A variety of stem cells exist in humans throughout life and maintain their ability to divide and change into multiple cell types. Different types of adult derived stem cells occur throughout the body, and reside within specific tissues that serve as a reserve pool of cells that can replenish other cells lost due to aging, disease, trauma, chemotherapy or exposure to ionizing radiation. When conditions occur that lead to the depletion of these adult derived stem cells the recovery of normal tissue is impaired and a variety of complications result. For example, we have demonstrated that when neural stem cells are depleted after whole brain irradiation a subsequent deficit in cognition occurs, and that when muscle stem cells are depleted after leg irradiation an accelerated loss of muscle mass occurs. While an increase in stem cell numbers after depletion has been shown to lead to some functional recovery in the irradiated tissue, such recovery is usually very prolonged and generally suboptimal.Ionizing radiation is a physical agent that is effective at reducing the number of adult stem cells in nearly all tissues. Normally people are not exposed to doses of radiation that are cause for concern, however, many people are subjected to significant radiation exposures during the course of clinical radiotherapy. While radiotherapy is a front line treatment for many types of cancer, there are often unavoidable side effects associated with the irradiation of normal tissue that can be linked to the depletion of critical stem cell pools. In addition, many of these side effects pose particular threats to pediatric patients undergoing radiotherapy, since children contain more stem cells and suffer higher absolute losses of these cells after irradiation.Based on the foregoing, we will explore the potential utility and risks associated with using human embryonic stem cells (hESC) in the treatment of certain adverse effects associated with radiation-induced stem cell depletion. Our experiments will directly address whether hESCs can be used to replenish specific populations of stem cells in the brain and muscle depleted after irradiation in efforts to prevent subsequent declines in cognition and muscle mass respectively. In addition to using hESC to hasten the functional recovery of tissue after irradiation, we will also test whether implantation of such unique cells holds unforeseen risks for the development of cancer. Evidence suggests that certain types of stem cells may be prone to cancer, and since little is known regarding this issue with respect to hESC, we feel this critical issue must be addressed. Thus, we will investigate whether hESC implanted into animals develop into tumors over time. The studies proposed here comprise a first step in determining how useful hESCs will be in the treatment of humans exposed to ionizing radiation, as well as many other diseases where adult stem cell depletion might be a concern.
Radiotherapy is a front line treatment used in California for many types of cancer, including brain, breast, prostate, bone and other cancer types presenting surgical complications. Treatment of these cancers through the use of radiation is however, often associated with side effects caused by the depletion of critical stem cell pools contained within non-cancerous normal tissue. While radiotherapy is clearly beneficial overall, many of these side effects have no viable treatment options. If we can demonstrate that human embryonic stem cells (hESC) hold promise as a safe therapeutic agent for the treatment of radiation-induced stem cell depletion, then cancer patients may have a new treatment for countering many of the debilitating side effects associated with radiotherapy. Once developed this new technology could position California to attract cancer patients throughout the United States, and the state would clearly benefit from the increased economic activity associated with a rise in patient numbers.
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.