The generation of adult induced pluripotent stem cells (iPS) has stirred much excitement in the stem cell biology field, as it is would be potentially be possible for every person to have a personalized stem cell that could be used to cure degenerative diseases and immune deficiencies. However, rapid progress to actual human clinical trials of iPS-based therapies is hindered by the lack of preclinical data on the efficiency of the production of immune system cells from iPS, and also the ability of iPS cells to stimulate the recipient’s immune system after transplantation. The immune system is trained during development to recognize what is self versus foreign, and therefore iPS cells derived from a patient, and then transplanted back into the same patient, are not expected to induce an immune response. However, the mechanisms that occur in cells that are “reprogrammed”, such as iPS cells, could result in the expression of proteins on the iPS cell to which the patient immune system could react. Furthermore, there is some evidence that iPS cells will not be able to be produced from all patients, and iPS cells from a mismatched donor may need to be transplanted instead. In this case, the immune system of the recipient will have to be educated to accept the mismatched iPS as “self”. Building on the strategies that result in successful organ transplantation currently used in the clinic, we will study the immune response to blood cells derived from a “safe” virus-free human iPS cell line. First, we will compare existing culture protocols to derive blood stem cells from iPS, by measuring their function and identifying their “immunogenicity profile” to predict if iPS-derived blood cells can induce an immune response. To test the ability of these human iPS-derived blood cells to survive after transplantation, we will transfer them into mice that have been engineered to contain a human immune system. This model will allow us to predict the fate of the iPS-blood cells in an actual patient. It is our goal that the proposed work will result in iPS culture methods that are highly efficient in the generation of blood stem cells. In addition, we aim to obtain important preclinical data on the immunogenicity of iPS-generated cells and their ability to function and contend with the recipient immune system after transplantation. This information could facilitate and propel forward the use of iPS cells in clinical therapies.
Statement of Benefit to California:
Results from the proposed research project will benefit the State of California and its citizens at several levels. Direct Impacts: This research project aims to target a potential barrier to stem cell therapies via induced pluripotent stem cell (iPS): the avoidance of immune rejection in the patient. Even though iPS are not thought to stimulate an immune response if transplanted back into the patient from which the iPS was derived, this has not been tested formally. Furthermore, iPS may not be able to generated from all patients, and thus iPS from mismatched donors might be used. If immunological tolerance to iPS-derived grafts is not achieved, the therapeutic iPS is destroyed and disease persists. The immune system is formed from hematopoietic (blood) stem cells, and our research goals are to establish protocols which promote engraftment of iPS-derived blood stem cells in a recipient to induce immunological tolerance. If successful, this will provide preclinical data to demonstrate that immunological tolerance to iPS-based therapies can be achieved. As immunological tolerance is important for all potential iPS-therapies, this work can have broad applications to a wide diversity of stem-cell based therapies and diseases. The work will also have indirect impacts outside of the research, such as notoriety to CIRM as the funding agency for this groundbreaking research, and be the springboard for improvements in health care, increase in tax revenues, and improvements in education for California residents. Health Care: If successful, this research could facilitate clinical trials for iPS-based therapies. As this research is funded by CIRM, it is highly likely that Californians would be the primary recipients of therapies designed using our research results. Biotechnology: My research already relies on a number of products and tools manufactured and sold in the state of California. If successful, research will require a scaled-up version of protocols designed in our studies. This could attract new biotechnology companies in the state, boosting the tax revenue in the state. This in turn, will provide new jobs for California state residents. Education: Establishment of successful stem cell based-therapeutics in California will encourage institutions of higher education to promote science education to fill the jobs created by stem cell research. This will retain California students in the state that are interested in biomedical research and medical careers. Furthermore, it could attract out-of-state students seeking degrees that will allow them access to careers in stem cell research. It is envisioned that this will trickle down to the K-12 levels and provide funding to promote science education at all levels.
The goal of this proposal is to determine the immunogenicity of virus-free induced pluripotent stem cells (iPSC) in immunocompetent hosts and to test whether these cells can promote mixed hematopoietic chimerism via stable engraftment. The investigators first plan to establish the best method for human hematopoietic progenitor (HP) differentiation from iPSC by optimizing published protocols. The cells will then be immunologically profiled in vitro and tested for their ability to engraft and mature into all the blood lineages by transplantation into immunodeficient, and later immunocompetent, allogeneic, hosts. Allogeneic hosts will be followed for one year to determine if the transplanted iPSC-HP survive and establish lasting mixed hematopoietic chimerism, and induce specific immunological tolerance. Aim 3 will profile the immune response of donor and host immune cells to each other with the goal of identifying the specific immune barriers to immunological tolerance to iPSC-HP. The reviewers agreed that the proposal targets a relevant question, but that it lacks innovation and has limited potential impact. They appreciated the value of optimizing HP generation from iPSC, but felt that the proposed studies to achieve this were not innovative but rather an exercise in improving existing technology. Determining the immunogenicity of clinically relevant cells such as iPSC-HP could direct future development of iPSC based cell therapies; reviewers appreciated the potential impact of this goal. However, the panel questioned the rationale of using an allogeneic iPSC on multiple levels. One of the potential major advantages of iPSC lies in their autologous origin, which may render them less immunogenic than fully allogeneic stem cells. Allogeneic use of iPSC in immunocompetent hosts abrogates this advantage. If successful, the proposal would find that allogeneic iPSC-HP cannot evade the immune system and would provide preliminary insights regarding cell types mediating rejection. For these reasons, the proposal would benefit more from a focus on identifying less toxic, novel strategies to induce allogeneic tolerance than on predicting what is very likely to occur. Disappointment was expressed that the experimental plan provides no detailed strategy to address this important issue. The PI cites the difficulty of deriving and utilizing autologous iPSC in certain diseases as a rationale to study allogeneic iPSC-HP. In these cases, the group felt allogeneic iPSC offer no therapeutic advantage over more readily available hESC. Major technical obstacles and flaws in the experimental design decreased enthusiasm for the project. The reviewers noted that the success of the proposal depends upon the generation of adequate numbers of iPSC-HP, and that the PI’s experience, primarily with murine ESCs, may not translate to success in this effort. Reviewers felt that aspects of the proposed work were oversimplified and did not take into account the dynamic nature of the experimental system. For example, the applicant utilizes in vitro assays to predict potential immunogenicity of iPSC-HP, but potential changes in marker expression following transplantation or differentiation may limit the predictive value of these assays. Similarly, the vital dye proposed to track transplanted cells becomes rapidly diluted within fast dividing cell populations, and macrophages may consume the dye in vivo. Reviewers also expressed concern that the literature currently does not support significant engraftment of iPSC-HP, which presents a serious bottleneck to the proposal’s success. They were not satisfied the proposed modifications would resolve this problem. Additionally, the investigators neglected to discuss that the proposed in vivo model may not mount an adequate allogeneic graft rejection response. Finally, reviewers questioned whether the project could be completed in the allotted timeframe, preliminary data supporting the proposal was limited, and noted the lack of a statistical strategy to analyze results. The reviewers commented that the PI is a junior investigator with excellent training and demonstrated commitment to the proposed area of study. The PI plans to commit 15% effort to the project. However, the reviewers felt the research team is small and does not have the appropriate experience to overcome the proposal’s numerous significant problems. In summary, this proposal plans to use iPSC-derived HPs to induce mixed hematopoietic chimerism and tolerance for iPSC-derived tissues. The proposal suffers from a questionable rationale, technical challenges, a flawed experimental approach and an inexperienced research team all of which decrease its chances of success and severely dampened reviewers’ support.