To harness the therapeutic potential of stem cells for the treatment of diabetes novel technologies will need to be developed. First, stem cells will be have to be isolated and expanded. Second, the stem cells will have to undergo directed and controlled differentiation toward the beta-cell lineage. Finally, fully functional beta-cells must be isolated from the resulting complex mixes of cells. In this application, we propose to develop reagents that will assist in the further development of all three of these steps. We will generate a comprehensive panel of monoclonal antibodies directed against cell surface epitopes of murine, primate and human pancreatic cells. These antibodies will permit the fractionation of pancreatic cell suspensions using fluorescence activated cell sorting and/or magnetic bead panning. This approach can be used to isolate pancreatic stem cells (step 1) as well as for the purification of differentiated progeny before therapeutic transplantation (step 3). In addition, this proposal is designed to identify the network of genes controlled by the pancreatic transcription factor, PDX-1. Evidence suggests that the family of protein-coding genes under the control of PDX-1 defines many of the phenotypic characteristics of differentiated beta cells. Although a few of these genes have been identified, the full constellation of PDX-1 responsive targets is unknown. We propose that PDX-1 also drives the expression of non-coding genes that are equally important for regulating beta cell function (steps 2&3). Definition of both families of targets is required for a complete understanding of beta cell biology and pathophysiology.

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We propose the generation of a transgenic model of rapid inducible diabetes. The major aim of this proposal is to determine the origin of insulin-producing beta-cells in adult mice. In these animals, complete, or partial, selective destruction of pancreatic beta-cells will be induced, either during development, during postnatal growth or in adults, upon administration of an inducer drug. These animals will not be engineered to study the pathogenesis of Type 1 diabetes and the autoimmune mechanisms, nor inflammation; rather, they will be useful to accurately assess islet (beta-cell) neoformation in adult pancreas during the regeneration process that should follow the treatment with the inducer agent. They will also be used in reconstitution assays to determine the differentiation potential of injected stem/progenitor cells, whether i.v. or directly into the pancreas, derived either from pancreatic or extra-pancreatic tissues (e. g. hepatic, neural or hematopoietic stem cells, or ES cell clones). Incidentally, this transgenic model will represent a unique tool to study the involvement of beta-cells in pancreas homeostasis, besides their role of producing insulin.

Beyond the basic processes of organ growth and maintenance, this study may have implications for cell replacement therapy in diabetes and for all ethical issues related to the use of embryonic or adult stem cells. Once generated, these transgenic strains will be put into the “public” Beta Cell Biology Consortium (BCBC) mouse repository, so as to become available to the broad scientific community, as we have already done with other transgenic mice that we produced in the past.

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Diabetes mellitus is a significant health problem, affecting approximately 16 million people in the United States. Future therapeutic approaches to diabetes will benefit greatly from a complete understanding of the expression profile of the b-cell under normal and pathological conditions and the functional annotation of differentially expressed genes. The goal of this application is to pool the complementary expertise available in three laboratories for mining of an exciting new resource—the more than 7,700 unique cDNAs cloned by the prior NIDDK-funded consortium on “Functional Genomics of the Developing Endocrine Pancreas.” Our goals are three-fold: Aim 1 of this proposal will establish a large cDNA microarray enriched for genes expressed in the endocrine pancreas by combining the 7,700 non-redundant cDNAs described above with the 3,400 clones of our current PancChip 2.0. We will employ this microarray for the screen of six paradigms of perturbed b-cell function to identify candidate genes to be analyzed further in aims 2 and 3. Aim 2 will transfer 1,000 selected cDNA clones from our collection into the FLEXGene repository. This repository will allow for high-throughput transfer of cDNAs into multiple expression vectors. In addition, we will select antigens for the production of antisera to derive marker antibodies of b-cells and their precursors. In Aim 3 we will functionally evaluate 500 selected cDNAs for their potential role in b-cell biology. Clones transferred into the FLEXGene repository and sequence verified (Aim 2) will be subcloned into adenovirus vectors to allow for efficient transduction of INS-1 cells. In some cases, the sequence of differentially expressed genes will be used for design of interference RNA (RNAi) oligonucleotides to allow suppression of target gene expression. The effect of modulation of target gene expression will then be tested in various models of b-cell function. Candidate cDNAs that are positive in this screen will be further evaluated in adenovirus-transduced islets and/or transgenic animals. Genes identified in this fashion may become candidate drug targets or could be useful in development of surrogate b-cells for cell-based insulin replacement therapy. This project will serve as a valuable gene discovery effort that will complement the program implemented by the NIDDK-funded Beta Cell Biology Consortium. The new resources generated through this project will be made available to the NIDDK-funded biotechnology centers and the diabetes research community at large.

