Project Investigators:

Doug Melton

David Gifford

Lay Abstract:

Diabetic patients would benefit enormously from the availability of a limitless supply of insulin-producing beta cells for transplantation. Much current research is focused on producing beta cells from stem cells, with the goal of meeting the needs of patients for transplantable cells. To date, cell culture procedures have been developed that allow for the production of insulin-expressing, beta-like cells. However, these beta-like cells lack a key feature found in normal beta cells: they are unable to accurately secrete insulin in response to glucose. In this way, the cells resemble the beta cells found during embryonic development, which also do not respond to glucose in the same way as adult beta cells. Further, stem-cell derived cells that are transplanted into a mouse gain the ability to respond to glucose over a period of months, indicating that the cells have the capacity to gain glucose responsiveness over time. The goal of our research project will be to determine the genes and signals responsible for the maturation of a beta cell from a glucose non-responsive state to a glucose responsive state. We will accomplish this by studying in great detail the changes in gene expression and chromatin architecture that occur as normal embryonic beta cells gain the ability to respond to glucose, using the mouse as a model organism. We will perform similar studies on stem cell derived cells, to identify the differences between the immature cells that can be produced in culture versus glucose responsive cells that appear after several months of incubation in a mouse. Based on these studies, we will identify key regulators of the maturation process. We will then manipulate those regulators in immature stem cell derived beta cells in culture, with the goal of forcing them to adopt a mature phenotype suitable for transplantation.

Project Investigators:

David Serreze

Dale Greiner

Stephen Miller

Lay Abstract:

Type-1 diabetes (T1D) in both humans and the NOD mouse model is an autoimmune disease that results in the complete loss of insulin-producing ß-cells in the pancreas. Beta Cell Biology Consortium (BCBC) investigators are developing approaches to generate ß-cells as replacement therapy for individuals with T1D. However, a major remaining hurdle is to develop means to prevent the destruction of transplanted ß-cells by the same autoimmunity responsible for the original development of T1D. Furthermore, if not derived from the recipient, propagated ß-cells transplanted into T1D patients will also be prone to alloimmune transplantation rejection. Our proposal uses novel technologies to address the alloimmune and autoimmune barriers of ß-cell replacement in T1D patients. This proposal is based on exciting technology created by Dr. Miller in mouse models of multiple sclerosis (MS) and extended to autoimmune diabetes and islet transplantation by Drs. Miller, Luo, and Serreze. This technology encompasses treatment with white blood cells (leukocytes) to which ß-cell proteins targeted by the autoimmune response have been coupled by the cross-linking agent ethylene carbodiimide (ECDI). Dr. Miller has shown this cell-based therapy is effective in a mouse of MS, and Drs. Miller, Luo, and Serreze have recently shown it also blocks T1D development in the NOD mouse model. In addition, Dr. Miller currently has a clinical trial in MS underway in Germany based on this technology. We propose to extend the exciting observations in mouse models of autoimmunity by addressing two important barriers: 1) by replacing ECDI-antigen coupled leukocytes with ECDI-antigen coupled microparticles (thus eliminating the need for cells), and 2) by extending the observations made in the mouse to the human using unique strains of mice engrafted with human immune (and autoimmune) systems. These experiments will be based on novel strains of mice that have been genetically engineered to support engrafted human immune systems. Our experiments will determine whether microparticles bearing ECDI coupled antigens might ultimately be used to prevent or reverse T1D, permitting cure of disease by transplantation of ß-cells generated by investigators in the BCBC.

Project Investigators:

Jeffrey Karp

Charles Lin

Maike Sander

Lay Abstract:

Type 1 diabetes mellitus (T1DM) is currently an incurable disease that is burdening over 3 million patients, many of them children, and responsible for over $100 billion in annual healthcare costs. There is a significant need for a cure that can regenerate insulin independence and restore the patient’s quality of life. -islet cell transplantation shows promise, however the scarcity of cadaver tissue prevent it from becoming the standard treatment for T1DM. One of the most promising approaches being develop for reversing diabetes involves transplantation of pancreatic progenitors that differentiate into functional β-cells in vivo. However, very little is known about this process and only a limited number of tissue transplantation sites for these cells have been tested. Expanding our understanding of beta cell maturation in vivo and identifying new sites that are more permissive to beta cell progenitor transplantation should be useful for developing more efficacious treatments for T1DM.

