous dsRed-IRES-puromycin knock-in within the Sox17 locus into the endoderm lineage. Sox17-expressing cells were selected by fluorescence-assisted cell sorting (FACS) and characterized at the transcript and protein level. Treatment of ES cells with high concentrations of activin A for 10 days resulted in up to 19% Sox17-positive cells selected by FACS. Isolated Sox17-positive cells were characterized by defini- tive endoderm-specific Sox17/Cxcr4/Foxa2 transcripts, but lacked pluripotency-associated Oct4 mRNA and protein. The Sox17-expressing cells showed downregulation of extraembryonic endoderm (Sox7, Afp, Sdf1)-, mesoderm (Foxf1, Meox1)- and ectoderm (Pax6, NeuroD6)-specific transcripts. The presence of Hnf4α, Hes1 and Pdx1 mRNA demonstrated the expression of primitive gut/foregut cell-specific markers. Ngn3, Nkx6.1 and Nkx2.2 transcripts in Sox17-positive cells were determined as properties of pancreatic endocrine progenitors. Immunocytochemistry of activin A-induced Sox17-positive embryoid bodies revealed coexpression of Cxcr4 and Foxa2. Moreover, the histochemical demonstration of E-cadherin-, Cxcr4-, Sox9-, Hnf1β- and Ngn3-positive epithelial-like structures underlined the potential of Sox17-positive cells to further differentiate into the pancreatic lineage. By reducing the heterogeneity of the ES cell progeny, Sox17-expressing cells are a suitable model to evaluate the effects of growth and differentiation factors and of culture conditions to delineate the differentiation process for the generation of pancreatic cells in vitro.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.
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Augustine, Tanya Nadine. "The differentiation of hepatic stem cells into pancreatic endocrine tissue: the influence of pancreatic mesoderm." Thesis, 2008. http://hdl.handle.net/10539/5858.
Full textAbstract:
The use of adult hepatic stem cells for the treatment of diabetes, based both on the close
embryological association of the pancreas and liver, and on a putative shared tissue stem
cell, has been proposed by a number of studies. This study investigated the capacity of
hepatic oval cells to differentiate into pancreatic endocrine cells in the presence of
pancreatic mesoderm. The GaIN model of hepatic injury was used to induce oval cell
activation in Male Sprague-Dawley rats. A viable and significant oval cell population
could not however, be isolated and propagated in culture. In order to continue
experimentation, a PHeSC-A2 cell line, derived from normal adult porcine liver, was
cultured with quail pancreatic mesoderm in the GFRM-Ham s F12.ITS culture system.
Cells demonstrating positive immulocalization of the pancreatic markers, insulin and
glucagon, were identified as PHeSC-A2-derived, by visual assessment of their nuclear
morphology. Techniques used to confirm these results and preclude the derivation of the
pancreatic endocrine cells from pancreatic endodermal contamination, proved ineffectual.
The tentative results obtained in this study have lead to the following postulations: firstly,
the PHeSC-A2 cell line may possess a higher level of potentiality than previously
demonstrated; secondly, this potential may be due to the shared embryological origins of
the pancreas and liver, and thirdly, permissive signaling from pancreatic mesoderm may
have the capacity to induce the differentiation of hepatic oval cells into pancreatic
endocrine cells. Further research is required to confirm the results obtained in this study
and to substantiate the aforementioned propositions.
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Lu, Chung-Kuang, and 盧重光. "Investigating Transcriptional And Post-transcriptional Regulation in Pancreatic Cell Differentiation." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/ny6bpg.