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The ability to generate multiple cell types from embryonic stem (ES) cell in culture offers unprecedented opportunities to investigate the mechanisms that regulate the earliest stages of lineage commitment that lead to the development of the primary germ layers; ectoderm, mesoderm and endoderm. In addition, ES cells offer a novel and potentially unlimited supply of cells for transplantation for the treatment of a broad range of diseases. For the basic biological and therapeutic potential of the ES cell system to be fully realized, it is essential to first define the mechanisms that regulate lineage induction and tissue specification in this model. The goal of this proposal is to investigate the mechanisms that regulate endoderm induction and pancreatic specification in both mouse and human ES cell differentiation cultures. The overall approach is to focus the experiments in each of the first three aims on three specific stages of development. In the first aim, we will use a mouse ES cell line with the GFP cDNA targeted to the brachyury locus and human CD4 targeted to the Foxa2 (HNF3b) locus to define the signaling pathways required for the earliest stage of development, the establishment of a primitive streak-like population. Induction of a population comparable to the primitive streak represents the first step in the development of definitive endoderm. The focus of the second aim is to determine what factors regulate the development of endoderm and Pdx1+ pancreatic progenitors from the primitive streak-like cells. The third aim will define the conditions necessary for the maturation of Pdx1+ progenitors to functional insulin secreting cells. In the fourth aim, we will translate the findings from the studies with mouse ES cells to the human system. Approaches comparable to those used for differentiation of the mouse ES cells will be used to establish conditions necessary for the development of primitive streak-like cells, endoderm, pancreatic progenitors and insulin-secreting cells from human ES cells. For these experiments we will use the following human ES cell lines: ES02, ES03, ES04, TE-03, TE-06, MI01, UC01, WA01, WA07, WA09, BG02 and BG03. The outcome of these experiments will provide new insights into the regulation of endoderm induction and pancreatic specification in the ES cell differentiation model.

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Type 1 diabetes is characterized by the permanent loss of pancreatic insulin-producing beta cells. Our goal is to understand the mechanisms determining pancreatic beta cell mass, as a first step toward the development of regenerative therapies for diabetes. We will use transgenic mouse technology to examine the hypothesis that beta cells have a significant regenerative capacity, which is modulated by specific signals.

A fundamental problem in pancreas biology concerns the dynamics of beta cell mass regulation. While autoimmune or pharmacological destruction of beta cells appears to be irreversible, adult beta cell mass does respond to specific physiological cues. This raises the exciting possibility that beta cells have a significant regenerative capacity that could be used to treat diabetes.

The long-term objective of this project is to understand the mechanisms determining pancreatic beta cell mass. We will use transgenic mouse technology to quantitatively assess the magnitude of beta cell regeneration after defined beta cell ablation, to determine the cellular origins of regenerating beta cells and to characterize signals that govern beta cell homeostasis.[ Hide ]

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We propose that Nkx2.2 and NeuroD, two essential pancreatic transcription factors, differentially regulate the development and differentiation of individual islet cell populations. Nkx2.2 and NeuroD are both required for the development of α and β cells in the mouse pancreatic islet. Consequently, mice lacking either of these factors die within days after birth due to neonatal diabetes. Nkx2.2-null mice completely lack insulin-producing β cells and have a large decrease of glucagon-producing a cells. In place of these cell types, the Nkx2.2 mutant islet is filled with cells producing the hormone ghrelin. Extensive analysis has indicated that Nkx2.2 is responsible for the initial specification of α and β cells. Alternatively, NeuroD appears to play a later role in islet cell differentiation as NeuroD-null mice display poor differentiation and extensive cell death among developing β cells, disorganized α cells, and subsequent failure to form islets. We have demonstrated that Nkx2.2 and NeuroD play critical roles in pancreatic cell differentiation, therefore understanding their precise functions in the islet will be essential for understanding of the molecular mechanisms that regulate pancreatic cell fate decisions.