In a collaboration between Dr. Jeff Karp (BioEngineer), Dr. Maike Sander (Beta Cell Biologist), and Dr. Charles Lin (In Vivo Microscopist), new technologies will be developed to enable the delivery of immature beta cells to tissues that are highly amenable to non-invasive in-vivo imaging. This should greatly enhance the engraftment efficiency and survival of -cell precursors and enhance the ability to monitor the maturation of pancreatic progenitors to become glucose responsive. This effort should enable multiple collaborative opportunities with investigators in the beta cell biology consortium who are trying to shorten and improve the in-vivo maturation step of beta cell progenitors to fully functional beta cells in vivo.

Project Investigators:

James Wells

Maike Sander

Lay Abstract:

Currently, there are no methods to generate functional pancreatic beta cells from human pluripotent stem cells in vitro (Embryonic stem/ES or induced pluripotent stem/iPS are collectively referred to as pluripotent stem cells/PSCs). We hypothesize that this is because current methods use non-physiologic conditions such as growth in two-dimensional cultures. In the embryo, the pancreas develops from a structure called the gut tube, which is a tube of cells from which the pancreas, liver and lungs emerge. Development of the embryonic pancreas involves growth and branching of the tube, and the beta cells emerge from the branches and appear clusters of beta cells that remain in association with the branching epithelium until birth. Given that pancreas and beta cell development normally occurs within a 3-dimensional environment, it is it is possible that the non-physiologic nature of current methods to generate beta cells from PSCs makes them intrinsically limited.

To overcome these limitations we propose to use an entirely novel and more physiologic approach to generate pancreatic tissue from human PSCs, based on our recent success in generating 3-dimensional intestinal tissue in vitro (Spence et al., 2011. Nature). In this work, we developed a method that mimics the 3-dimensional embryonic conditions that favor intestinal growth. Under these conditions, PSCs underwent stereotypic 3-dimensional morphogenesis, similar to development of the fetal intestine, resulting in the formation of tissue in vitro that appeared and functioned like human intestinal tissue. This study suggested that mimicking “embryonic-like”, 3-D development favored the formation of functional tissue.

We will use an analogous approach to generate pancreatic tissue and beta cells from human PSCs. Our first aim is to generate 3-dimensional cultures of pancreatic tissue in a manner similar to normal embryonic development of the pancreas. Our second aim is to investigate the impact of 3-dimensional growth and morphogenesis of pancreatic organoids on the formation of endocrine progenitors and beta cell clusters. We hypothesize that 3-dimensional pancreatic epithelium will undergo morphogenesis reminiscent of pancreatic development, which will include formation of endocrine progenitors and clustering into neoislets containing functional beta cells.

Project Investigator:

Anna Moore

Lay Abstract:

Type 1 diabetes (T1D) results from destruction of insulin-producing pancreatic beta-cells by diabetogenic T cells. Various immunoregulatory therapies have been and are currently being evaluated for their utility in the prevention and treatment of T1D. However, the toxicity of most drugs and the risk associated with immune suppression tremendously limit the use of these agents. One of the reasons for the high toxicity/poor performance of these drugs is the necessity to inject high doses in order to achieve an adequate therapeutic effect.

We propose to overcome these difficulties by delivering therapeutics directly to the site of inflammation, i.e. to pancreatic islets. In order to do this we will exploit the innate leakiness and hyperpermeability of the inflamed islet vasculature that increases with the disease development. We will design small biodegradable nanosize carries with encapsulated drugs that can penetrate through the fenestrated islet blood vessels and release their content in a controlled manner directly into the pancreatic islet microenvironment. Furthermore, in addition to their drug payload these nanosized carriers will carry an imaging moiety, which will allow for monitoring and tracking of their accumulation in live animals using non-invasive imaging.

We expect that this approach will significantly increase the efficiency of existing therapeutics, decrease the toxicity and lower the injected dose. Finally, and most importantly, this approach will pave the way for delivery of other molecules (small organic and large natural compounds (proteins, DNA, RNA)) for beta-cell regeneration, regulation and immunoprotection.