Full textAbstract:
博士國立中正大學
分子生物研究所
102
During pancreas development, many transcription factors function coordinately to ensure normal differentiation of different cell types. In particular, Pdx1, Ptf1a, and Neurog3 play pivotal roles in the growth and lineage specification of pancreatic multipotent progenitor cells (MPCs). Ptf1a is essential for both exocrine pancreas development and maintenance of acinar cell differentiation. The bHLH transcription factor Neurog3 controls pancreatic endocrine cell fate specification in MPCs. However, additional factors which modulate the genetic network to complete the developmental process are much less noticed. Here, I investigate the roles of several novel factors that could be involved in the regulation of pancreatic cell differentiation. Based on a previous differential screening assay, Rbms3 (RNA binding motif, single strand interacting protein 3) was identified. RBMS3 contains two RNA-binding motif and bind tightly to A/U oligoribonucleotides, suggesting that it is an RNA-binding protein. I found that RBMS3 was specifically expressed in the pancreatic rudiment. Next, I showed that RBMS3 specifically bound to the 3’UTR of Ptf1a mRNA, but not the 3’UTR of Pdx-1. Ectopic expression of RBMS3 increased the expression of a reporter gene carrying the Ptf1a 3’UTR. Down-regulation of Rbms3 in the AR42J-B13 pancreatic exocrine cell line affected the expression of endogenous PTF1A protein and its downstream target genes. These results revealed a novel mechanism of post-transcriptional regulation in controlling exocrine pancreas development. On the other hand, two other factors that I investigated, Ccar1 (Cell cycle and apoptosis regulator 1) and Zfp668 (Zinc finger protein 668), appeared to be involved in regulating endocrine differentiation at the transcriptional level. These two factors were previously identified from a yeast two-hybrid screening for interacting with NEUROG3 in our laboratory. The interactions between NEUROG3 and CCAR1, ZFP668 have been further verified using GST pull-down assay and Co-IP experiment. CCAR1 protein has been shown to play the role as a co-activator of nuclear receptors (NRs) to recruit mediator complex. Interestingly, from luciferase reporter gene assays, we found that CCAR1 was required for NEUROG3 to activate the expression of the reporter genes containing Neurod1 promoter. Furthermore, when endogenous levels of Ccar1 were reduced by a shRNA transduction in the PANC-1 pancreatic ductal cell line, NEUROG3-induced the expression of its target genes and the transdifferentiation program were compromised. These results suggest that CCAR1 is one of the competence factors that enable pancreatic progenitor cells to respond to the induction by NEUROG3 and differentiate into endocrine cells. ZFP668 belongs to the krüppel C2H2-type zinc finger protein family and contains 16 C2H2-type zinc fingers, indicating its role as a DNA-binding protein. So far, the function of Zfp668 has remained entirely unexplored. To understand the role of ZNF668 in pancreatic endocrine cell differentiation, I performed an immunohistochemical staining of ZFP668 expression and found that ZFP668 is abundantly expressed in mouse embryonic pancreas. I next demonstrated that ZFP668 was a nuclear protein that physically interacted with NEUROG3 and CCAR1, respectively. I also verified that ZFP668 could cooperate with NEUROG3 in activating Neurod1 and Insm1 expression by using luciferase reporter gene assays. Likewise, down-regulation of endogenous Zfp668 in the PANC-1 cell inhibits the transdifferentiation program initiated by NEUROG3. On the other hand, ZFP668 does not affect the stability of NEUROG3 protein. The results described above may elucidate the transcriptional regulation of NEUROG3-mediated pancreatic endocrine cell differentiation.
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Thatava, Tayaramma. "Differentiation of bone marrow stem cells into functional pancreatic insulin-producing cells /." 2007. http://www.gbv.de/dms/bs/toc/526576839.pdf.
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"Isolation, characterization and differentiation of pancreatic progenitor cells from human fetal pancreas." Thesis, 2007. http://library.cuhk.edu.hk/record=b6074338.
Full textAbstract:
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.
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Chan, Shun-Hung, and 詹蕣璜. "Functions of HIF2 alpha and NeuroD in Zebrafish Pancreatic Beta-Cell Differentiation." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/k7tsdx.
