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 hESC can induce immune tolerance. This is a three year application that proposes to 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 work of Dr. Hans Kierstad (who is a collaborator on this project) 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 underlies and/or is responsible for tissue repair. The proposal is based on preliminary experiments which have identified that components of the TLR and NF-kB pathways 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 NFkB is activated in oligodendrocytes differentiating from hESCs. She also proposes that TLR-2 signaling may play a role and shows expression of this at different stages of oligodendrocyte differentiation.
This proposal is from a young investigator who appears to be working without sufficient stem cell advisers. The preliminary data is weak and the experimental design does not establish that the NFkB/TLR pathway 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 Specific Aim 1 the PI will investigate the cytokines and chemokines controlled by NFkB expression 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 when co-cultured with primarily neuronal cells pre-exposed to toxic stimuli (glutamate and hydrogen peroxide) 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 NF-kB activation and TLR-2 expression profiles. It also is not clear whether these are new or established in vitro assays. Unfortunately, the preliminary data also do not demonstrate a direct connection between NF-kB activation and/or innate immune toll-like receptor expression and the in vivo effects observed. Therefore, these data do not directly support the hypothesis to be tested in Aim 1 and the proposed experiments would be considered risky. This section is short in technical detail, as indeed is much of the research design.
In Specific Aim 2, the applicant will explore whether hESC can prevent neurodegeneration by a mechanism similar to immune tolerance. She will explore whether hESC secrete IL-10 and TGF-beta as they differentiate into oligodendrocytes, and in addition will study the immunosuppression molecule soluble human leukocyte antigen-G. 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, in particular recent data from Chen, et al., AJT 2006, which demonstrates that the engagement of TLR prevents transplantation tolerance; the overall premise and hypothesis is not tenable in light of the work by Chen, et. al. and others. The experiments are designed simply to determine whether hESC-derived oligodendrocytes express IL-10, TGF-, and soluble HLA-G both 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 NFkB and TLR-2 expression corresponds to different stages of oligodendrocyte differentiation. Time in culture is not sufficient to define differentiation. She 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 Dr. Kierstad at UC-Irvine. Second, the applicant has requested three years of support unlike all the other applicants, and this perhaps reflects on a lack of confidence that this work will lead to further discovery since in general, the experiments proposed could be accomplished in one year and do not require three years.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Dr. Campbell is an assistant professor at University of California-Irvine and has worked entirely at UC Irvine throughout her career. The candidate received her PhD in 1999 and was post-doc from 2000-2002 in neurotoxicology. From 2002-2006 she was an Assistant Adjunct Professor, also working in neurotoxicology on aluminum and its neurodegenerative effects, with both clinical and basic science work on neural inflammation. In 2006, she was appointed Assistant Professor in the Department of Pharmaceutical Sciences at Western University Health Sciences. Her publication record is slim, and the lack of exposure to other academic environments during various phases of research training is considered a weakness.
Dr. Campbell has not worked with hESCs but will be working under the mentorship of Dr. Kierstad to learn hESC culture methods at the UC-Irvine stem cell core facility. The career development plan appears to be carefully outlined to involve mentorship by Dr. Kierstad, which involves training in hESC culture methods, and biweekly meetings comprised of a team of stem cell researchers from a variety of institutions in Southern California.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: A letter of support from Dr. Henriksen, the new Vice President for Research and Biotechnology, states that he is making major new initiatives in research at Western University 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 a newcomer to stem cell research, this is critical. Although she states that Hans Kierstead will mentor her, the PI may need more continuous mentorship that he 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 her intent to make a career in stem cells, in this proposal she lacks the appropriate guidance.
