Project Investigator:

Yi Zhang

Lay Abstract:

Type 1 diabetes (T1D) is an autoimmune disease in which the insulin secreting beta cells in the pancreas are destroyed by the patient's own immune system. Because insulin is required for glucose homeostasis, patients with T1D must rely on exogenous insulin injection to survive. However, insulin injection cannot prevent chronic and devastating complications, such as kidney failure, blindness, and heart attack. A cure for T1D will require restoring functional beta cell mass by either regeneration of beta cells in the pancreas or by exogenous replacement of insulin secreting cells through transplantation. Increasing evidence suggests that islet cell transplantation can allow patients with T1D to achieve insulin independence. However, the extreme shortage of donor tissue and the necessity for chronic immunosupression makes it impossible for this treatment to be used for the general diabetic population.

Two recent advances have raised the possibility that generation of patient-specific islet-like cells suitable for transplantation may be possible. The first advance is the differentiation of human ES cells to islet-like cells capable of glucose regulated insulin secretion. The second advance is the generation of hESC-like induced pluripotent stem (iPS) cells from human somatic cells. By combining these two techniques, we have recently demonstrated that insulin-secreting cells can be generated from human skin fibroblasts. Despite this encouraging result, several obstacles have to be overcome before the value of this technique can be realized clinically. In this proposal, we will focus on two issues:
1) generation of clinically safe patient-specific iPS cells;
2) development of new protocols for efficient differentiation of iPS cells into functional islet-like cells.

Completion of the proposed studies will greatly facilitate reaching our final goal of developing a beta cell replacement therapy for diabetes.

Project Investigators:

Dale Greiner

Leonard Shultz

Lay Abstract:

The challenge of curing of type I diabetes (T1D) can be divided into two main issues: the absence of pancreatic beta cells and the autoimmune attack that destroys them. Our overall goal is simple: to create an experimental system to ‘watch' human diabetes develop and then pinpoint the molecules and cells responsible. The project outlined here provides one of the two critical tools required for successful accomplishment of this goal. The linked ARRA Signature Project submitted by Dr. Doug Melton will provide the first critical tool. He will focus on generating human induced pluripotent stem (iPS) cells from normal and diabetic (DiPS) donors and differentiating them into the critical components of the system: beta cells, hematopoietic stem cells, and thymic epithelium. Our linked ARRA Signature Project will generate the second critical tool: novel genetic stocks of "humanized" mice to permit the reconstruction of human diabetes in murine hosts using human DiPS-derived tissues generated by the Melton laboratory. These hosts will function as in vivo "vessels", into which all the human components can be engrafted. The humanized mouse models will be based on our novel stock of humanized mice that will be crossed with our spontaneous and inducible diabetic stocks of mice and with our novel genetic stocks of HLA transgenic mice representing ~80% of the major HLA class I and class II genes that are important genetic determinants of type 1 diabetes. We propose 4 goals. 1) To create and characterize novel immunodeficient stocks of hyperglycemic mice and 2) use them to generate appropriate HLA expressing diabetic stocks. 3) To develop embryonic stem cells that will permit rapid generation of knockin and knockout mice. 4) To initiate establishment of the "proof of concept" model system using human hematopoietic stem cells, thymus, and pancreas. First, we will generate, characterize, and validate a novel immunodeficient mouse stock in which diabetes is readily inducible by injection of a low dose of diphtheria toxin. Second, HLA-transgenic stocks of unique immunodeficient mice will be developed by genetic breeding using HLA-Tg stocks available in our labs. Third, we will generate embryonic stem cells and use "knockin" and "knockout" technology to delete and replace genes that will improve human immune system function in the immunodeficient mice. Fourth, to provide the "proof of principal" for the model system, we will establish the human hematopoietic stem cell/thymus/pancreas system in immunodeficient mice as the hyperglycemic stocks are being developed and validated. This will provide a system in which each individual fetal tissue component can be replaced by HLA-matched iPS-derived cells and tissues, permitting controlled individual validation of the function of each of the iPS-derived cell populations. We envision that the HLA haplotypes of patient specific DiPS lines will determine the number of model systems we develop. For this purpose a large panel of individual DiPS lines will be generated by the Melton ARRA Signature Project. We will develop optimal hosts for the differentiated derivatives of these pluripotent stem cells in our ARRA Signature Project. These two linked SRRA Signature Program proposals will provide a large panel of DIPS lines and the in vivo tools required to assess their diabetogenic function.

Project Investigators:

Doug Melton

George Daley

Lay Abstract:

We propose to reconstruct human type1 diabetes (T1D) in a mouse. This project, if successful, will enable us to watch diabetes develop, using human cells, and understand the initiation and development of the disease. In particular, it will allow us to identify the cells and genes responsible for the immune attack on pancreatic beta cells.

This can be thought of as a "reconstruction project": we want to find an experimental system wherein we can watch human T1D develop. To that end, it requires making all the cell types involved in T1D, including immune cells, beta cells, and cells of the thymus, so that the entire human disease T1D can develop in a mouse. In this case, the mouse will be humanized (cf project by Greiner/Schultz) to serve as "test tube" wherein the human cellular components can interact. In addition to the principal aim of finding out which cells and genes have the defects that cause T1D, we will eventually use iPS cells from a number of T1 diabetics. This will allow us to find out how many different ways a patient can or does develop T1 diabetes. In all, if this project is successful, it will allow us to design new treatments to induce tolerance and may lead to new ideas for prevention of T1D.

The present proposal is aimed at creating the starting material, the starting cells, needed for the overall goal of reconstructing T1D in a mouse. In order to make all of the cellular components, one needs to begin with a stem cell. We will use induced pluripotent stem cells (iPS cells) from patients with T1D so that the cells contains all the genes that are involved in the disease. To create diabetic iPS cells, (DiPS), we begin with skin biopsies from patients and convert those skin cells into pluripotent stem cells.

The DiPS cells have to be differentiated into all the cellular components involved in T1D. This includes beta cells, thymic epithelia and cells of the immune system. Studies on how to convert DiPS into beta cells and thymic epithelia are covered by other proposals, as is the creation of the humanized mouse. This proposal concerns a key component: the immune system. To this end, we propose to convert DiPS into hematopoietic stem cells.