Funding opportunities

Control Mechanisms Governing Human ESC Self Renewal and Differentiation

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00145
Funds requested: 
$2 507 692
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
All of the diverse cells in a human body contain the same “library” of genetic information stored in the form of DNA molecules. Over the past 40 years, scientists have made considerable progress in understanding how the cell reads DNA to decode the information carried in our chromosomes. This process of gene expression is exquisitely regulated and drives the formation of all the differentiated specialized cell types within organs such as brain, breast and skeletal muscles. The experiments described in this proposal are directed at understanding the regulation of gene expression in both self-renewing and differentiating stem cells. These experiments represent a critical first step in the quest to propagate human embryonic stem cells and to derive differentiated cells from them for the purposes of disease therapy. In preliminary studies, our laboratory has identified a novel protein complex, SCC, required for activating genes needed to maintain stem cell self-renewal – an essential defining property of stem cells. Here we propose to characterize the molecular components of this complex as an important step in our efforts to reliably grow human stem cells in a reproducible manner. At the same time we hope to determine whether these same key proteins are also active in cancer cells which may complicate the use of stem cells in human therapeutic applications. Thus, regulatory factors such as SCC are potential targets for drugs aimed at increasing or decreasing the ability of stem cells to divide. A second major focus of the proposal is to understand the network of gene activity needed for the differentiation of dopamine-producing neurons, the brain cells that are lost in Parkinson’s disease. Our collaborators have devised a method to grow dopamine neurons from human embryonic stem cells in culture. Here we propose biochemical and molecular biological studies to identify the genes active in these dopamine neurons, with the goal of improving the efficiency of dopamine neuron culture and subsequently, the development of new therapies for Parkinson’s disease. Finally, we will investigate the regulatory proteins required for gene activity in muscle cells. We will use biochemical assays to understand the different activities of a key regulatory protein, TAF3, in immature and differentiated muscle cells, and will use cell culture strategies similar to those employed for dopamine neurons to differentiate muscle cells from human embryonic stem cells in culture. Many of the muscular dystrophies are the consequence of mutations in genes expressed specifically in muscle cells. The proposed studies could lead to strategies for drug design or treatment for these conditions.
Statement of Benefit to California: 
The ultimate goal of these studies is the development of new therapies for diseases that are fundamentally the result of inappropriate levels of cell division and differentiation. The proposed experiments will determine how gene activity is controlled, either to maintain a renewing population of stem cells, or to direct differentiation of specific mature cell types implicated in human disease. For example, Parkinson’s disease and muscular dystrophies arise because the body is unable to replace damaged cells, dopamine neurons and muscle cells respectively. Conversely, breast cancer, another disease targeted by the proposed experiments, is the result of misregulated cell division and the failure of the newly produced cells to assume an appropriate location or function. Because all biological activities; cell division, differentiation and function, are the result of differential gene expression, an understanding of the gene regulatory networks controlling these processes will be crucial for drug development and testing. Thus, this proposal will not only advance the science of stem cells but also provide the technological platform for establishing new bio-pharma enterprises based on the development of disease intervention using original cell based assays for drug discovery . The proposed experiments will benefit the people of the State of California both directly and indirectly. In the short term, the research will support the training of four post-doctoral scholars and one graduate student, three lifelong California residents and two new residents who have moved here specifically to participate in this project. CIRM funding will also enable us to expand our collaboration with a European laboratory, bringing new stem cell technologies and expertise to the State. In the long term, the proposed work will likely reveal gene activities that are essential for the establishment, survival and maintenance of stem cells as well as differentiated cell populations. These studies will likely reveal previously unknown potential drug targets that will allow the screening of novel classes of drugs. For example, one possible target is the stem cell coactivator complex SCC. By increasing SCC activity, it may be possible to enhance the expansion of stem cell populations. Conversely, if SCC activity is necessary for the perpetuation of breast cancer stem cells, this would be an attractive target for drugs aimed at decreasing cancer cell potency. This project will also result in the refinement of the methods for engineering dopamine neurons and muscle cells. Such cells could be used directly in stem cell therapy. The possibility of culturing these cells in significant quantities under defined conditions in vitro, in combination with a detailed understanding of their gene regulatory networks, will also open up new ways to screen for drugs useful in the treatment of Parkinson’s disease or muscular dystrophy.
