New Faculty I
$2 420 218
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:
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 .
SYNOPSIS: Expression of the transcription factor NANOG is required for maintaining self-renewal and pluripotency of human embryonic stem cells (hESC), while overexpression of NANOG leads to feeder-independent hESC self-renewal. The PI proposes to investigate how NANOG interacts with different cofactors by characterizing the structure and function of different NANOG domains and identifying the proteins bound to NANOG at active and repressed loci in hESC. This will allow the characterization of domain-specific NANOG-binding proteins. This proposal has three specific aims. In Aim 1, the PI intends to characterize NANOG protein domains, alone and in combination. An in vitro reporter assay will be used to determine the transcriptional activity of a panel of NANOG deletion mutants on promoters that are active either during hESC self-renewal, such as those from OCT4 and NANOG itself, or during differentiation, such as those from GATA6 and GATA4. This will be followed by testing the activity of these NANOG mutants in hESC. In aim 2, NANOG-binding-proteins will be identified by either co-immunoprecipitation (IP), using candidate binding partners, or in an unbiased fashion by liquid chromatography and mass spectrometry. Finally, in Aim 3, the PI will attempt to produce recombinant TAT-NANOG proteins from full-length NANOG and deletion mutants for delivery to hESCs, in order to study their effect on self-renewal and differentiation. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: Although NANOG has been described as a key regulator of mouse ESC and human ESC self-renewal, surprisingly little is known about the relationship of NANOG protein structure to its function. This proposal aims to elucidate the molecular mechanisms mediated by NANOG, 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, cookie cutter 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. Aim 1 will test deletion NANOG mutants for their transcriptional activation/repression potential in reporter assays and test their capacity to maintain self-renewal under differentiation conditions in hESC. These are important experiments to further define the role of NANOG in these processes. The PI has interesting preliminary data on the effect of NANOG deletion mutants on OCT4 promoter activity, which makes the aim seem feasible. This is a very straightforward approach and should yield very useful information. Aim 2 will identify binding partners of NANOG. These are important experiments, which, however, are not particularly innovative and quite competitive. Preliminary NANOG IP data support the feasibility of the approach. It is not clear how the PI will validate the identified interacting proteins, e.g. functional studies in hESCs should be performed. Aim 3 will attempt to produce recombinant TAT-NANOG protein from full-length NANOG and deletion mutants to study their effect on self-renewal. It is not quite clear what additional information the TAT-NANOG approach, if successful, will provide when compared to the conventional overexpression system of Aim 1. Preliminary data suggest that full-length TAT-NANOG can enter the nucleus of cells. However, there is no mention of whether this 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 PRINICIPAL INVESTIGATOR: The reviewers provided different assessments of Dr. Lutzko’s qualifications. One point of view was that she is a talented investigator with many important contributions to her field, as shown by her list of publications. This reviewer had no hesitation affirming that she and her team are perfectly qualified to perform all the specific aims of this grant. Conversely, another reviewer felt that Dr. Lutzko has no proven track record as first or last author in the hESC / NANOG field, although she is a co-author on publications using hESCs and lentiviral vectors. Her major expertise is in the blood system. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: Children’s Hospital of Los Angeles is the best institution for 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. This is a well-written proposal with three interesting aims that address issues directly relevant to hESC research. The preliminary data on NANOG and the focus of the grant are significant. However, the proposal was not deemed innovative, which may be especially problematic because the NANOG field is very competitive and many laboratories are pursuing this subject. Furthermore, the PI has no prior experience in this field and lacks the required molecular biology prowess; in other words, the track record of the PI is not of high enough quality to convince the reviewers that she 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.