Human embryonic stem cells (hESCs) possess the ability to become specialized cells such as brain, cardiac, and pancreatic cells. However, despite the excitement surrounding ESC research, important issues surrounding immunogenicity have not been fully addressed and strategies to prevent the immune system from rejecting transplanted hESCs remain largely untested. A viable alternative to hESC therapies is the use of induced pluripotent stem (iPS) cells, which are non-pluripotent cells (i.e. skin, neural, adipose) “reprogrammed” to a hESC-like state. In theory, iPS cells would not face the same barriers such as immune response and rejection, because iPS cells can be derived from and transplanted back into the same person. However, no study has investigated whether iPS cells will indeed evade immune recognition if transplanted in such a manner. Therefore, it will be essential to investigate the immune properties of iPS cells, and our proposal aims to address three issues that are critical to understanding the immunogenicity of iPS cells and the development of strategies to promote long-term survival of transplanted cells.
Our first goal is to characterize the immunogenicity of iPS cells. We will measure the expression level of numerous genes and proteins which are recognized by the host immune system and can stimulate an immune response. To “reprogram” a cell into an iPS cell requires that either genes or proteins be inserted into the cell. This can be accomplished by viral, non-viral, or protein-based techniques. The immunogenicity of iPS cells may vary based on the technique employed. We will generate iPS cells by all three techniques and compare the immunogenic profile of cells generated from each technique in an attempt to identify the least immunogenic approach.
Our second goal is to study iPS cells in an environment which most closely resembles that found in a human. We will use a live animal model termed a “humanized” mouse model to study the immune response of human immune cells against human iPS cells. A “humanized” mouse is generated by transplanting human immune system progenitor cells into an immunodeficient mouse. Through this model, we will examine the same gene markers profiled in the first goal, while concurrently monitoring the immune response to iPS cells transplanted into a foreign immune environment.
Our third goal is to develop methods by which transplanted iPS cells may survive in the long term. To maximize the potential of success we will target both the transplanted cells and the host immune system. To make the transplanted iPS cells less offensive to the immune system, we will force the expression of molecules that inhibit and decrease the expression of molecules which activate the immune system. To blunt the response of the host immune system to the transplanted iPS cells, we will deliver an immunosuppressive regimen which prevents T-cell activation against foreign cells.
Statement of Benefit to California:
At present, the most urgent problem in transplantation is the chronic lack of suitable donor organs and tissues. As the population ages and life span increases, demands for organs and tissue therapies will only increase. One alternative to organ transplantation is cell therapy, which promises to replace, repair, or enhance the biological function of damaged tissue or diseased organs. Thus, a prerequisite for cell transplantation is to find a renewable source of cells that could be used in humans to regenerate damaged tissues and organs. In recent years, human embryonic stem (hESCs) have been heralded as the cell population best suited for this purpose. However, standard methods for hESC derivation requiring embryo destruction come under ethical and political controversies. In addition, the potential immunological rejection of allogeneic transplanted hESCs represents another major barrier precluding their therapeutic application. These concerns have stimulated an intense search for alternative sources of pluripotent stem cells. One alternative that can theoretically circumvent these concerns is induced pluripotent stem (iPS) cells. iPS cells are assumed to be non-immunogenic if they are therapeutically transplanted into the same patient from whom they were derived. However, they have never been studied in any immunocompetent animal model, and it remains unknown whether they will be immunologically rejected. Therefore, it is critical for scientists to further understand the immunogenic properties of iPS cells. Specifically, we must find out which molecules iPS cells possess that could stimulate an immune response so as to develop techniques to remove these molecules or avoid the immune response altogether.
We believe that answering these questions will lead to significant advances in iPS cell research and novel therapies. We have assembled a multidisciplinary team of experienced investigators to attack the challenges of this project. At the same time, we will train and mentor a new generation of bright students and junior scientists in the areas of technology development and iPS cell biology. This ensures that an essential knowledge base will be preserved and passed on into the foreseeable future.