Review Summary: 
SYNOPSIS: This proposal is focused on identifying and characterizing the components of transcriptional regulatory machinery that are used by hESCs to maintain a proliferative self-renewal state and to differentiate into dopaminergic neurons and muscle cells. The study brings together expertise in transcriptional regulation and stem cell differentiation. The Principal Investigator (PI) is a well-established investigator and pioneer in the field of in vitro transcriptional control models and intends to apply these techniques to hESCs in culture to understand self-renewal and differentiation. Specific aims are: 1) to understand the role of SCC/Oct4/Sox2/Nanog in maintenance of hESCs in the undifferentiated state, 2) to study the gene regulatory networks involved with hES differentiation towards dopaminergic (DA) neurons and myotubes. Aim 3 focuses on cancer stem cells. The methods include in vitro transcriptional model systems as well as other methods including mass spectroscopy, microarray expression analysis, chromatin immunoprecipitation, shRNAi knockdowns and transient transfection of reporter genes. Collaborations are in place for studying regulation of LMX1A and in lineage-specific differentiation to dopaminergic neurons and skeletal muscle cells, respectively. IMPACT AND SIGNFICANCE: This proposal aims to take a variety of transcriptional approaches towards understanding the pluripotency and differentiation of hESC lines. The impact of this research would be to acquire a better understanding of the mechanisms governing these processes in hESC. Specifically, the proposed studies are likely to reveal new knowledge and advance our understanding of how Oct4, Sox 2 and Nanog interrelate to control self-renewal by applying a comprehensive experimental plan to test their hypothesis. This proposal contains three fairly distinct specific aims. The potential impact of the first aim depends upon the specificity of the so-called stem cell co-activator (SCC) complex for stem cells and/or stem cell transcription factors. Identification of the components of the complex and the inhibition of their activity in hESC offers the potential to identify novel regulators of stem cell characteristics. Collaboration with investigators at the Karolinska Institute provides a foundation for the examination of the role of Lmx1a interacting proteins and target genes as hESC differentiate towards dopaminergic (DA) neurons. The effects of Lmx1a have already been demonstrated by preliminary experiments, and identification of downstream targets and interacting proteins will help determine how Lmx1a directs DA differentiation as well as additional factors important for this process. This approach could produce information that directly affects production of DA neurons for therapeutic applications. Knowing more about the differentiation of ES cells to DA neurons and muscle would have obvious benefits for the study or treatment of diseases in which these cells are lost. The hypothesis, which is supported by the strong preliminary data for Aim 1, is original and the application of an in vitro transcriptional model system to hESCs is innovative. QUALITY OF THE RESEARCH PLAN: The research plan for Aim 1 and parts of Aim 2 are considered strong, whereas the plan for Aim 3 is considered weak. Aim 1 focuses on the identity of SCC protein components, specificity of interaction with Oct4-Sox2 complexes, and association of SCC with chromatin. The feasibility of this aim relies upon the biochemical purification of the SCC complex, which is shown with preliminary data. The impact relies upon function in stem cells; ideally, one would like to have data suggesting that SCC is specific to ESC or Oct-Sox complexes. While the potential for SC co-activator is very high, a reviewer questioned the naming of "SCC", and considered this name a little premature, as the specificity of the complex has not yet been demonstrated. However, even if SCC is not really a stem cell specific factor, its characterization would provide valuable information regarding basic molecular mechanisms of Oct-Sox transactivation. The reagents necessary for successful ChIP analysis of SCC have not been generated yet. The strong preliminary data for Aim 2 showing the effects of forced Lmx1a expression on the differentiation of mouse and human ESC towards DA neurons provides a foundation for the first of two subaims. ChIP-on-chip experiments will be used to identify sites bound by Lmx1a during hESC differentiation into DA. Pull-down experiments and co-immunoprecipitation assays will be used to identify Lmx1a-interacting proteins. In contrast, the second subaim to examine TAF3 in myogenic differentiation is not supported by data shown in this proposal. Although development of protocols to promote MyoD-induced myotube differentiation from hESC is stated as another subaim, no research plan towards developing a protocol or rationale is provided. Data are summarized for Aim 3, but not shown, to suggest that Oct4, Sox2 and Nanog are expressed in some breast cancer cell lines, “but that the levels and pattern of expression are distinct from those observed in ESCs.” Without seeing the levels of expression, it is difficult to interpret what this statement means with respect to the potential of any of these factors to affect cancer cell characteristics. That said, several previously published manuscripts support the re-expression of Nanog and Oct4 in several tumors, including breast cancers. An assessment of Nanog/Oct4/Sox2 expression in a full spectrum of human breast cancers will test the robustness of the correlation; however, the