Full textAbstract:
碩士國立臺灣海洋大學
生物科技研究所
102
NeuroD is a basic helix-loop-helix (bHLH) transcription factor that plays important roles in vertebrate CNS, retina and pancreas development. Overexpression of neurod in in Xenopus embryos induces early neuronal differentiation. Patients with Neurod mutation suffer MODY6 diabete syndrome due to dysfunctional pancreas. The pancreas β cell development is mediated by NeuroD. Previous studies demonstrated that HIF2α plays critical functions in CNS development. Depletion of HIF2α inhibited neurod expression and aboragged neuronal differentiation due to loss of birc5a expression. It is unclear whether neurod expression in pancreas β cell is also mediated by HIF2α. Here I demonstrated that the neurod expression in pancreatic cells is not mediated by HIF2α. Depletion of HIF2α did not prevent neurod transcription in pancreas cells. Furthermore, hif2α knockdown did not inbite insulin transcription in pancreas β cell. Conversely, most of neurod transcription in CNS regions was inhibited in hif2α morphants. In conclusion, this study demonstrated the neurod gene is regulated by different mechanism in neural cells and pancreas β cell.
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Blyszczuk, Przemyslaw [Verfasser]. "Differentiation of embryonic stem cells into pancreatic insulin-producing cells / von Przemyslaw Blyszczuk." 2004. http://d-nb.info/97560032X/34.
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Liou, Shian-Wen, and 劉獻文. "Investigating a novel factor that collaborates with Ngn3 in mediating pancreatic endocrine differentiation." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/30964606548893289929.
Full textAbstract:
碩士國立中正大學
分子生物研究所
99
Previous studies have shown that several transcription factors are critical regulators of pancreatic development, such as PDX1, Ptf1a and Ngn3. In above mentioned, Ngn3 is especially important as a bHLH transcription factors that involved in development of all endocrine cell lineages and has been designated as a marker of islet precursor cells. To identify proteins which interact with Ngn3 and possibly modulate the activity of Ngn3 down-stream gene to regulate endocrine cell differentiation during pancreatic development. Previously, our laboratory has used yeast two-hybrid system to screen a mouse embryonic (E10.5) pancreatic bud cDNA library using Ngn3 as a bait. After an intensive screening, Zinc finger protein 668 (Zfp668), which contain 16 C2H2 type zinc finger motif, had been identified to interact with Ngn3. In my study, I have verified that Ngn3 has a direct binding with Zfp668 through GST pull-down assay, and subsequently used co-immunoprecipitation to prove the Ngn3-Zfp668 interaction in mammalian cells. To further dissect Zfp668 for motifs responsible for interacting with Ngn3, a series of GST-tagged Zfp668 truncate protein were purified. I found that the 80~164 and 278~542 amino acid region of Zfp668 are the shortest fraction to mediate the interaction with Ngn3. Significantly, I have also validated that Zfp668 could affect the expression of Ngn3 down-stream target (NeuroD) by luciferase reporter assay. Ectopic expression of Ngn3 and Zfp668 in PANC-1(human pancreatic carcinoma) cells with lentivirus also lead to the same conclusion. In addition, the stable PANC-1 cell line with Ngn3 or Zfp668 overexpression clone were established to determine the biological function of Zfp668.
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Churchill, Angela Josephine. "Spatiotemporal and Mechanistic Analysis of Nkx2.2 Function in the Pancreatic Islet." Thesis, 2016. https://doi.org/10.7916/D84M94N2.
Full textAbstract:
Pancreatic beta cell specification is a complex process, requiring proper function of numerous transcription factors. Nkx2.2 is a transcription factor that is crucial for beta cell formation, and is expressed early and throughout pancreatic development. Nkx2.2-/- mice display complete loss of the beta cell lineage and defects in the specification of other endocrine cell types, demonstrating the importance of Nkx2.2 in establishing proper endocrine cell ratios. Recent studies have also demonstrated a role for Nkx2.2 within the mature beta cell to maintain identity and function.
