Project Investigators:

Pedro Herrera

Dale Greiner

Alvin Powers

Leonard Shultz

Lay Abstract:

Over the first two years of the HUMANE CBP, we have developed four immunodeficient mouse models and two immunocompetent mouse models that can be induced environmentally or genetically to become diabetic. Two of the immunodeficient models have been fully validated for their ability to be engrafted with mouse and human islets, and the other two are now successfully backcrossed onto a specific strain of immunodeficient mice. Backcrossing of the two immunocompetent strains is continuing. Progress for the first two years of the CBP is summarized in our Progress Report and highlights the successful accomplishment of each of the milestones proposed. Importantly, all four immunodeficient models have been engineered with the long-term anticipation that they will facilitate studies of human beta cell development and function in the presence of functional human alloreactive or autoreactive immune system
As the needs of the BCBC continue to evolve, we propose in the next year of the HUMANE CBP to complete the validation of the novel models we have developed, and incorporate additional goals that our CBP is uniquely positioned to accomplish. Based on extensive consultation with BCBC Program Directors, we have restructured the CBP and propose to incorporate an additional investigator, Dr. Alvin Powers at Vanderbilt University.
Participants in this Collaborative Bridging Project combines:
1) The expertise of Dr. Pedro Herrera in β-cell development and production of mouse models to study developmental intermediates and regeneration
2) The expertise of Dr. Leonard Shultz in the generation and validation of humanized mouse models for studies of T1D and other diseases
3) The expertise of Dr. Dale Greiner in islet transplantation and T1D.
4) The expertise of Dr. Alvin Powers in islet transplantation and human beta cell physiology.
Goal 1 will perform validation experiments on immunodeficient mice that develop spontaneous diabetes. We will use these models to determine the optimal site(s) for implantation of islets and dissociated beta cells. Goal 2 will validate newly developed unique immunodeficient and immunocompetent models of beta cell ablation that can be induced to become diabetic based on beta cell expression of the diphtheria toxin receptor. Goal 3 will evaluate the effect of diphtheria toxin on human islets in vitro and in vivo and establish a novel model in which the effects of immunosuppressive agents on human islets can be evaluated. Goal 4 will perform a pilot study with Dr. Gordon Keller to evaluate the ability of human embryonic stem cell-derived beta cells engraft and function in the newly developed model systems.

Project Investigators:

Ray MacDonald

Anne Grapin-Botton

Mark Magnuson

Palle Serup

Kenneth Zaret

Lay Abstract:

Project Investigators:

Kenneth Zaret

Ondine Cleaver

Gerard Gradwohl

Harry Heimberg

Gordon Keller

Eugene Kolker

Lay Abstract:

The pancreatic islet is an endocrine gland; thus, the developing beta cells must coordinate their specification and differentiation with the vasculature so that the final architecture of the islet and pancreas enables rapid and efficient sensing of circulating glucose and secretion of insulin into the bloodstream. Although certain organisms such as zebrafish can survive early development without a vasculature, genetic and functional studies have shown that amniotes, including mammals, require proper vascular development and direct interactions with vascular endothelial cells for beta cell development and islet function. Research from our laboratories and others has shown that endothelial cells help promote endocrine and beta cell development at multiple discrete steps, including the initial differentiation of Ptf1a gene-positive pancreatic progenitors from an initial pool of Pdx-1 gene positive endoderm progenitors; the induction of insulin gene expression from a later pool of Ngn3 gene positive endocrine progenitors; and the normal function of the islet in adult animals. Given the known complexity of beta cell development and the difficulty in reconstituting proper glucose-sensing, insulin secreting cells from stem cells, additional steps of beta cell development may require endothelial cell interactions. To address these issues, in preliminary studies we showed that the Ptf1a induction step of the Pdx-1 positive endoderm, where endothelial cell interactions are crucial, could be reconstituted with secreted endothelial cell proteins in an in vitro explant assay with tissues from flk-1-/- embryos, which lack endothelial cells. Further reiterative cycles of biochemical fractionation and explant screening, followed by proteomic analysis, identified netrin-4 as a secreted endothelial factor that is sufficient to completely replace the effect of endothelial cells in co-cultures. Subsequent analyses of netrin-4 expression in embryogenesis show that the protein is normally induced just prior to the time of Ptf1a induction and is necessary, in the explant assay, for coordinate induction of various early pancreatic regulatory genes. Others in the BCBC have found that netrin-4 promotes endocrine and later insulin expression in mouse embryonic stem cells. Given these successes, we propose to repeat the above screening and proteomic approach to discover endothelial secreted molecules that could: 1) induce later endocrine and insulin expression in embryo pancreatic explant cultures; 2) promote the differentiation of Pdx-1 positive beta cell progenitors that arise after pancreatic duct ligation in adult animals; 3) induce different early genes and developmental states in flk-1-/- embryonic explants, compared to our previous studies; and 4) promote the differentiation of human embryonic stem cells from the Pdx-1 positive stage to later stages of beta cell development.

