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RN1-00528-1: Characterization of Immune Responses in Human Embryonic Stem Cells
Recommendation: Not recommended for funding
Public Abstract (provided by applicant)
Approximately 250,000 people in the United States are diagnosed with multiple sclerosis (MS). In patients with this condition, the white sheaths known as myelin that cover nerve fibers are erroneously destroyed by the body’s own autoreactive immune system. Patients diagnosed with MS fall into a spectrum ranging from benign, where individuals diagnosed may lead normal lives, to severe, where patients are progressively debilitated. The goal of this proposal is to determine if stem cells, at various phases of differentiation to myelin-producing oligodendrocytes, have characteristics which could not only replenish the lost myelin, but also confer a healing microenvironment for dying nerve cells. The focus will be on two beneficial immune-based processes. First specific aim will determine if innate immune responses, necessary for healing damaged tissue, are present in human embryonic stem cell (hESC)-derived oligodendrocytes and establish if these processes provide a microenvironment conducive to nerve cell survival. Innate-immune responses have been shown to underlie tissue repair of dying cells. Our preliminary experiments have identified that components of this pathway are present in hESC, particularly at the later phases of differentiation to mature oligodendrocytes. Levels of factors involved in this process will be evaluated. To further establish the potential of hESC to create a regenerative microenvironment for damaged cells, dying nerve cells will be grown with hESC-derived oligodendrocytes. Cell viability will be determined using a commercially available cell toxicity 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 prevent destruction by 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 hESC-derived oligodendrocyte cell populations 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 with minimal chances of host rejection.
Statement of Benefit to California (provided by applicant)
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 human embryonic stem cells 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.
Review
SYNOPSIS: This proposal will explore if human embryonic stem cell (hESC)-derived oligodendrocytes activate signaling pathways involved in innate immune responses that in turn can promote neuronal survival in the context of neurodegenerative disease. It will also explore whether hESCs can induce immune tolerance, and characterize the possible mechanisms by which hESCs at various phases of differentiation towards oligodendrocytes could confer a regenerative microenvironment. The proposal is largely based on the published work of a collaborator showing that oligodendrocytes derived from hESCs can promote endogenous neuronal regeneration and functional improvement after spinal cord injury in animal models. The hypothesis here is that the functional improvement that is achieved in this model is due to the upregulation of innate immune responses which underly and/or are responsible for tissue repair. The proposal is based on preliminary experiments which have identified that components of a specific molecular signaling pathway (a transcription factor and its activating receptor) are present in hESCs, in particular in later phases of differentiation to mature oligodendrocytes. The long-term clinical application of this research is to develop better cell-based therapies based on hESCs that may benefit patients with multiple sclerosis and spinal cord injuries.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The proposed research will explore the role that oligodendrocytes (ODCs) derived from hESCs may play in regulating the immune neuroenvironment in disease and potentially provide neuroprotection to dying neurons and/or axons. The concepts on which the proposal is based, including the potentially interesting concept of the restorative microenvironment, are of considerable interest. The applicant has shown that a specific transcription factor is activated in oligodendrocytes differentiating from hESCs. S/he also proposes that signaling through a second member of this molecular pathway may play a role and shows expression of this second member at different stages of oligodendrocyte differentiation. This proposal, however, is from a young investigator who appears to be working without sufficient stem cell advisors. The preliminary data is weak and the experimental design does not establish that the signaling pathway in question is specifically activated in oligodendrocytes and functions to protect dying neurons. Further, the experiments focusing on the induction of immune tolerance by hESC are very limited and will not directly analyze the immune response induced by these cells in a neurodegeneration model.
In the first aim, the Principal Investigator (PI) will investigate the cytokines and chemokines controlled by expression of the specified transcription factor and as measured using Northerns, Westerns, and ELISA. The applicant will then determine the functional impact of expression of these inflammatory molecules, and whether hESC-derived ODCs can preserve neuronal cell viability and prevent inflammatory molecule-mediated cytotoxicity. The experimental design is not developed in a cogent way and in general seems biased. For example, it is not clear if these are purified ODC cultures or whether other cell types within the cultures are responsible for the observed molecular activation and expression profiles. It also is not clear whether these are new or established in vitro assays. Unfortunately, the preliminary data do not demonstrate a direct connection between the specified transcription factor activation and/or the innate immune receptor expression and the in vivo effects observed. Therefore, these data do not directly support the hypothesis to be tested in the first aim, and the proposed experiments would be considered risky. This section is short in technical detail, as indeed is much of the research design.