This thesis work investigated the timing of pancreatic beta cell specification and the mechanism of this process. In these studies, Nkx2.2 was ablated specifically within the Ngn3-expressing endocrine progenitor population in vivo. These mice displayed defects similar to Nkx2.2-/- mice. Surprisingly, the disruption of endocrine cell specification did not require loss of expression of multiple essential transcription factors known to function downstream of Nkx2.2, including Ngn3, Rfx6, and NeuroD1. While these factors are all necessary for beta cell specification, their preserved expression did not rescue beta cell formation. ChIP-Seq analyses also revealed co-occupancy of Nkx2.2, Rfx6, and NeuroD1 near endocrine-related genes, suggesting Nkx2.2 may cooperate with its downstream targets to regulate beta cell fate. These results have revealed a unique requirement for Nkx2.2 during a critical window of beta cell development.
In addition, the role of a conserved domain of Nkx2.2, the specific domain (SD), was assessed using Nkx2.2SDmutant mice. Transcriptional profiling of Nkx2.2SDmutant endocrine progenitors revealed a critical role for the SD domain in regulating the transcription of endocrine fate genes early in the process of endocrine differentiation. In addition, beta cell-specific deletion of the Nkx2.2 SD domain resulted in hyperglycemia, glucose intolerance and dysregulation of beta cell functional genes. This suggests the SD domain is important for mediating Nkx2.2 function within the beta cell to maintain glucose homeostasis.
Together, these results have elucidated a critical developmental window for beta cell specification and demonstrated an essential role for Nkx2.2 and specifically its SD domain in this process. Furthermore, these studies suggest that beta cell transcription factors may also regulate endocrine fate in a combinatorial manner, and exert changes within the endocrine progenitor lineage. These findings have provided us with a better understanding of in vivo pancreatic development, and will improve current research efforts to differentiate beta cells in vitro from hPSCs.
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Thatava, Tayaramma [Verfasser]. "Differentiation of bone marrow stem cells into functional pancreatic insulin-producing cells / von Tayaramma Thatava." 2007. http://d-nb.info/983842833/34.
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(11134677), Shiqi Tang. "Differentiation of Cav1.2 and Cav1.3 pharmacology and role of RyR2 in pancreatic beta-cell electrophysiology." Thesis, 2021.
Find full textAbstract:
The L-type VGCC subtypes, including subtypes Cav1.1-1.4, have been shown to play critical roles in various cellular activities, including muscle contraction, hormone secretion, and neurotransmitter release. Recent research indicates the potential involvement of Cav1.3 in various neurological and psychiatric disorders, such as the early onset of Parkinson’s disease and substance abuse disorders. Non-selective L-VGCC subtype blockers such as dihydropyridines (DHPs) are used to treat hypertension and angina because they potently inhibit Cav1.2, but no selective Cav1.3 inhibitors have been developed yet. We resolved the molecular determinants to differentiate Cav1.2 and Cav1.3 in response to DHP nifedipine. Nifedipine IC50 for Cav1.2 and Cav1.3 are 22nM and 289nM determined by whole-cell patch-clamp. We identified two significant amino acids, Cav1.3/M1030 to Cav1.2/V1036 in the transmembrane IIIS5 and Cav1.3/S1100 Cav1.2/A1106 in the extracellular IIIS-3P loop, to differentiate the subtype affinity to nifedipine.
We found that the Cav1.3/II-III loop fused to eGFP decreased glucose-activated action potential (GSAP) frequency by ~80% in the pancreatic β-cell. We introduced several synthetic peptides, and peptide P3-1 from C-terminal induced a -16mV shift in V1/2 inactivation with an EC50 of 231nM. P3-1 contains a protein kinase G (PKG) phosphorylation site (RRISE) required for PKG inhibition of Cav1.3 current but not conserved in Cav1.2. We found that the shift in V1/2 inactivation induced by co-expression of Cav1.3 with the Cav1.3/II-III loop/GFP requires the presence of a Cavβ subunit, and Cavβ3 also exhibits selectivity over other β subunits. Significantly, P3-1 shifts the Cav1.2 inactivation to a more positive voltage when co-expressed with either Cavβ2a or Cavβ3, demonstrating the ability of P3-1 to differentiate Cav1.2 and Cav1.3 in a Cavβ-dependent manner.
