Currently there are no clinical medical procedures, and limited surgical approaches, available to people who suffer from critical limb ischemia (CLI). This is a vascular disease that is due to poor blood flow in the legs and is often a complication of diabetes. CLI affects 1% of the population over 50 and necessitates approximately 100,000 amputations per year in the US. One promising use of embryonic stem cells is to use them to repair the blood vessels of patients who suffer from CLI. If our research is successful, we will develop a new therapeutic stem cell based intervention for those who suffer from CLI, and open the door for new clinical applications of embryonic stem cells. Our research is based on the discovery of a new type of drug that can create immune system tolerance to transplanted cells and tissues that are usually recognized as foreign by the immune system. When human stem cells are injected into recipients to regenerate damaged tissues, they are usually rejected. Stem cell transplants will not be widely used unless immune rejection of mismatched cells can be overcome. The basis of the research in this proposal is our discovery that a naturally occurring molecule called adenosine inhibits the activation of immune cells in animals. Moreover, we have succeeded in making synthetic adenosine analogs as new drugs that are superior to natural adenosine in potency and stability. We plan to test these new adenosine drugs to determine if they enhance tolerance to human adult and embryonic stem cells. This will be done in two ways: we will prepare cells of the human immune system and mix them with stem cells derived from a different individual. Normally this will cause immune rejection and killing of the stem cells by the cells of the immune system. We will determine if the new adenosine-like drugs inhibit the rejection process. In a second series of experiments we will inject human stem cells into mice with tissue damage caused by CLI and determine how long the human cells survive and how well they repair these injuries. We expect our experiments to show that treatment with adenosine-like drugs enhances tolerance of the immune system to stem cells which in turn enhances blood flow and tissue repair. If these experiments are successful we will plan a clinical trial within 4 years to determine if adding adenosine drugs in combination with human stem cells improves clinical outcome in patients with critical limb ischemia. We also expect that our strategy with adenosine analogs will generally inhibit immune rejection and open the door for extensive use of embryonic and adult stem cells not only for CLI but also for many other diseases in the field of regenerative medicine.
Statement of Benefit to California:
Critical Limb Ischemia (CLI) is an advanced form of peripheral vascular disease causing approximately 100,000 amputations per year in the US. Currently there are no medical interventions for CLI and surgical approaches, which are applicable to a minority of patients, usually generate only short term benefit. Trials using autologous cell-based therapies have demonstrated clinical improvement, however such approaches have several drawbacks, including limited wide-scale applicability and intrapatient variability of cell quality. While cell based angiogenesis protocols appear superior to transfection of pro-angiogenic genes, the mechanisms of cell based therapy for CLI are currently under dispute. Recent studies suggest the growth factors secreted by the implanted cell population play a major role in angiogenesis, and that differentiation into EC is not even required in some cases.
The application of allogeneic ES-derived angiogenesis stimulating cells in treatment of CLI would have several advantages: A) CLI is such a debilitating condition that the risk/benefit ratio may justify clinical translation of ES derived cells: B) Cellular implantation in CLI patients would occur in the leg, thus monitoring and excision of potential teratoma/ectopic tissue is feasible; and C) Therapeutic effect using a variety of adult stem cell/progenitor cells has been observed, even in allogeneic settings: the growth factor release of ES-derived cells is anticipated to exert a more potent therapeutic effect as compared to previous approaches.
In this proposal we develop a new application for drugs that have a role in adenosine receptor signaling and are already in clinical use, as we believe they have the potential to modulate the immune system and inhibit stem cell rejection. If successful in our proposal objectives this model will form a solid basis for the rapid development of a clinical trial at UCSD involving human stem cell therapy to treat critical limb ischemia. This could transform the medical options available to the estimated 1% of the US population over 50 years of age, a large portion of which live here in the State of California and suffer from this devastating medical condition which is responsible for 100,000 leg amputations performed each year.
Recent studies indicate that agonists to the adenosine A2A receptor (A2AR) can suppress innate and adaptive immunity. This is a proposal to determine whether A2AR agonists can produce sustained immune tolerance to human stem cell antigens and enhance human stem cell engraftment. Aim 1 would determine whether A2AR agonists could be used to inhibit human cytotoxic T cells from killing human embryonic stem (ES) cell-derived hemangioblasts (ES-H) and human endometrial regenerative cells (ERCs) in vitro. Aim 2 would examine the effects of using A2AR agonists to prolong the survival of human ERCs in a preclinical critical limb ischemia (CLI) xenotransplantion model. Aim 3 investigates the effects of A2AR agonists on human hemangioblast engraftment, and on their ability to promote revascularization, in the CLI model.
Reviewers agreed that exploring the effect of A2AR agonists in inducing tolerance in stem cell grafts is innovative and creative. They also appreciated the potential impact of this study on clinical translation of stem cell therapies, particularly for CLI. If the project were successful, it would identify an FDA-approved drug for further testing in human studies of stem cell allogeneic grafts.
Despite the project’s novelty and compelling rationale, the reviewers had serious reservations about the experimental design and project scope. First, they were critical of the proposed use of ERCs in these experiments. ERCs are similar to mesenchymal stem cells, which have been shown to display immunomodulatory activity that may affect allogeneic graft rejection on their own. Second, reviewers also questioned the choice of peptides for the in vitro tolerance induction assays, as the proposed markers are characteristic of pluripotent ES cells but are down-regulated or absent in differentiated ESC derivatives or in MSCs. The applicant provided no preliminary data that these markers were expressed in ES-H or ERCs, or that cytotoxic lymphocytes recognizing these peptides could be produced. Third, reviewers expressed serious reservations that the mouse xenograft model would provide interpretable results, since there is very little evidence that rejection of human cells in the mouse model depends on HLA expression. The proposal would have been much stronger had the applicant proposed to perform these transplants in a humanized mouse model. Finally, reviewers found the scope of the proposal to be too narrow and were concerned that the entire project relied on achieving positive results from the in vitro experiments proposed in Aim 1. Furthermore there is very little proposed exploration of the mechanism of A2AR modulation of the immune response and little discussion of alternative experiments or approaches.
The reviewers praised the PI’s expertise in immune tolerance, autoimmune disease, and in utero transplants. Although the PI’s publication record is reasonable, it is not extensive and did not indicate leadership within the stem cell field. The two collaborating scientists, who are experts with A2AR agonists and immune responses, were recognized as strengths of the proposal. The research environment is outstanding.
In summary, this proposal explores the role of A2AR agonists in inducing tolerance in stem cell grafts. Reviewers concluded that critical flaws in the experimental design outweighed this project’s innovative rationale.