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Deciphering intracellular signaling mechanisms in initial differentiation of hESC

Funding Type: 
Basic Biology I
Grant Number: 
Funds requested: 
$1 380 818
Funding Recommendations: 
Not recommended
Grant approved: 
Public Abstract: 
It has been widely appreciated that human embryonic stem cells and somatic stem cells are expected to be sources of cells to regenerate or rejuvenate damaged tissues, yet the research work on human embryonic stem cells has been hindered by the difficulties in manipulating their self-renewal versus differentiation properties in vitro. This is largely due to lack of understanding of the molecular and cellular basis for maintenance of human embryonic stem cell pluripotency, the ability to duplicate itself and also give rise to other types of cells with specialized functions such as blood cells, neural cells in the brain and skeletal muscle cells. Like many other cell types, the functions of human embryonic stem cells are regulated by extracellular signals. A unique intracellular signal transduction mechanism can interpret various extracellular signals, guiding the cell to adjust its activities for adaptation to the changes in the environment. One important biochemical mechanism for signal transduction is reversible phosphorylation on proteins, in which a phosphate can be added or removed from proteins. This reversible process is facilitated by two groups of enzymes, i.e. kinases that can add a phosphate on a protein and phosphatases that remove a phosphate from a protein. The applicant discovered a phosphatase that operates in the initial step of intracellular signal transduction more than a decade ago, published in Science, 1993. Notably, his group has found that removal of this phosphatase promotes mouse embryonic stem cell self-renewal. More recently, the applicant has also observed that this phosphatase has a similar function in human embryonic stem cells. On this project, the applicant and his colleagues will use their expertise on stem cells to investigate and illuminate the biological properties of human embryonic stem cells. Based on the database accumulated from other research groups, this laboratory will do experiments and develop a better set of biological markers for identification of human embryonic stem cells. Finally, the applicant and his colleagues will develop new reagents that can be used to amplify the human embryonic stem cells without changing its property in culture for basic research and clinical application.
Statement of Benefit to California: 
This applicant must say at the outset that I am very proud of being an American, in particular a Californian. Californians have the tradition of stimulating an entrepreneurial spirit, thus making the state ahead of others to launch novel and risky ventures, particularly in the biotechnology field, and in the stem cell research. In the 21st century, there is no doubt that the research work on human embryonic stem cells will take the lead in the advancement of biomedicine and biotechnology. Progress in this type of research will benefit people suffering from many different types of diseases such as cancer, neurodegenerative diseases and cardiovascular disorders. On the proposed project, the applicant and his colleagues will take advantage of decade-long research experience in molecular and cell biology, particularly on mouse and human embryonic stem cells. We will carefully investigate the human embryonic stem cell properties, to understand better how these cells can duplicate themselves and also produce other cell types with specialized functions, such as blood cells, neural cells and skeletal muscle cells. This information will be instrumental for future research and application of human embryonic stem cells in and outside of California. Based on experimental results from this and other laboratories, we will be able to develop a better set of biological markers for identification of human embryonic stem cells. Finally and most importantly, we will design and produce new reagents that can be widely used in culturing and amplification of human embryonic stem cells for basic research and clinical application. All this will benefit tremendously Californians and people in this country, and in the world.
Review Summary: 
This goal of this proposal is to elucidate the mechanisms by which a specific phosphatase molecule coordinates the actions of multiple signaling pathways in order to promote differentiation in human embryonic stem cells (hESC). In the first Aim, the applicants will use various approaches to down-regulate the expression of this phosphatase and determine the effects on relevant signaling pathways as well as on certain aspects of stem cell behavior and function. As a secondary goal, they propose to identify substrates that are targeted by this molecule. For the second aim, the applicants will explore the role of a specific histone acetyltransferase in the epigenetic control of hESC differentiation and determine how this activity is coordinated with that of the relevant phosphatase. Finally, the applicants propose to screen for and optimize chemicals that modulate the activity of the phosphatase under investigation. Candidate reagents will be evaluated for their ability to support hESC propagation in an undifferentiated, pluripotent state. In general, the reviewers acknowledged that this application offers an innovative angle for addressing key issues in stem cell biology. However, the described approaches were criticized for being standard, broadly based, and lacking in critical details. As a result, the reviewers feared that the overall impact would be marginal. One reviewer indicated that all three aims lacked compelling rationale and seemed at odds with the intent of the preliminary data. For example, while this effort focused on the mechanisms by which the relevant phosphatase contributes to hESC biology, the preliminary studies stressed the contrasting activities of this molecule when comparing the mouse to the human context. Thus, it was not entirely clear how premises derived from the mouse work were translated to relevant, testable hypotheses in the human system. In essence, this proposal was viewed as an investigation into the mechanism of a specific phosphatase rather than a study geared towards addressing critical questions in stem cell biology. This notion was underscored by the reviewers’ apprehension that the relevant molecule may only contribute indirectly or even tangentially to such processes. The reviewers expressed significant concerns about the feasibility of this proposal, which was somewhat difficult to evaluate based on the superficial descriptions provided by the applicants. Importantly, several aims lacked a clear working hypothesis or sense of prioritization. For example, Aim 1b offered a list of potential phosphatase targets, several of which are controversial, but an unsatisfying framework for how these would be strategically or systematically evaluated. Similar problems were noted for Aim 1c, which focused on upstream signaling pathways but offered no specific insights as to how their functional significance would be determined. Although the premise of Aim 2 was clearer, some reviewers felt that it was based on an assumption that was not adequately supported by the preliminary data, i.e. that a deficiency of the histone acetyltransferase activity would alter acetylation patterns when the level of a cofactor remain unchanged. One reviewer indicated that this rationale would have been more compelling if the applicants had demonstrated that the cofactor levels are insufficient for maintaining the expected epigenetic phenotype. Aim 3 was thought to be the strongest, but the applicants neglected to articulate or justify many of the details surrounding the nature of the assays to be conducted, whether any counter-screens against other phosphatases would be utilized, or the number of hits that would be needed to ensure that the desired specificity could be obtained. Further concerns were raised about Aim 3b, where the active concentration of the compound to be optimized was judged to be unsuitably high and almost certain to render off target effects. The reviewers would also have appreciated a more compelling rationale for why the proposed cocktail of inhibitors in Aim 3c might prove more effective for maintaining hESC pluripotency than use of competitive antagonists. Finally, the applicant’s general discussion of pitfalls and alternative approaches was found to be lacking. The reviewers described the principal investigator as one of the pioneers of relevant phosphatase biology with the expertise and qualifications to conduct the proposed research. The collaborators and staff were also thought to be highly qualified, although one reviewer perceived some naïveté in the area of medicinal chemistry based on experiments proposed in Aim 3b. Overall, this application represents a somewhat unique angle on how standard approaches could be used to address fundamental questions in stem cell biology. However, despite a strong research team, the lack of compelling rationale and paucity of experimental details left the reviewers with little hope that the field would be substantially impacted.
  • Ali Brivanlou

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