Failure of pancreatic β-cells to secrete enough insulin to maintain glucose homeostasis is a hallmark of Type 2 diabetes. However, the consequences of the dysregulation of the endoplasmic reticulum (ER) Ca2+ channel ryanodine receptor-2 (RyR2) in pancreatic β-cells are not fully understood. Therefore, we characterized the electrical activity in INS-1 in which RyR2 has been deleted via CRISPR/Cas9 gene editing. We observed a decreased level of IP3 receptor binding protein (IRBIT) in RyR2KO INS-1 cells and generated IRBITKO INS-1 cells. VGCC current density in RyR2KO doubled compared to controls and was also elevated in IRBITKO compared to control cells. All HVA Ca2+ channels were upregulated, determined by fractional current blocked by nifedipine. We also found that GSAP frequency is doubled by RyR2 deletion due to failure to activate apamin sensitive SK (small conductance calcium-activated potassium) channels.
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Wang, Sui. "The Myt1 and Ngn3 feed-forward expression loop drives pancreatic islet differentiation in the mouse." Diss., 2009. http://etd.library.vanderbilt.edu/available/etd-11262009-124114/.
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Kuen, Janina. "Influence of 3D tumor cell/fibroblast co-culture on monocyte differentiation and tumor progression in pancreatic cancer." Doctoral thesis, 2017. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-156226.
Full textAbstract:
Pancreatic cancer (PC) remains one of the most challenging solid tumors to treat with a high unmet medical need as patients poorly respond to standard-of-care-therapies. Prominent desmoplastic reaction involving cancer-associated fibroblasts (CAFs) and the immune cells in the tumor microenvironment (TME) and their cross-talk play a significant role in tumor immune escape and progression. To identify the key cellular mechanisms induce an immunosuppressive tumor microenvironment, we established 3D co-culture model with pancreatic cancer cells, CAFs, monocyte as well as T cells. Using this model, we analysed the influence of tumor cells and fibroblasts on monocytes and their immune suppressive phenotype. Phenotypic characterization of the monocytes after 3D co-culture with tumor/fibroblast spheroids was performed by analysing the expression of defined cell surface markers and soluble factors. Functionality of these monocytes and their ability to influence T cell phenotype and proliferation was investigated. 3D co-culture of monocytes with pancreatic cancer cells and fibroblasts induced the production of immunosuppressive cytokines which are known to promote polarization of M2 like macrophages and myeloid derived suppressive cells (MDSCs). These co-culture spheroid polarized monocyte derived macrophages (MDMs) were poorly differentiated and had an M2 phenotype. The immunosuppressive function of these co-culture spheroids polarized MDMs was demonstrated by their ability to inhibit autologous CD4+ and CD8+ T cell activation and proliferation in vitro, which we could partially reverse by 3D co-culture spheroid treatment with therapeutic molecules that are able to re-activate spheroid polarized MDMs or block immune suppressive factors such as Arginase-I. In conclusion, we generated a physiologically relevant 3D co-culture