Recommended if funds allow
Statement of Benefit to California:
SYNOPSIS: Dr. Charles Limoli from UC Irvine proposes to study the effects of irradiation on phenotypes of implanted hESC. Radiation selectively kills neural progenitor cells and the experiments will determine whether local irradiation changes the phenotypes of hES cells transplanted into the tissue. In addition, he plans to examine the potential carcinogenic risk of implanting hESCs within irradiated brain or leg. SIGNIFICANCE AND INNOVATION: This is a very innovative and original application, involving a commonly occurring clinical situation. Irradiation is frequently used to treat cancers of the brain and other tissues. A now popular theory of stem cell interaction with tissues is the concept of niches where stem cells interact with other cells in the tissue. It would be of interest to see what irradiation does to implanted stem cell fates and differentiation. Previous results from the author suggest that irradiation of the brain is likely to result in more of the implanted stem cells becoming or producing more astrocytes. This would be of interest, especially since few studies have analyzed the fate of implanted multipotent cells in irradiated tissues. The application raises several important points relevant in general to ES cell transplantation. Will ES cells form tumors? Are they more likely to form tumors in irradiated tissues than in non-irradiated tissues? Can ES cells be used to replenish stem cell populations in an irradiated brain? STRENGHTS: The investigator is an experienced and productive investigator who has published 54 articles, many of them since he received his Ph.D. in 1994. Many of the studies have to do with genomic instability and the effects of such instability on cell death and fate. The application is familiar with the proposed methods, and is quite detailed both in the review of previous work and also the experimental approach that is being proposed. For example, the discussion of the irradiation dose indicates that the applicant has put quite a lot of thought into the radiation needed to deplete different tissue of stem/progenitor populations. There are several strong aspects of the experimental plans as well. For example, the use of athymic nude rats will substantially decrease potential problems from cross-species tissue rejection. In addition, the hESCs will be labeled with eGFP to ease identification of the cells; the applicant will be examining the cells over a one-month period and one can reasonably expect that the eGFP label to remain for this period. They will use HLA antigen staining as well. WEAKNESSES: The proposed studies have a number of potential weaknesses. One general weakness is that it isn’t clear that all of the transplanted hES cells will differentiate along neural lineages. The PI should broaden the assessment of the transplanted cells to include other non-neural lineage markers. It will be important to know if there is any mesenchymal or endodermal differentiation, for example, as they may have profound effects on transplantation. Similarly, if tumors arise, they may not necessarily be neuroectodermal in nature. Other general weaknesses include the failure to discuss the possible effect of irradiation on "niche" cells in the tissue. Many studies are not identifying specific cells in tissues that must interact with stem cells and instruct the cells concerning what cells they should produce or differentiate into. Also, the applicant does not consider the possibility that hESC may fuse with host cells and they do not propose any means of detecting fusion events. Finally, the authors are not looking at when the cells will be implanted after irradiation. It would seem that this is a very important factor and they will, sooner or later, have to look at this. The Progeny of eGFP expressing cells may show less expression. The applicant does not discuss the effect of proliferation versus differentiation on eGFP expression. It is likely that they will need to do HLA immunohistology on all the sections, to ensure that they are not missing cells The experiments for the second specific aim of potential carcinogenic risk of hESC implants are very thoughtful, but are weaker from the viewpoint of experimental design. One weakness of the second set of experiments to examine carcinogenicity of the hESC implants is that applicant will be studying nude athymic rats. They will be studying the rats at 6 and 12 months periods. While use of these immune-incompetent rats is necessary to eliminate immune rejection as a factor in the experiments, it is unclear that results from athymic rats can be extrapolated to immune-competent humans, particularly in the experiments examining carcinogenic risk. As long as this weakness in kept in mind, the results should be interesting. A second but relatively minor weakness is the lack of specific criteria and likely insensitivity of the assay for "cancer" formation. The applicant admits that the conclusions are necessarily limited due to small sample size (16 rats per group) and study of only one genetic strain. DISCUSSION: This strong application raises the important question of whether hESC can be used to replenish irradiated NPCs. The PI implies that the transplanted hESC will differentiate along neural lineages, but there is no reason to believe that this is the only lineage. There were several other recommendations for small changes in the experimental protocol that would improve the proposal: 1) The authors should systematically assess several populations of cells in the irradiated brain, including astrocytes, microglia, oligodendroglia, neurons, and inflammatory cells (macrophages). It would be important to ascertain that irradiation have similar effects on all the tissues and may provide insights into the stem cell "niche" in brain. 2) The applicants should do BRDU labeling in all the experiments to look for newly mitotic cells. This will provide important information that will help them interpret the results. It may also help them evaluate the number of dividing cells. The applicant has experience with BRDU. 3) It is likely that the applicants will need to do HLA immunohistochemistry in all the experiment because eGFP expression may be changed by proliferation and differentiation. 4) The applicants need to do a time series of implants at different times after irradiation.