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RN1-00553-1: Mechanisms of NANOG Function in hESC
Recommendation: Recommended if funds available
Scientific Score: 61
First Year Funds Requested: $480,466
Total Funds Requested: $2,420,218
Public Abstract (provided by applicant)
Human embryonic stem cells (hESC) have been proposed as a renewable source of tissue for regenerative medicine applications. One obstacle to the development of hESC based therapies is that hESC spontaneously and randomly differentiate (or change) into non-specific cell types in the laboratory. The main reason for this random differentiation is that we don’t completely understand the basic biology of these cells and mechanisms that keep the stem cells renewing themselves in the lab (called self-renewal). In fact the most reliable method to keep human embryonic stem cells alive in the laboratory is to culture them on cells taken from mouse embryos. This techniques requires a significant amount of time to make these mouse cells and test them, as well as leading to significant batch-to-batch variety in quality, as well as the possibility of introducing animal viruses or other pathogens.
In the proposed studies, we will investigate one gene that we know is very important for stem cells to make more stem cells (self-renew)- called NANOG. Despite the importance of NANOG to stem cells, there is relatively little know about the physical structure of this gene or protein, or how it works, and what other genes and proteins it interacts with to keep stem cells renewing. We will then use the data generated in our studies to generate specific growth or ‘self-renewal’ factors that can be added to the embryonic stem cells and reliably keep them undifferentiated. If successful, the development of such growth factors will make all aspects of human embryonic stem cell research more reliable and efficient. This in turn will increase the time that researchers have to developing therapeutic protocols, rather than spending a large amount of their time and energy to keep their cells alive and renewing. Furthermore, it will facilitate the scale-up of stem cell applications from small scale laboratory studies. Thus, our studies have the potential to impact on all human embryonic stem cell research, and increase the rate of development of all stem cell based therapies. The development of a growth factor(s) that can replace mouse cells, and reliably keep the stem cells alive would prove very useful to stem cell researchers, as well as increase the safety of the cultures for therapeutic purposes.
Statement of Benefit to California (provided by applicant)
One of the biggest hurdles in human embryonic stem cell research is that the cells spontaneously and randomly differentiate into a variety of non-specific cell types in culture. These non-specific cell types have little, if any, therapeutic potential. It requires a high degree of technical training to minimize this random differentiation, and even then is not preventable with currently available growth factors and techniques. Our project will study the biological processes that keep stem cells renewing. We will then use this information to evaluate new growth factors that will keep human embryonic stem cells in an undifferentiated state. If successful, these growth factors will increase the efficiency of all human embryonic stem cell research, by dramatically decreasing the amount of time spent keeping human embryonic stem cells alive and undifferentiated. In turn, this will allow researchers to spend more time and effort developing stem cell therapies for human disease, and increase the rate of development of stem cell therapies .
Review
SYNOPSIS: Expression of the regulatory factor that is the subject of this proposal is required for maintaining self-renewal and pluripotency of human embryonic stem cells (hESC), while its overexpression leads to feeder-independent hESC self-renewal. The PI proposes to investigate how this factor interacts with different cofactors by characterizing the function of its different domains and identifying the proteins that bind to it. This will allow the characterization of domain-specific factor-binding proteins.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: Although the factor under investigation has been described as a key regulator of mouse ESC and human ESC self-renewal, surprisingly little is known about the relationship of its protein structure to its function. This proposal aims to elucidate the molecular mechanisms mediated by this factor, thereby advancing the field of stem cell research.
Overall, this is a well-written proposal with three important aims that stand on their own, and with interesting preliminary data on many of the proposed projects. However, the proposal lacks innovation. The collection of safe, standard projects listed in this proposal will inevitably add to our current knowledge. Enthusiasm was dampened somewhat, not only because of lack of risk-taking, but also by the limitation of the study to presidential cell lines, which are known to be very unstable and suffer from genetic abnormalities.
The investigator proposes to perform a structure / function analysis of the regulatory factor and to identify its binding partners. These are important experiments to further define its role in self-renewal, although they are not particularly innovative and quite competitive. Preliminary data support the feasibility of the proposed approaches. It is not clear, though, how the PI will validate the identified interacting proteins, e.g. functional studies in hESCs should be performed.
Finally, the PI will attempt to produce recombinant regulatory factor proteins for efficient delivery to hESC in order to study their effect on self-renewal. It is not quite clear what additional information the approach proposed for this aim, if successful, will provide when compared to the conventional overexpression system of the earlier proposed studies. Preliminary data on the recombinant protein are limited and there is no mention of whether it has any biological effect. Furthermore, controls, such as the stability of the mutant proteins and their potential resistance to post-translational modification, have not been addressed.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The reviewers provided different assessments of the applicant’s qualifications. One point of view was that s/he is a talented investigator with many important contributions to his/her field, as shown by the list of publications. This reviewer had no hesitation affirming that the applicant and his/her team are perfectly qualified to perform all the specific aims of this grant. Conversely, another reviewer felt that the applicant has no proven track record as first or last author of articles related to the analysis of hESC or the regulatory factor under investigation, although s/he is a co-author on some relevant publications.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The applicant institution is well-suited to support these studies, encompassing all of the necessary resources and centers to successfully execute the proposed specific aims. Institution and faculty mentors support the PI’s engagement in hESC research and collaborators have committed to assist with biochemical analyses.
DISCUSSION: There was general agreement amongst the reviewers in assessing the strengths and weaknesses of this grant proposal. This is a well-written proposal with three interesting aims that address issues directly relevant to hESC research. The preliminary data on the regulatory factor and the focus of the proposal are significant. However, the proposal was not regarded to be innovative, which may be especially problematic because the field engaged in the analysis of the factor is very competitive and many laboratories are pursuing this subject. Furthermore, the PI has no prior experience in this field and does not have sufficient molecular biology expertise; in other words, the track record of the PI is not of high enough quality to convince the reviewers that s/he will be successful in this field. More specifically, questions were raised about the choice of cells, since the PI intends to use presidential cell lines only, even though they are known to be problematic. Furthermore, the use of transient transfections in structure / function analyses was deemed inappropriate. This analysis should be performed in the right functional setting.
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