To define the specific roles of Nkx2.2 and NeuroD in each of the islet cell types and their respective roles in the function of the mature α and β cells, we have generated floxed-conditional knock-out mice for Nkx2.2 and NeuroD. We will use these mice to delete each gene in developing or mature α or β cells to determine their respective roles in each cell type. In addition, we will determine the precise molecular mechanisms of Nkx2.2 and NeuroD function in each individual cell type by introducing targeted mutations into each gene using the ES cell cassette exchange approach (Magnuson). Finally, preliminary data suggests that Nkx2.2 and NeuroD have intersecting affects on ghrelin cell expression in the islets. We will determine the epistatic relationship between Nkx2.2 and NeuroD in the regulation of the ghrelin cells and the other islet cell types by generating Nkx2.2/NeuroD double null mice. Given that cell replacement therapy for Type 1 diabetes will heavily depend on our understanding of the factors involved in development of pancreatic beta cells, our research will have a significant impact on molecular and cellular approaches to generating functional endocrine cells for curing Type 1 diabetes. [ Hide ]

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The goal of this proposal is to advance technologies and understanding about the mechanism of pancreatic beta cell development in embryogenesis and adult islets, and to promote the application of such advances to islet regeneration and stem cell biology by other members of the Beta Cell Biology Consortium (BCBC). I propose to bring two technologies to the BCBC; one will identify new and unanticipated transcription factor-based regulatory events that are relevant to endocrine development, and the other will define endothelial cell signaling factors that promote endocrine differentiation and pancreatic stromal cell survival. In our first Aim, we will generate in vivo footprinting data and related information on pro-endocrine transcription factor genes at different stages of pancreatic development and aging, to better define regulatory events that govern endocrine progenitor cell differentiation and beta cell regenerative capacity. By disseminating data to members of the BCBC, collaborators can help us define new factor-regulatory sequence interactions and use the information to monitor, predict, and ultimately perturb beta cell generation from stem cells and during islet regeneration. This approach is complementary to existing genetic studies, which give terminal phenotypes but not information about genetic regulatory mechanisms. In our second Aim, we will use existing endothelial cell lines and create new ones from mouse embryos to identify endothelial signaling factors that promote endocrine progenitor differentiation and pancreatic stromal cell survival. Since endothelial cells and stromal cells both control pancreatic growth, these studies are intended to reveal new signaling molecules that control islet development and regeneration. We also plan to link Aims 1 and 2 by investigating transcription factor occupancies at pro-endocrine genes in response to endothelial signals. By sharing technology and information from our work with the BCBC consortium, our basic developmental studies will be more rapidly translated to develop cures for diabetes.

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This is a renewal application in response to RFA-DK-04-017 for a program that will explore the molecular and gene regulatory events that occur during pancreas development. To achieve the goal of creating a replenishable source of b-like cells for transplant therapy of diabetic patients, it is necessary to understand the growth properties and cellular differentiation capacity of pancreatic precursor cells, and to be able to direct these cells towards a b-cell phenotype. The goal of this program, which consists of four individual projects and one core (administrative), is to gain a deeper understanding of the gene regulatory processes that occur near the midpoint of murine pancreogenesis. We will create and characterize a variety of new mouse models in order to determine how different gene regulatory processes impact the fate of immature pancreatic progenitor cells. We expect that these studies will contribute to a better understanding of how b-cell differentiation differs from that of other endodermal cell types.

Project 1 (MacDonald) will examine the interface of Notch-signaling and the key transcription factor PTF1a for resolving the islet versus acinar cell fate from common embryonic progenitors.

Project 2 (Magnuson) will use cassette exchange technology in mouse ES cells to better understand how the Delta/Notch signaling pathways affect both the multipotency and cell fate decisions of pancreatic progenitor cells.

Project 3 (Stein) will determine the special role that MafA plays to direct the expression of genes that define b-cell identity.

Project 4 (Wright) will analyze the role of several key gene transcriptional networks, and how they affect endocrine cell differentiation within the pancreas.