model, which can be used as a promising tool to study complex cell-cell interactions between different cell types within the tumor microenvironment and to support drug screening and development. In future, research focused on better understanding of resistance mechanisms to existing cancer immunotherapies will help to develop new therapeutic strategies in order to combat cancerBei Bauchspeicheldrüsenkrebs handelt es sich um eine maligne Tumorerkrankung, deren Behandlung Ärzte noch immer vor große Herausforderungen stellen und die zur dritthäufigsten krebsbedingten Todesursache der westlichen Welt zählt. Desmoplastische Reaktionen im Tumorgewebe sind hierbei ein besonderes Merkmal dieser Erkrankung. Dabei spielen tumor-assoziierte Fibroblasten sowie unterschiedliche Zellen des Immunsystems und deren Interaktionen eine essentielle Rolle hinsichtlich Tumorwachstum und der Herunterregulation des Immunsystems. Um zelluläre Mechanismen, die ein immunsuppressives Tumormilieu induzieren, zu identifizieren, entwickelten wir ein 3D Ko-Kultur Modell mit Bauchspeicheldrüsenkrebszellen, tumor-assoziierten Fibroblasten sowie Monozyten und T-Zellen. Mit Hilfe dieses Modells konnten wir den Einfluss von Tumorzellen und Fibroblasten auf den Phänotyp und das Verhalten von Monozyten untersuchen. Dazu wurden Monozyten in einer 3D Tumorzell/Fibroblasten Ko-Kultur kultiviert und differenziert, um anschließend die Expression definierter Zelloberflächenmarker und löslicher Faktoren zu analysieren. Des Weiteren wurde das Verhalten dieser 3D Ko-Kultur differenzierten myeloiden Zellpopulation sowie ihre Fähigkeit den Phänotyp von T Zellen und deren Proliferation zu beeinflussen untersucht. Die 3D Ko-Kultur der Monozyten zusammen mit den Tumorzellen und den Fibroblasten führten zur Produktion immunsuppressiver Zytokine und Chemokine, wodurch die Differenzierung der Monozyten in M2-ähnliche Makrophagen induziert wurde. Diese durch die 3D Tumorzell/Fibroblasten Sphäroide polarisierten aus Monozyten herangereiften M2-ähnlichen Makrophagen besaßen außerdem immunsuppressive funktionelle Eigenschaften, indem sie in der Lage waren, die Aktivierung und Proliferation von autologen CD4+ und CD8+ T Zellen in vitro zu inhibieren. Die Suppression sowohl der CD4+ als auch der CD8+ T Zellen konnte durch die Behandlung therapeutischer Moleküle, die die Re-Aktivierung der immunsuppressiven 3D Sphäroid polarisierten Makrophagen stimulierten oder suppressive Faktoren wie Arginase-I blockierten, wieder aufgehoben und die T Zell Proliferation teilweise wiederhergestellt werden. Unser etabliertes 3D Ko-Kultur System repräsentiert ein vielversprechendes physiologisch relevantes Modell, welches genutzt werden kann, um Zell-Zell Interaktion und Kommunikation im Tumormilieu zu untersuchen und dadurch die Wirkung von Medikamenten zu verbessern. Ein gezieltes besseres Verständnis von Tumorresistenz Mechanismen gegen bereits bestehende Immun Therapien fördert die Entwicklung neuer therapeutischer Ansätze zur Bekämpfung von Krebs
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Yi-Hsien, Fang, and 方議賢. "Investigating the influence of ZFP668 on pancreatic endocrine cell differentiation using ex vivo tissue culture system." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/55928346031382170753.