In a second aim, the applicant will explore whether hESCs can prevent neurodegeneration by a mechanism similar to immune tolerance. S/he will explore whether hESCs secrete certain cytokines as they differentiate into oligodendrocytes, and in addition will study a molecular factor involved in immunosuppression. The idea that hESC-derived oligodendrocytes may play a role in the therapy of demyelinating disorders beyond remyelination is extremely interesting. Indeed, the role that inflammation plays in remyelination or other restorative features of repair is being investigated in depth by others. However, the proposal presented here lacks the detail required to provide confidence that it will succeed, and the experiments are based on a questionable hypothesis that is not strongly supported in the literature. That is, why would these injected cells alter immune functions of the recipient or promote tolerance? In fact, the PI has failed to consider the role of these molecules in allograft rejection, and in particular recent data which demonstrates that the engagement of the molecular signaling pathway to be studied in Aim 1 prevents transplantation tolerance; the overall premise and hypothesis is not tenable in light of this work. The experiments are designed simply to determine whether hESC-derived oligodendrocytes express the cytokines and molecular factor of interest at the RNA and protein level, but are not designed to determine if the hESC-ODC mediated regeneration is dependent on local or systemic cytokine production. It is possible that the dominant microenvironmental phenotype could depend, at least to some degree, on the type, extent, stage and/or chronicity of the injury. The microenvironment and host tissues may also impact the responses or activity of the cells in vivo. Therefore, in vitro responses and expression profiles may not exactly correlate with functions in vivo, and in vivo responses are not tested in this application.
The preliminary data, while interesting, are poorly presented. One reviewer did not feel that the applicant had clearly shown that activation of the molecular signaling pathway corresponds to different stages of oligodendrocyte differentiation. Time in culture is not sufficient to define differentiation. The PI would have to show that this corresponds to acquisition of mature oligodendrocyte markers and expression of myelin protein(s). There are two other limitations in this proposal. First, the intended experiments are entirely dependent on the availability of hESC-derived ODCs from a collaborator. Second, the experiments proposed could be accomplished sooner than the timeframe requested.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: This PI is an Assistant Professor who had worked entirely at one institution prior to 2006. The candidate received a PhD in 1999 and was a post-doctoral fellow from 2000-2002 in neurotoxicology. From 2002-2006 s/he was an Assistant Adjunct Professor, also working in neurotoxicology, with both clinical and basic science experience. In 2006, s/he was appointed Assistant Professor at the applicant institution. The publication record is slim, and the lack of exposure to other academic environments during various phases of research training is considered a weakness.
The applicant has not worked with hESCs but will be working under the mentorship of a collaborator to learn hESC culture methods at a stem cell core facility. The career development plan appears to be carefully outlined to involve mentorship by the collaborator, which involves training in hESC culture methods, and biweekly meetings comprised of a team of stem cell researchers from a variety of neighboring institutions.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: A letter of support from the Vice President of Research states that major new initiatives in research are underway at the applicant institution, and that stem cell research will be located in a laboratory of excellence. While the institution provides reasonable evidence of a commitment to enlarging their stem cell research environment, and full support and space for research will be provided to the investigator, it is likely that the candidate will be very isolated in this environment that appears to lack a critical mass of stem cell scientists. As this PI is a newcomer to stem cell research, this is critical. Although the PI states that the collaborator will mentor him/her, the PI may need more continuous mentorship that this person likely can supply.
DISCUSSION: The PI has identified an interesting question. It is an interesting idea to explore the importance of oligodendrocytes in regulating the immune environment, and to generate oligodendrocytes from human ES cells to cure Multiple Sclerosis. Unfortunately, the proposal is poorly described and lacks technical details, and the investigator suffers from lack of mentorship. While the PI is clear about his/her intent to make a career in stem cells, in this proposal s/he lacks the appropriate guidance.
The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:
- None