Project Investigators:

Gordon Keller

Mark Magnuson

Chris Wright

Peter Zandstra

Lay Abstract:

One potential new therapy for type I diabetes is the transplantation of functional beta cells into diabetic patients. While attractive, this approach for treating diabetes suffers from a severe shortage of transplantable cells. Embryonic stem (ES) cells and the more recently generated induced pluripotent (iPS) cells offer a novel, unlimited source of many different cell types including pancreatic beta cells. For stem cell-derived beta cell transplantation to become a reality, it is essential to develop appropriate technologies to efficiently and reproducibly direct the differentiation of stem cells to generate insulin-producing cells. The studies outlined in this application address a number of important issues regarding the generation of functional beta cells from ES and iPS cells. In the first aim of these studies, we propose to apply a newly modified differentiation protocol to different stem cell populations and compare their ability to form insulin-producing cells in tissue culture. Such comparative studies are essential to determine if the current methods are sufficiently robust to promote and support pancreatic differentiation from a broad range of stem cell lines. The goal of the second aim is to genetically modify an ES cell line to enable us to isolate virtually pure populations of pancreatic progenitor cells and insulin producing cells. These isolated cell populations will be transplanted into diabetic mice to determine if they can function to cure the diabetes. In the final set of studies, we propose to develop new technologies that will enable us to generate large numbers of insulin producing cells from these stem cell populations, numbers sufficient for transplantation into large animals and ultimately for clinical trials in humans.

Project Investigators:

Markus Grompe

Klaus Kaestner

Christian Stoeckert

Lay Abstract:

In order to produce functional insulin producing β-cells for the cure of diabetes it is necessary to know all of the genes expressed in these cells inside a normal human pancreas. It is also important to know how the other pancreatic cell types (other hormone producing cells and cells making digestive enzymes) are regulated, because proper β-cells should not produce these other genes. In this work we produce the first comprehensive catalog of all genes active or inactive in five major human pancreatic cell types, including β-cells.

Project Investigators:

Philip Streeter

Gordon Keller

Lay Abstract:

Stem/progenitor cells offer the promise that transplantable beta cells may be generated in the laboratory. For this promise to be realized, these cells will need to be isolated and cultured, they will need to be differentiated to become beta cells, and the differentiated cells may need to be isolated from complex cell mixtures following cell culture. In this work, we propose to develop reagents directed against cell surface molecules expressed by beta cell precursors. These reagents will be used to accelerate progress in the development of methods for generation of transplantable beta cells.

Project Investigators:

Lori Sussel

Kristin Artinger

Didier Stainier

Lay Abstract:

In this collaborative study we propose to characterize a newly identified pancreas transmembrane protein, TM4SF4. We originally identified TM4SF4 in a screen for genes that are regulated by the essential pancreatic regulatory protein Nkx2.2. In previous studies, we demonstrated that Nkx2.2 is critical for the production of all insulin-producing β-cells and most glucagon-producing α-cells. In the absence of Nkx2.2, TM4SF4 is up regulated and α and β-cells fail to form. We have now determined that the TM4SF4 protein is highly conserved throughout evolution and is expressed in the liver, pancreas and intestine. Our preliminary data from both the mouse and zebrafish experimental systems suggest that TM4SF4 functions to regulate the number of insulin and glucagon cells during embryonic development. The purpose of this collaborative study is to bring together an interdisciplinary team of scientists to characterize the function of TM4SF4 in the developing and regenerating pancreas using the complimentary zebrafish and mouse model systems. Based upon what is known about TM4SF4 function in the liver and our initial studies in the pancreas, we hypothesize that TM4SF4 must be down regulated to allow for the optimal expansion of the α and β-cell populations in the islet. Furthermore, inhibition of TM4SF4 may enhance the production of α and β-cells during islet regeneration. This pilot project will not only provide information on the molecular function of TM4SF4 in the islet, but will indicate whether we can use the inhibition of TM4SF4 as a diabetes therapy to promote the formation of new islet cells.