source of the cancer cell lines to be used was not given. Forced expression and RNAi experiments will be performed to test the functional effects of these transcription factors on cancer cell differentiation, proliferation, and apoptosis. These are straightforward experiments that should produce some results; however they do not directly test a hypothesis that Nanog, Oct4 or Sox2 affect important cancer stem cell characteristics, such as maintenance of tumor-forming capability in authentic tumor cells. The proposed examination of effects of transcription factor expression in CD44+ CD24- breast cancer stem cells is not supported by an experimental plan. It is not a trivial feat to manipulate transcription factor expression in authentic breast cancer stem cells isolated from human tumor specimens. In general, the research plan for the first aim and the first part of aim 2 is considered strong. This series of studies brings new investigators into the hESC field and collaborations appear to be in place both with the group from the Karolinska Institute and Dr. Martin, a cancer stem cell biologist at the University of California, Berkeley. One reviewer describes the research plan as diffuse and in many cases, lacking in clear deliverables. A number of experimental approaches to be used are barely described, if at all, although the topics of pluripotency and differentiation of ES cells into a variety of cell types and of cancer are considered of significant interest. STRENGTHS: Numerous strengths of this application are identified by the reviewers. This is a senior and well-established PI, a pioneer in the field of transcriptional regulation. The collaboration established with hESC investigators, Dr. Ericson and Dr. Perlmann, are also considered strengths providing mutual benefit. Thus, the research team is considered excellent. An additional strength is the original hypothesis and novel description of a potentially important stem cell co-factor. The project entails a comprehensive analysis of transcriptional regulation by multiple methodologies in hESCs and several differentiation lineages in experiments for which the laboratory has expertise. The plan is supported by strong and relevant preliminary data, particularly for aims 1 and 2, demonstrating a likelihood of being able to achieve these goals. The DA neuron component of the project seems particularly well thought out, topical, and likely to generate results of interest. A potentially important, novel SCC protein complex has been purified. Examination of Lmx1a-mediated differentiation of hESC into DA is supported by good preliminary data from differentiation of mouse and human ESC. These data contribute to a solid rationale for identifying Lmx1a interacting proteins and downstream target genes. The topic areas are relevant to CIRM and only a subset of the work could be funded by the NIH. WEAKNESSES: One reviewer describes this proposal as diffuse, seemingly to fund many projects that already exist in the PI's laboratory and are ongoing, rather than a distinct research proposal. Muscle experiments seem relatively weak compared to DA neuron experiments. Experimental plans for several subaims are not described adequately. In general the proposal is considered somewhat diffuse also due in part to the use of multiple different lines and both mouse and human systems (hESCs, mouse C2C12 cells, and Ntera-2 cells, multiple human breast cancer cell lines, embryonic mouse midbrain tissue). The rationale for the studies in breast cancer cell lines (Aim 3) is based on parallels between ES cell growth and cancer stem cell growth, however this aspect of the overall project is descriptive, considerably weaker than other parts of the proposal, and does not incorporate any hypothesis about the stem cell co-activator (SCC). One weakness was in the presentation of preliminary data. One reviewer comments that the lack of figures supporting Aim 3 presents significant obstacles in assessing the feasibility and impact of that portion of the research plan. Objectives that had no experimental plans or alternatives described adversely affect the scoring of this proposal. Specifically, Aim 2, subtask 3 is not fleshed out, and there is no experimental plan or alternatives. Aim 3 has the same problems, which drove down the reviewer's general enthusiasm. One reviewer suggests that the third aim, which could be funded by other mechanisms, be dropped from the application and that aim 1 and the second part of aim 2 be fleshed out in order to generate a stronger proposal. Other minor weaknesses include some inconsistencies in the research design vs. preliminary data with regard to LMX1A vs. B and the absence of a demonstrated use of specific immunoprecipitation grade antibodies against LMX1A which will be necessary to perform chip assays proposed in Aim 2. Aim 3 does not require CIRM funding per se and could be funded by NIH. DISCUSSION: There is general agreement that while there are strengths to this application, there also are weaknesses. All reviewers agree that the PI is a leading expert in the field of transcriptional regulation and that aims 1 and first part of 2 are strong and well-developed with good preliminary data. However, the second part of aim 2 (myogenic differentiation) and aim 3 (cancer stem cells) are considered weak. One reviewer considers these latter studies barely, if at all, stem cell experiments per se. One reviewer commented s/he was hesitant to support this type of grant writing, but that this is a strong PI who should be encouraged.
Conflicts: 

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