In this application, the principal investigator (PI) proposes to investigate the immunological properties of induced pluripotent (iPS) cells and test their engraftment potential. The theoretical advantage of iPS cells is the potential for autologous transplantation and avoidance of immune rejection. However, the applicant suggests that the personalized medicine approach might not be economically feasible or a practical approach for treatment of acute diseases and injuries and suggests that HLA-typed iPS cell banks might be a necessity for clinical application. In this event, it is critical to characterize immunogenicity of iPS cells and develop strategies to promote transplant tolerance. The applicant proposes three aims. In Aim 1, the immunophenotype of iPS cells will be characterized by profiling iPS cell expression of immunogenic genes in cells that have been reprogrammed using several different techniques and at various stages of reprogramming and differentiation. Aim 2 explores the immune response after iPS cell transplantation in an allogeneic humanized mouse model. Aim 3 attempts to promote engraftment of allogeneic iPS cells through modulation of the host immune system to induce tolerance and the donor cell population to decrease immunogenicity of the graft.
Reviewers noted several innovative aspects of this proposal. The proposed approaches to achieve tolerance have been studied extensively in experimental organ transplantation models but have not been applied previously to iPS cell-derived tissues. Additionally, the comparison of immunogenicity of iPS cells derived by different methods is worthwhile and innovative. The applicants utilized innovative approaches to generate starting iPS populations, proposed novel single cell comparisons of cells reprogrammed using different techniques, and included a creative application of microfluidics. However, while reviewers agreed that identification of a candidate gene or pathway that regulates iPS cell rejection would have a substantial impact on inducing tolerance to iPS cells, reviewers had were not confident of the proposal’s potential impact. The reviewers felt the proposal’s impact hinged on the critical assumption that the generation of autologous iPS cells will not be a practical clinical solution; this issue has not yet been fully evaluated, and so, the impact of allogeneic iPS cell immunity is unclear. Further reducing the potential impact was the applicants’ plan to include analyses of cell populations that are not clinically relevant: teratoma-forming partially reprogrammed iPS cells. Reviewers also suggested that investigating whether autologous iPS cells still possess immunogenicity would have been helpful and would have strengthened the study’s impact.
The reviewers raised several concerns regarding the proposal’s rationale. The rationale for the profiling of undifferentiated and partially differentiated iPS cells was unclear and, as mentioned, lessened the potential impact of the proposal. Reviewers felt that profiling iPS cells under different conditions of generation only makes sense if one assumes that all cell types might be useful clinically, differences exist in the rejection process for each variety of the cells, and one can choose the least immunogenic version for clinical use. Reviewers suggested that in order to advance the clinical application of human iPS cells, it would be more important to select and analyze a hierarchy of discrete progenitors and terminally differentiated cells that are likely to be transplanted into patients and compare them to undifferentiated cells or fibroblast cells.
Reviewers also expressed concerns regarding the proposed research plan. Although the aims seemed achievable, the reviewers criticized the descriptive nature of Aim 1, noting it lacked clear goals and clinical relevance. Although reviewers acknowledged that proposed experiments would provide relevant data, they criticized the absence of a formula for quantifying which iPS cells are less immunogenic. Such a formula would be critical in determining which iPS cells to use subsequently in Aims 2 and 3, and development of a quantitative assessment of immunogenicity appears essential for the project’s success. Furthermore, alternative plans for inducing tolerance in Aim 3 were inadequately addressed, and the proposal lacked detail on how decisions would be made concerning which pathways to pursue.
Reviewers had concerns about the project’s feasibility and, in particular, the use of the proposed humanized mouse model. For the successful completion of Aims 2 and 3, human T cells in this model must be able to mount an allogeneic response to the human iPS cells. While the applicant provided evidence of this in the supplementary data, alloreactivity in this model system has not yet been validated, and alternative plans are inadequately addressed. Further, it has not been demonstrated that this model is an accurate representation of the patient setting, and the proposed immune modulation strategies are unlikely to yield useful information unless this is shown to be the case.
The reviewers praised the quality of the PI and research team. The PI has a strong publication and patent record and impressively productive. He/she has assembled an excellent group of investigators with complementary expertise in stem cell biology, immunology, transplantation, and imaging, and the team has all the expertise, equipment, and facilities to accomplish the goals of the project. Reviewers noted that the proposal would have benefited from additional effort by the co-investigators whose commitment ranged from 1-3%.
In summary, although the PI, the research team, and the project’s innovation are excellent, the proposal’s potential impact is limited by unclear rationale, a questionable experimental plan, and lack of appropriate alternate plans.