Full textAbstract:
碩士國立中正大學
生物醫學研究所
104
The pancreas is a glandular organ in the digestive system and endocrine system of vertebrates. It controls blood glocuse stability. Transcription factors such as PDX1、Ptf1a(P48) and Ngn3 function coordinately to regulate pancreas development during embryonic development.. Neurogenin3 (Ngn3) is a member of the basic helix-loop-helix family. It controls the development and formation of the endocrine cells ‚such as α cells、β cells、ε cells、δ cells、PP cells. Previous study has shown that Zinc finger protein 668 (ZFP668)，which contains 16 C2H2 type zinc finger motif can interact with Ngn3. ZFP668 has been identified to interact with Ngn3 by GST-pull down assay and co-immunoprecipitation (Co-IP) experiment. Our previous study has also shown that ZFP668 could regulate the expression of Ngn3’s downstream target genes such as NeuroD1 and Insm1. The aim of my project, is to clarify whether ZFP668 could modulate endocrine cells differentiation. I used β cell line- NIT1 and ex vivo embryonic pancreas bud culture as my experimental model. First‚ I suppress the ZFP668 expression by lentivirus infection in both model. After ZFP668 is knocked down, I analysed the Ngn3 downstream genes expression in NIT1 cells. On the other hand‚ I also dissected the embryonic pancreas bud from mouse embryos, infected with lentivirus and then cultured the cells for seven days. Finally, I used immunostain to detect the lentivirus infected cells which co-expressed endocrine cell markers. Then, I investigated whether knockdown ZFP668 can affect endocrine cells differentiation. In order to further confirm whether ZFP668 can affect endocrine cell differentiation, I established the ES cells differentiation model. I induced ES cells to differentiate into insulin-producing cells and analysed the expression of pancreatic endocrine markers during differentiation. The current results show that endoderm markers can be detected, and some endocrine cell markers can also be deceted during ES cell differentiation process. These results confirmed that ES cell can be induced to differentiate into pancreas like cell. However, different conditions for the induction, including culture medium and different combination of growth factors for treating ES cell, need to be explored to improve the efficiency of pancreatic cell differentiation at final stage.
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Wu, Yi-Chuan, and 吳宜娟. "Effects of Lotus Leaf Methanolic Extract on Pancreatic Lipase Activity and Differentiation of 3T3-L1 Preadipocytes." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/36430238472993842798.
Full textAbstract:
碩士臺灣大學
食品科技研究所
95
Obesity is the most common nutritional disorder and it is considered to be a risk factor associated with the development of the major human diseases, including cardiovascular disease, diabetes, and cancer. It is an important topic in the world of public health and preventive medicine. It has close relations with lipid absorption and pancreatic lipase activity. The differentiation of 3T3-L1 preadipocytes plays an important role on the mechanism of adipogenesis. In the research about lotus (Nelumbo nucifera Gaertn.) leaf methanolic extract (LLM) on pancreatic lipase activity and differentiation of 3T3-L1 preadipocytes is rare. The aim of the present study was to assess the effects of LLM on pancreatic lipases activity and differentiation of 3T3-L1 preadipocytes. LLM were screened for inhibitory effects on pancreatic lipase (PL) activities as well as on triglyceride (TG) accumulation in 3T3-L1 preadipocytes to find the effective fractions, subfractions and active compounds that having the anti-obesity trend. Six flavonoids were separated from LLM, including quercetin, quercetin-3-O-glucoside, quercetin-3-O-glucuronide, quercetin-3-O-galactoside, catechin and kaempferol-3-O-glucoside. In pancreatic lipase activity-inhibiting assay, quercetin exhibited significant inhibitory effects at the concentration of 50 μM with 75% pancreatic lipase activity v.s control (p<0.05). In 3T3-L1 preadipocyte experiment, subfractions 1, 3 and 4 of LLME (LLM ethyl acetate partition) markly inhibited about v 25% of lipid accumulation in 3T3-L1 cells at the concentration of 50 μg/mL. Moreover, quercetin and quercetin-3-O-galactoside were found to inhibit TG accumulation in 3T3-L1 cells at the concentration of 50 μΜ with 15% and 18%. Subfraction 3 of LLME could reduce expression of PPARγ and C/EBPα mRNA, especially mRNA expression-inhibiting of C/EBPα is more apparent than PPARγ. Consequently, LLM could reduce pancreatic lipase activity, inhibit the differentiation of 3T3-L1 preadipocytes and decrease TG accumulation in 3T3-L1 cells.
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Lehnert, Lasse [Verfasser]. "Morphological and molecular characterization of human pancreatic adenocarcinoma cells undergoing duct-like differentiation / vorgelegt von Lasse Lehnert." 2000. http://d-nb.info/972055460/34.
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