Human embryonic stem cells (hESC) provide a promising future therapeutic approach for replacement of diseased or damaged tissue. This proposal addresses the development of therapeutic strategies that can specifically aid two different neurological disorders. Multiple sclerosis (MS) is a chronic condition which affects the brain and spinal cord. In patients with this condition, the myelin sheaths that cover nerve fibers are erroneously destroyed by the body’s own immune system. In spinal cord injuries, the resulting inflammation causes severe damage to myelin-producing cells. The goal of this proposal is to determine if stem cells, at various phases of differentiation to myelin-producing cells, have characteristics which could not only replenish the lost myelin, but also confer a healing microenvironment. The focus will be on two beneficial immune-based processes. First specific aim will determine molecular signaling pathways involved in innate immune responses in hESC and establish if these processes provide a microenvironment conducive to tissue regeneration. Innate-immune responses have been shown to underlie tissue repair gene expression by dying cells. Our preliminary experiments have identified that components of this pathway are present in human embryonic stem cells (hESC), particularly at the later phases of differentiation to mature myelin-producing cells. Levels of factors involved in this process will be evaluated in hESC. To further establish the potential of the hESC to create a regenerative microenvironment for damaged cells, inserts of stressed and dying cells will be co-cultured with hESC. Cell viability, with or without co-exposure to hESC, will be determined using a commercially available cell cytotoxicity assay. The hypothesis to be tested is that hESC can provide a restorative microenvironment for stressed or dying cells and this involves up-regulation of innate immune responses. In the second specific aim, we will evaluate whether hESC can down regulate harmful inflammatory immune responses by a process used by fetal cells to escape the maternal immune response. This process is called ‘immune tolerance. To determine if hESC have the ability to provide an immune privileged microenvironment (similar to the fetus) components of this response will be determined in hESC. The hypothesis to be tested is that hESC can subvert destructive immune responses by processes similar to those that underlie immune tolerance. The long term objective of these studies is to design oligodendrocyte populations derived from hESC that provide an ideal microenvironment for promotion of cell survival, regeneration, and immune tolerance. These can eventually be used therapeutically in chronic demyelinating diseases such as MS or acute traumatic spinal cord injuries with minimal chances of host rejection.
Statement of Benefit to California:
The proposed research will benefit California and its citizens in several ways. First, the results may aid in the design of effective therapies based on hESC that can benefit patients with multiple sclerosis or spinal cord injury. For the citizens of California who are suffering from these disorders, the development of these stem cell-based therapies will alleviate some of the pain and hardships associated with their condition. Second, successful development of stem-cell based therapies will lighten the economic burden for the state of California, which provides health care services to its citizens. Third, the results gained from the studies proposed may lead to intellectual property rights which can be used as a foundation for creating new biotechnology companies. This will not only create jobs but will also contribute to the economic growth of the state of California.
SYNOPSIS: Dr. Arezoo Campbell from the Western University of Health Sciences proposes to determine whether and how hESC implants can provide a "healing microenvironment". This proposal is designed to test whether oligodendrocytes derived from human embryonic stem cells (hESCs) can influence the pathologic microenvironment into which they are transplanted based on their effects on the immune response. In the first part of the work, the applicant will explore innate immune responses in hESCs at different stages of differentiation. In particular, the NF-kB/Toll-like Receptor-2(TLR-2) will be measured. hESCs will be exposed to media from neuronal culture exposed to LPS. Secondly, the PI will ascertain whether hESC are "immune-tolerant" by looking for cytokines such as IL-10, TGF-b, and soluble immunosuppressive HLA-G antigen to test the hypothesis that hESC may subvert destructive immune responses by processes similar to those that induce immune tolerance. The "long-term objective" of the studies is to design oligodendrocyte populations derived from hESC that not only remyelinate but provide "an ideal microenvironment" for promoting cell survival, regeneration, and immune tolerance. SIGNIFICANCE AND INNOVATION: This is an innovative and original proposal that presents interesting and novel ideas. The ability of cells, in this case oligodendrocytes that are derived from hESCs, to both remyelinate axons and influence the microenvironment in a positive way is intriguing. The proposed experiments might also help address other questions that are presently being asked about the possibility of transplanted neural stem cells influencing the inflammatory background into which they are transplanted and the immune response. STRENGTHS: This is a thoughtful proposal which asks interesting questions and which has some preliminary data to support the hypotheses. The subject of immune effects of hESC cells is very important, and the proposed experiments are feasible. WEAKNESSES: The experimental plan may not work in part as LPS is unlikely to damage neurons because neurons do not usually express TLR4 or CD14, which are essential for LPS binding. This is a serious and significant technical concern. Also, the fact that cytokine release from hESCs was undetectable by ELISA is concerning. More importantly, the proposal contains few definitive experiments that would test some of the novel ideas proposed. It is of interest to determine what cytokines are produced by hESCs but little details are provided concerning how the hESCs will be prepared. The applicant does not seem to have much experience with cell culture and particularly stem cells. There are great ideas presented, but more details of the experiments should be given since it is not clear that the ideas will be tested rigorously. The PI might consider using known compounds that will damage neurons such NMDA, kainic acid or nitric oxide, etc.. In addition, before moving to Western blot or immunostaining for cytokines, which is perhaps ever more difficult, the applicant could begin by determining RNA levels. DISCUSSION: There was no discussion further following the reviewers' comments.