Funding opportunities

Mechanisms underlying the diverse functions of STAT3 in pluripotent stem cell fate regulation

Funding Type: 
New Faculty I
Grant Number: 
RN1-00580
Funds requested: 
$2 419 403
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Embryonic stem (ES) cells are derived from very early embryos. ES cells can be maintained in culture indefinitely while retain the ability to make any type of cell in the body. These properties make ES cells a very powerful tool to address basic biology questions. ES cells also offer an important renewable resource for future cell replacement therapies for many diseases such as Parkinson’s disease, spinal cord injury, etc. However, before the full potential of ES cells can be exploited in the clinic, we need to understand more about their biological properties so that we can control their fate towards either self-renewal or differentiation into a specific cell type required for cell replacement therapy. STAT3 is a major player in controlling the fates of a variety of cell types including ES cells. Recently we demonstrated that STAT3 has diverse and distinct roles in regulating cell fate in both mouse and human ES cells. In mouse ES cells, STAT3 is involved in cell adhesion, cell growth/survival and maintenance of self-renewal. Interestingly, STAT3 seems to have opposite roles in human ES cells. It induces growth arrest and differentiation of human ES cells. Why does the same factor play such diverse and contradictory roles between these very similar cells? The answer may lie on how STAT3 is in action. STAT3 is present in every type of cell. It contains six distinct functional regions. STAT3 can directly induce the expression of many genes. STAT3 can also cooperate with other proteins to regulate gene expression. We recently derived STAT3-/- ES cells in which the STAT3 gene was removed. These cells will provide us a powerful tool to dissect STAT3 function. We will first determine the role of each of its six functional regions. Then we will try to understand why they function differently. Is it because they induce different sets of genes, or because they cooperate with different partners? Understanding how STAT3 works is important for us to control the fate of ES cells, and for their eventual clinical application.
Statement of Benefit to California: 
Human embryonic stem (ES) cells can reproduce themselves in a culture dish. They can also give rise to every cell type in the body. In the future, human ES cells may hold the key to replacing cells lost in many devastating diseases such as Parkinsons. Before human ES cells can be used clinically, however, we must learn more about how to control their fate. STAT3 is a key player in regulating ES cell fate. STAT3 is also involved in the pathogenesis of diverse human cancers. In this proposed research, we will use a unique tool developed by us to understand STAT3's function. Our work will lead to a better understanding how human ES cell fate is regulated, which will be an important step towards achieving the therapeutic potential of human ES cells. We also expect that our research will have a great implication in developing effective cancer therapies against novel STAT3 targets identified in this study.
Review Summary: 
SYNOPSIS: The overall goal of the proposed research is to understand how STAT3-mediated signaling regulates diverse and distinct functions in pluripotent mouse embryonic stem (mES) cells, and determine whether STAT3 also plays a role, presumably a different one, in human ES (hES) cells. Unlike mES cells, hES cells do not depend on STAT3 to maintain pluripotency but instead differentiate in response to its activity. Three specific aims are proposed. Aim 1 will define the key/minimum domains of STAT3 required for each of its specific functions in mES cells. A series of point mutations will be introduced into each of the 6 functional domains of STAT3 and their functions tested by introducing them into STAT3 knock-out cells, assessing self renewal, proliferation and differentiation. Aim 2 will characterize the underlying mechanisms of ES cell fate regulation by STAT3. Using the results generated in Aim 1, STAT3-specific binding partners will be identified along with down-stream transcriptional targets. Aim 3 will investigate the function of STAT3 in two types of stem cells, stem cells derived from mouse post-implantation epiblast (mEpiSCs), which, like hES cells, do not require STAT3 signalling for maintenance of pluripotency, and hES cells. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: This is an interesting, well-written, detailed research application to study a molecular pathway that promotes pluripotency in mES cells, but not in post-implantation mouse epiblast cells, nor in hES cells. Importantly, Dr. Ying developed a 3i culture system that enabled the derivation of STAT3 mutant mES cells for the first time. The STAT3 mutant ES cells show reduced substrate attachment, proliferate much more slowly, undergo massive cell death, and differentiate rapidly upon removal of the 3i medium even in the presence of LIF and feeder cells. The PI will test how STAT3 regulates these diverse cell behaviors using structure / function analysis in mES cells and then test whether STAT3 plays a role in hES cells. The innovation comes from the STAT3 mutant mES cell line he developed as a postdoc. Otherwise, the experiments use largely standard techniques. The experiments on hES cells are not clearly defined. Reviewers made a few specific suggestions relating to experimental protocol, such as including controls for level of protein expression or possible problems with folding of mutant proteins in the structure / function experiments of Aim 1, and to refine the timing of the tissue culture / microarray experiments to bias toward identification of direct rather than indirect STAT3 targets in Aim 2. Overall, this is a logical, yet ambitious, set of experiments. The experiments proposed in Aims 1 and 2 would be appropriate for an RO1, but Aim 3 makes the proposal more relevant to CIRM. QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Dr. Ying is clearly a talented scientist. He received his PhD in China, and trained as a postdoc with Austin Smith (1999-2006) in the Institute for Stem Cell Research, University of Edinburgh. His postdoctoral work resulted in the publication of 2 high profile research papers and 3 techniques papers. Dr. Ying is now in the Center for Stem Cell and Regenerative Medicine, USC, as an Assistant Professor in the Department of Cell and Neurobiology. He plans to focus his research career on understanding the molecular circuitry of both mouse and human ES cells that governs the choice between self-renewal and lineage commitment. His work to date on STAT3 signaling typifies these goals. He holds a CIRM-funded grant to study self-renewal of hES cells that started in July, 2007. In his Career Development Plan, Dr. Ying states that supervision of students and postdocs is not only a responsibility but a vital part of his own success in science and for his entire career. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: Dr. Ying has 1200 sq feet of laboratory space in the Center for Stem Cell and Regenerative Medicine at USC, along with access to ample nearby core facilities. DISCUSSION: The discussants judged the applicant to be an accomplished stem cell scientist, who trained with Austin Smith, and can already be regarded as a leader in California stem cell research, making important contributions to the stem cell field. For instance, he has currently an interesting paper under review at Nature. The focus of the proposal to investigate LIF-mediated STAT3 signaling was viewed as an important subject to pursue, but the discussants voiced 2 different opinions about the relevance of this work to hESC. Since hESC differ from mESC in that they do not require STAT3-mediated signaling to maintain their stemness, one panelist questioned the value of studying this pathway in mouse or human cells, anticipating little advance in understanding how stemness is controlled in hESC. However, others felt that it is likely that some STAT3 pathway components are conserved between human and mouse cells and that actually studying the differences between these 2 species is likely to yield important information in general on how stemness is regulated.
Conflicts: 

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