Although stem cell-based therapies offer tremendous hope for treatment of a variety of human diseases, the full potential of this approach will not be reached unless we can achieve stem cell graft acceptance. The main barrier to reaching this goal is that we do not fully understand how the immune system of graft recipients causes the transplanted cells to be rejected. If we could understand this process, it would be possible to develop new approaches to prevent, or circumvent, graft rejection. Our previous work in organ transplantation has identified certain cells and molecules in the immune response that are critically involved in graft rejection. We have also identified methods that promote long-term organ graft survival in donor-recipient transplant combinations that would ordinarily result in graft rejection. Here we plan to determine what the specific cells and molecules are that cause an immune response and rejection of transplanted stem cell grafts. We will analyze this process using three different types of stem cell grafts to understand how the graft itself, and where it is placed, contributes to the outcome of transplantation. On the basis of these findings we will develop new methodologies to help the recipient immune system accept the transplanted stem cell grafts as if they were not foreign, but instead were “self”. Finally, the efficacy of our strategies will be tested in a model of liver disease to determine if we can achieve long-term survival of foreign stem cell transplants that ultimately restore liver function. These studies have the potential to improve the outcome of stem cell transplantation in general, as well as to expand treatment options for people with liver disease or liver failure.
Statement of Benefit to California:
Liver diseases are a significant cause of morbidity and mortality in the United States and are disproportionately evident in California. A recent study by the Institute of Medicine estimates that one in fifty Americans has viral hepatitis, a major cause of liver cancer and the leading cause of liver transplants nationwide. Twenty percent of all Americans on waiting lists for liver transplantation reside in California yet there is a dramatic shortage of transplantable organs to treat these patients. Stem cell therapies have the potential to overcome the shortage of available donor livers, and ultimately to provide treatment and disease cures that could save the lives of California children and adults with liver disease. The liver is one of the least immunogenic organs however, paradoxically, isolated allogeneic hepatocytes are readily rejected by the immune system. Our studies will define the immune response to stem cell-derived allografts that could promote hepatic regeneration for Californians with liver disease. We will use this information to develop strategies to achieve long term survival of stem cell transplants. Over the next twenty years, annual medical costs for people with liver disease are projected to increase dramatically, thus it is essential that effective therapies be developed soon. Our proposed studies will help to preserve California’s leadership role in research in stem cell biology, liver disease, and transplantation and stimulate growth of the California biotechnology and pharmaceutical industries. Successful completion of our work will lead to therapies to improve the health of Californians with liver disease and will have broad applicability in realizing the goal of applying stem cell-based therapies to regenerative medicine.
This proposal seeks to define the allogeneic immune response elicited towards three stem cell types, embryonic stem cells (ESCs), ESC-derived hepatocyte-like cells (ESC-HC) and bone-marrow stem cells (BM-MSC). To this end, the expression of immune-related molecules prior to transplant, as well as graft survival and the host anti-graft immune response will be analyzed. The involvement of innate immune mediators family will be evaluated by genetic approaches. In addition, the participation of specific T cell subsets will be confirmed in mouse models, in which particular cells types, including regulatory T cells will be compared for their efficacy in establishing long-term allogeneic tolerance. Last, the functional relevance of tolerance-inducing strategies will be tested in a mouse model of tyrosinemia type I. Although not particularly innovative, reviewers felt that the research proposal addresses important issues related to innate or adaptive immune components that may modulate long-term survival of stem cell allogeneic grafts in vivo. Particular emphasis is given to NK cells, dendritic cells and T cells, due to their well-documented role in initiating and propagating allo-reactive immune responses. The approach was considered for the most part adequate, but not groundbreaking, as much of the work proposed for Aim 1 has been characterized already and Aim 2 proposes to test established methods of tolerance induction in the mouse model and offers no novel approach. Reviewers noted that undifferentiated ESCs would not be used clinically because of teratoma formation. Also, MSCs are already being transplanted allogeneically in large clinical trials and because of immuno-modulatory activities appear not to be rejected. Therefore, tolerance to ESC-derived specialized allogeneic cells is where the impact would reside and what should be the focus of the proposal. The impact of these studies may rest with the potential of ESC-derived hepatocytes to enhance liver function. Overall, reviewers felt that the rationale is justified and convincing. Although the project would likely not be completed in the 3-year timeframe, the proposed plans are explained clearly and the milestones that are provided make sense. However, reviewers had several concerns with the experimental plan. First, most analyses measuring graft survival rely on poorly quantitative histological techniques depicting GFP-expressing stem cells. Real-time PCR-based analyses for GFP-specific amplicons performed on total DNA and RNA extracted from livers and kidneys would have provided additional and more quantitative readouts on the absolute and relative number of GFP-carrying stem cells. Second, the possible issues that may prevent efficient and quantitative lentivirus-driven GFP expression in the 3 stem cell populations is not discussed. The preliminary data pertaining to neuronal cells do not predict which percentage of the proposed stem cells will be GFP positive at the time of injection. Third, the possibility that GFP itself may elicit an immune response, as one would expect based on work by others, was not addressed and therefore important that GFP-specific immune responses be measured. Fourth, in order to avoid immunity to a given marker, reviewers suggested that stem cell tracking might be better accomplished molecularly using genetic markers derived from the respective allogeneic strain or by introduction of specific congenic markers into the appropriate mouse background prior to stem cell injection. Fifth, the PI proposes to use BALB/c mice as recipients and C57BL6 mice as graft donors. These studies should be repeated using C57BL6 mice as recipients, to provide more stringent conditions. Sixth, quantitative and qualitative analyses of intrahepatic immune responses must be primarily performed by flow cytometry on leukocytes isolated from known amounts of liver tissue, as it is regularly done by others. The proposed histological approach is not as informative and does not allow for the many FACS-based functional assays to be performed on the cells of interest. The PI and the Co-Investigators have adequate experience in immunology, stem cell research and transplantation tolerance to conduct the proposed project. The PI has the necessary qualifications to lead and conduct the proposed research. Overall, reviewers appreciated the well-written proposal, which aims to better understand the immune response to stem cell grafts. The project brings together appropriate expertise in stem cells and transplantation immunology but a focus on the cell populations that are likely to be used for cell therapy would have enhanced the potential impact of these studies. The project did not offer much novelty in the study or proposed induction of tolerance to stem cell grafts. The experimental plan presented mostly feasible studies, but did not consider better approaches to circumvent possible issues and obtain quantifiable data that would lend to meaningful results.
- Bruce Blazar