Project Investigators:

Maike Sander

Klaus Kaestner

Bing Ren

Lay Abstract:

One major hope for diabetes therapy is to cure the disease by generating transplantable replacement β-cells from patient-derived pluripotent cells. While some progress has been made, it is still not possible to generate functional β-cells in the culture dish. The main bottleneck for improving in vitro protocols for the generation of β-cells is the absence of cell lines, in which the correct induction of β-cells or their precursors can be monitored easily. Enhancers are small modules within the genome, whose activity is highly cell type-specific. Therefore, identification of enhancers that are specifically active in β-cells and their precursors will allow us to monitor induction of the correct cell types in the culture dish. We propose to identify pancreas- and β-cell-specific enhancers and to build cell lines from patient-derived pluripotent cells, in which pancreas and β-cell induction can be rapidly monitored. This will allow for high throughput screens to find molecules that can guide patient-derived pluripotent cells to develop into β-cells. The reagents generated under this proposal will also enable the direct monitoring and imaging of transplanted cells in animals. Such experiments will inform us of how cells mature into fully functional β-cells after transplantation.

Project Investigators:

Klaus Kaestner

Benjamin Glaser

Lay Abstract:

The cells that produce insulin for the body, called beta-cells, are essential to the control of blood sugar, and are either completely missing or functionally insufficient in type 1 and type 2 diabetes, respectively. The proposed project attempts to coaxed mature human beta-cells into dividing multiple times to increase the functional beta-cell mass and thus insulin production. If successful, the project could be moved to a practical application relatively quickly, and provide a new avenue towards diabetes treatment.

“Experiments of nature”, or naturally occurring genetic mutations that result in specific phenotypes, provide unique insight into human physiology. These genetic events are inherently random and therefore, by correlating the genetic defect with observed physiology, totally unexpected mechanisms can be discovered. When such observations are made in human diseases, the likelihood that discoveries can be directly translated into the clinic is greatly enhanced. The rationale behind this proposal is based on the observation in humans with a specific, well-defined albeit rare genetic disease that results in non-neoplastic β-cell replication. One of the candidate proteins, p57, is not expressed in rodent β-cells, meaning that this observation could never have been made in rodents and that all studies must be done in human tissue. By transplanting islets into immune deficient mice, human β-cell replication can be assessed for extended periods of time, allowing for in depth studies of proliferation, survival and function of the resultant β-cells.

Previous studies manipulating the expression of cell-cycle components in rodent islets may have had limited clinical relevance, given the apparent major differences between replication control in rodent and human islets. The uniqueness and originality of this study stem from its very nature as a “bedside to bench and back to the bedside” project based on proven human physiology.

Project Investigators:

Matthias Hebrok

Eugene Kolker

Lay Abstract:

Differentiation of insulin-producing beta-cells from human stem cell populations holds great promise with regard to therapeutic intervention of patients suffering from insulin dependent diabetes. While great advances have been made in recent years, the current cell culture protocols do not allow to fully replicate the situation during pancreas organ development when the developing beta-cells receive signals from surrounding tissues, including the mesenchyme, that guides their development and expansion. Here, we propose to test the hypothesis that supplying these additional signals to late stages of human stem cell differentiation protocols will support the formation of endocrine progenitor cells and the differentiation and expansion of insulin-producing beta-cells. This will be accomplished by taking advantage of a novel transgenic mouse that provides the opportunity to isolate and purify pancreatic mesenchyme cells at important stages of endocrine progenitor and beta-cell development. Purified mesenchymal cells will be analyzed by proteomic analysis to identify signals produced by these cells that could guide beta-cell formation. The approach will be an iterative process where proteomics analysis of the development/postnatal stages producing the most cells will performed first (e17.5, postnatal 2 weeks), followed by proteomics and bioinformatics analysis to characterize candidate proteins. Subsequent analysis will include earlier embryonic time points to develop an overview of how the expression of secreted proteins changes over time during pancreas development and beta-cell maturation. Candidate proteins will then be tested in established hESC to pancreas differentiation protocols for their activity to promote formation of endocrine progenitors and insulin-producing beta-cells.