Year 1

The goal of our project is to study and develop immunological approaches to overcome rejection of embryonic stem cells (ESCs) and other cells and tissues derived from these pluripotent cells. We utilize animal modeling to study the impact of the immune system on survival and rejection of ESCs since we are better able to study specific cell populations and strategies involved in the underlying biology. We have further developed a strategy to study the survival and trafficking of injected cells using light emission in a process called bioluminescent imaging (BLI). This imaging approach is accomplished by introducing a light emitting protein called luciferase, generated from the North American firefly into cells of interest. In particular we have generated ESCs which express luciferase and therefore emit light. Using BLI we can study the growth and survival of these transplanted ESCs in living animals. Using this approach rejection and engraftment can be readily and non-invasively assessed. In the past year we have developed all of the necessary ESCs from different genetic backgrounds that express luciferase and can be imaged. We have demonstrated that animals with intact immune systems reject these transplanted cells over several weeks whereas animals with crippled immune systems do not. A goal of the research proposal was to study naturally occurring T and natural killer – T (NK-T) cells with known capacity to regulate immune responses. The goal is to develop strategies whereby injection of these regulatory cells will impact the rejection of the transplanted ESCs. Our initial studies have demonstrated that the simple co-injection of the regulatory T cells with the ESCs was not sufficient to prolong survival of the ESC grafts. This is not unexpected since the regulatory T cells may also be rejected by the recipient immune system. Current experiments are to study the survival and trafficking of the regulatory T cells in animals with and without ESCs and to explore ways to enhance survival of these grafts through manipulation of the immune system of the recipient animals. One approach under current study is to utilize a preparative regimen developed at Stanford including total lymphoid irradiation (TLI) and anti-lymphocyte serum (ALS) which in both animals and patients is very well tolerated and reduces immune function such that engraftment of foreign cells is possible. We are exploring the impact of TLI/ALS with and without the regulatory T cells and NK-T cells on ESC survival and engraftment. The ultimate goal is to develop approaches that will be translatable to the clinic to enhance the engraftment and survival of the transplanted ESC tissues. We have made excellent progress in the first year of funding and developed all of the necessary ESC lines emitting light and performed all of the control experiments demonstrating the immunological rejection of the ESCs. In the next 2 years we are well poised to study and develop immunological approaches to enhance ESC survival.