Stem cells are the primitive cells that give rise to the different tissue types in the body. In a way, stem cells are the universal cells from which all cells are derived. Their unlimited proliferation and differentiation potential raises the prospect that stem cells could be used as therapeutical tools offering hope for millions who suffer from debilitating diseases and conditions for which there are limited or no treatments including: neurological disease, cardiovascular disease, autoimmune diseases, diabetes, and osteoporosis. Furthermore, stem cells may serve as diagnostic tools, cancer perhaps being one of the most promising areas. But before these potential applications become a reality, we need to have a better understanding of the mechanisms by which human embryonic stem cells renew themselves indefinitely. Our knowledge of the cellular and molecular mechanisms that control self-renewal and pluripotency of human embryonic stem (hES) cells is rather scarce, and most of the information comes from comparisons with their murine counterparts. Based on published evidence by our group and others and our preliminary results, we hypothesize that the self-renewal of hES cells depends on the establishment of distinct cell populations (compartments). We propose that such cell compartments are specified by a crosstalk between the Notch and Wnt signaling pathways and maintained, at least in part, by regulating the relative activities of p53 and p63 and describe three Specific Aims towards this goal. The successful completion of the scientific program proposed in this application would be of the highest significance both for our understanding of basic stem cell biology and for the implementation of strategies of regenerative medicine. While our understanding of the network of transcription factors associated with ES cell self-renewal has increased significantly in the recent years, very little is known about the cellular bases of ES cell self-renewal. Our preliminary results indicate that the Notch and Wnt signaling pathways play essential roles for hES cell self-renewal. Here we propose to analyze the specific cell behaviors and compartmentalization dynamics within colonies of undifferentiated hES cells controlled by Notch and Wnt signaling in the context of hES cell self-renewal, and which are the downstream mediators of those effects. Uncovering the specific cell behaviors controlled by extrinsic factors in the context of hES cells will enable us to understand, modulate, and perhaps induce the self-renewal of pluripotent cells.The implications might be far reaching for the implementation of strategies in the fields of diagnosis and treatment of human degenerative diseases.
Statement of Benefit to California:
The long term goal of this research grant proposal is to understand the mechanisms by which human embryonic stem cells renew themselves indefinitely. . Uncovering the molecular factors implicated in self renewal and pluripotency will enable us to understand, modulate, and perhaps induce the self-renewal of pluripotent cells.The implications might be far reaching for the implementation of strategies in the fields of diagnosis and treatment of human degenerative diseases. Neurological and cardiovascular disorders, autoimmune diseases, diabetes, cancer and osteoporosis strike no less than 10 million Californians each year, causing an incalculable personal toll and an annual economic cost of billions of dollars in medical expenses and lost productivity. Thus, one benefit that will be derived from this area of research is the generation of specific tools and methods for reducing medical costs and increasing the quality of life and level of productivity of afflicted Californians. A second key benefit derived from this research grant proposal is the training of new scientists to serve as educators and researchers for the future, many in the burgeoning area of stem cell biology for which the State of California has emerged as a world's leader. Finally, the discoveries derived from innovative and multidisciplinary research on hES cells described in this proposal, are likely to lead to important new areas of intellectual property that are essential for creating high quality jobs in the biotechnology and pharmaceutical industries in California.
SYNOPSIS: The PI proposes to study the roles of Notch and Wnt signaling in hESC proliferation and maintenance of markers of pluripotency. The studies are well-motivated, well-designed, and firmly based on the PIs preliminary data. The PI has all the cellular and molecular reagents in hand, or in close proximity. The studies should yield clear answer to the questions that he asks. IMPACT AND SIGNIFICANCE: This proposal will investigate the role of 2 well-characterized developmental signaling pathways (Notch and Wnt) in the self-renewal and differentiation of hES cells. The importance of these pathways in ES cells has been controversial and may differ between mouse and human ES. These studies could potentially clarify this controversy and shed light on basic mechanisms of ES maintenance/pluripotency. This information would be useful in optimizing strategies for ES culture and directed differentiation. In addition, this proposal will generate a battery of hES reporter lines using fluorescent protein expression to indicate Notch or Wnt signaling and to discriminate ES differentiation. These lines will be useful to many investigators in many types of future studies. QUALITY OF THE RESEARCH PLAN: The goal of this research is to understand the role of Notch and Wnt signals in hES biology. In particular, the applicant posits a novel hypothesis that hES self-renewal decisions are influenced by the establishment in hES cultures of distinct cell compartments that segregate differentiated and undifferentiated cells, and that this segregation is modulated by Notch/Wnt. To test this hypothesis, the applicant will generate an extensive battery of genetically-modified hES cells. These include reporters of Notch and Wnt signaling, reporters of differentiation, and lines in which Notch or Wnt signaling can be inducibly regulated by expression of constitutively active or dominant negative components of these signaling pathways. The applicant's preliminary data indicates his abiltiy to culture and transduce/transfect hES, and he has significant prior expertise in the study of these pathways during vertebrate development. He will then assay these lines for their ability to self-renew and differentiate, and to "compartmentalize" in mosaic cultures. An overriding concern in the application is that the proposed readout of "compartmentalization" is unclear - how exactly will this be scored? Also, no preliminary data demonstrating compartmentalization is provided. In addition to this major issue, in several instances, important details regarding the experimental design are lacking. For instance, in Aim 1, which stages of colony formation will be assayed? At what stages will transcriptional arrays be performed? And then in Aim 3, how will some of these targets will be selected for further analysis? (This adds quite a bit to an already ambitious proposal.) Finally, in generating all these reporter lines, it will be essential to confirm that selection does not alter the properties of the cells. Although they are not described, karyotyping, and controlled in vitro and in vivo pluripotency assays will be important to help address the concern that any observed differences among lines is not a reflection of additional changes/mutations introduced by selection. STRENGTHS: The proposal addresses a potentially important and controversial issue relating to pluripotency of hESC and will generate many useful reagent cell lines. The proposal could identify novel methods for promoting hESC self-renewal or directing their differentiation. The investigator is productive with expertise in developmental embryology and developmental signaling pathways, has recruited collaborators with expertise in hES derivation and culture, and has a sound plan for interaction with them. It is highly likely that Wnt and Notch are involved in some manner in pluripotency and the proposal has strong preliminary data. WEAKNESSES: This is not a terribly ambitious proposal, given the scope of the problem. Unless one of these signal transduction pathways generates a home run (which seems unlikely), then the field will not be so very far further along. Although it is hard to fault someone for a focused application, given the skill level of the lab and the PI, some more exploratory experiments would have added enthusiasm. DISCUSSION: The hypothesis that colonies have compartments is unproven and unclear. There is no data about compartmentalization and no clear definition of it. The proposal is very focused on one pathway and is "myopic" about others. Although the experiments are feasible and the pathway is likely to be important, the proposal is not very original. A more ambitious proposal would have generated more enthusiasm.