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Islet cell transplantation represents a logical approach to a curative treatment for Type 1 diabetes mellitus. However, even if the obstacles associated with transplantation, such as immunosuppression, are overcome in the future, the supply of islets remains a major problem. Clearly, novel strategies are critically needed to provide sufficient islet material for all potential patients. Embryonic stem cells (ES) offer a promising source for in vitro generation of beta cells, but before this will become reality, several challenges should be surmounted. One major issue is the limited knowledge of the developmental processes that govern patterning, growth, and differentiation in the developing embryo. The central theme of this Program is to investigate the biology of pancreatic progenitor cells. We will generate novel mouse strains and use these to investigate how pancreatic progenitor cells (PPC) are specified and how the formation of endocrine progenitor cells (EPC) is regulated. Additionally, we will investigate the mesenchymal signals that control the proliferation and endocrine lineage commitment of the PPC as well as the signals that direct the proliferation and differentiation of the EPC. We will investigate the mechanisms by which Ngn3 induce generic endocrine- and endocrine subtype-specific differentiation events as well as the mechanisms by which genes such as Nkx6.1, Arx, and Pax4 regulate endocrine subtype specification. Lastly, we will test whether the mechanisms used in the embryo for regulation of endocrine development are also utilized for beta cell formation in adults after partial pancreatic duct ligation, or whether beta cells under these circumstances are formed purely by replication of pre-existing beta cells. To accomplish our objectives we are building on previously established collaborations between the different investigators, and we are expanding these to include the generation of novel tools that will be shared across project boundaries, with other BCBC investigators, and with the community at large. In particular, in this proposal we will generate novel mouse lines and use these to investigate the cellular and molecular mechanisms that regulate beta cell development.

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This purpose of this application is for Vanderbilt University to continue to serve as the Coordinating Center (CC) for the Beta Cell Biology Consortium (BCBC), a team science initiative that has been in existence since September 2001. The mission of the BCBC, as defined by RFA DK-01-014, is to facilitate interdisciplinary approaches that will advance our understanding of pancreatic islet development and function with the long-term goal of developing a cell-based therapy for insulin delivery. In the second phase, the BCBC will undertake a variety of investigator-initiated studies while also placing a major emphasis on reagent-generating activities. Thus, for this team of scientists to be able to generate new antibodies and genetically modified mice, while also maintaining focus on the scientific needs that lie at the heart of determining how to develop a cell-based therapy for insulin delivery, a CC has been developed over the past two years. The CC is now fully positioned to provide financial, organizational, and informatic support for the wide variety of scientific activities that will be encompassed by the BCBC.

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To harness the therapeutic potential of stem cells for the treatment of diabetes novel technologies will need to be developed. First, stem cells will be have to be isolated and expanded. Second, the stem cells will have to undergo directed and controlled differentiation toward the beta-cell lineage. Finally, fully functional beta-cells must be isolated from the resulting complex mixes of cells. In this application, we propose to develop reagents that will assist in the further development of all three of these steps. We will generate a comprehensive panel of monoclonal antibodies directed against cell surface epitopes of murine, primate and human pancreatic cells. These antibodies will permit the fractionation of pancreatic cell suspensions using fluorescence activated cell sorting and/or magnetic bead panning. This approach can be used to isolate pancreatic stem cells (step 1) as well as for the purification of differentiated progeny before therapeutic transplantation (step 3). In addition, this proposal is designed to identify the network of genes controlled by the pancreatic transcription factor, PDX-1. Evidence suggests that the family of protein-coding genes under the control of PDX-1 defines many of the phenotypic characteristics of differentiated beta cells. Although a few of these genes have been identified, the full constellation of PDX-1 responsive targets is unknown. We propose that PDX-1 also drives the expression of non-coding genes that are equally important for regulating beta cell function (steps 2&3). Definition of both families of targets is required for a complete understanding of beta cell biology and pathophysiology.

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Investigators within the NIDDK Islet and Autoimmunity Branch (IAB), along with intramural and extramural collaborators, are involved in several ongoing projects to assess the capacity of putative beta cell progenitor cells to differentiate into functionally relevant and physiologically regulated insulin producing cells. Toward that end, and depending upon the progenitor cell source, IAB investigators have developed a mouse model (described at previous BCBC retreats) specifically designed to promote in vivo differentiation of the putative beta cell progenitor. For instance, in that mouse, immune mediated destruction of the endogenous beta cells can be carefully regulated such that the killing takes place over weeks (to create an increasing need for new beta cell function) and such that new beta cells created from the progenitor would not be susceptible to the immune mediated killing. We have also developed a non-human primate model (also described at previous retreats) for testing potential islet progenitors- and experiments testing cells grown in vitro by our intramural collaborator (Dr. Marvin Gershengorn) are underway. Last, we have two clinical protocols, one under way and one in the late planning phase, to test whether pancreatic insulin producing capacity can recover in patients with long standing type 1 diabetes mellitus. With regard to the latter clinical trials, we are careful to not list as an endpoint recovery of beta cell mass, since we acknowledge that mass cannot, as of today, be measured. We are also, therefore, testing a novel imaging technique we believe holds promise for assessing beta cell mass in vivo.

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