Basic Biology II
Despite the enormous potential for human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) for development of new treatments for human disease, there still remain important gaps in our knowledge about the molecular mechanisms regulating establishment and maintenance of the pluripotent state. Improved understanding of fundamental mechanisms regulating pluripotency could improve the ability to establish pluripotent stem cells, in understanding how to maintain them in the undifferentiated state and how to differentiate them into specific cell lineages. The research proposed here seeks to provide a fundamentally better understanding of pluripotency and how it is controlled in hES cells and closely related iPCs. Maintenance of stem cells is known to be controlled by a group of core proteins that keep them in an undifferentiated state. When these proteins are downregulated they undergo differentiation into specialized cell types. Little is known about how the master regulatory circuitry is regulated other than feedforward positive interaction between the three core regulatory factors. Here we propose to study another protein that interacts with these core regulatory proteins and that may be a key regulator of their activity. These studies can expand our definition of the core stem cell regulatory circuitry. Through the research proposed here we will obtain a better understanding of the molecular processes at work when pluripotent ES cells decide to commit to lineage specific differentiation. For example, what genes must be turned off or on to achieve differentiation into specific lineages? How do chromatin modifications contribute to this regulation? This could lead to improvements in culture of hES cells and in methods for making iPSCs. A better understanding of these features could help better control these cells for use in regenerative medicine. Because hES cells are derived from the human embryo, these studies will also contribute important insights into human embryonic pre-implantation development.
Statement of Benefit to California:
A primary goal of Proposition 71 is to translate basic stem cell research to clinical applications. The disability and loss of earning power and personal freedom resulting from a disease or disorder are devastating and create a financial burden for California in addition to the suffering caused to patients and their families. Therapies using human embryonic stem cells (hESCs) and the related induced pluripotent stem cells (hiPSCs) have the potential to change millions of lives. Using hESCs and hiPSCs as models of disease will help us understand the underlying causes of disease and likely aid in the development of drugs to treat those diseases. However, for the potential of these cells to be realized, we need a better understanding of how they can be grown and what factors regulate the growth and self-renewal of the stem cell population. Maintenance of stem cells in an undifferentiated state is still problematical and long term growth of stem cells can be associated with appearance of genetic alterations some of which have previously been associated with cancer development. Moreover, understanding the mechanisms regulating stem cell growth will be important not only in maintaining stem cells but also in understanding how to drive their differentiation into more specialized cells. Finally, understanding the factors that support stem cell growth will be important for understanding the risks of transplanting stem cells and their differentiated derivatives into patients. Therefore, the raison d’etre for the proposed research is to provide a fundamentally better understanding of how hESCs and hiPSCs grow and self-renew. Anticipated benefits of our research to the Citizens of California include: 1. Development of improved methods for growing pluripotent stem cells and developing new cell-based treatments for a variety of diseases and disorders. 2. Development of improved understanding the risks of transplantation of stem cell-derived cells into patients and therefore improving the safety of stem cell-based transplantation. 4. Improved methods for understanding normal development of the early embryo 6. Transfer of new technologies and intellectual property to the public realm with resulting IP revenues coming into the state 7. Creation of new biotechnology spin-off companies based on generated intellectual property 8. Creation of new jobs in the biotechnology sector. It is anticipated that, in the long term, the return to the State in terms of revenue, health benefits for its Citizens and job creation will be significant.
EXECUTIVE SUMMARY The overall goal of this proposal is to elucidate the function of the Wnt pathway effector, T cell factor-3 (TCF3), in human embryonic stem cells (hESCs). Recent studies suggest that TCF3 represses expression of key pluripotency factors and becomes down regulated rapidly as ESCs differentiate. The applicant proposes to build on these investigations by exploring potential mechanisms by which these events lead to an exit from the pluripotent state. In the first Aim, tools will be developed and employed to define the molecular mechanisms responsible for TCF3 down regulation during differentiation of hESCs. For Aim 2, the applicant will investigate the function of TCF3 in the context of the larger stem cell regulatory network. Reviewers agreed that this application addresses an important need to understand the regulatory circuits that control hESC pluripotency and cell-specific differentiation. While some did not find the underlying hypothesis particularly novel, they appreciated the mechanistic focus and found the diverse, integrated research strategy to be creative. Reviewers believed that if successful, the knowledge gained from this effort could lead to improved methods for propagating undifferentiated hESCs as well as more efficient means of generating induced pluripotent stem cells (iPSC). Some reviewers questioned the impact of this outcome, given that it is already possible to routinely and reproducibly generate iPSCs in labs around the world. Others noted that there is no guarantee that such improved methods would circumvent the need for retroviral transduction of pluripotency factors. Despite these caveats, most were convinced that the mechanistic insights to be gained from this study would be sufficiently interesting to move the field forward. The reviewers found the research plan to be well written and organized with a clear discussion of objectives and timelines. The preliminary data were compelling, both supporting the underlying rationale as well as demonstrating the core competencies of the team. While most of the needed tools were in place, some reviewers worried about the dependence of certain experiments on antisera and constructs that have yet to be generated or validated. They would have appreciated a more detailed discussion of alternative plans should these primary strategies not come to fruition. In addition, a number of minor technical details regarding the experimental design were omitted, such as the number of modifications to be tested by mutational analysis, and the criteria by which cell lines would be selected for teratoma studies. While these weaknesses were noted, reviewers believed that such deficiencies could be overcome, and that this project would be successful. The reviewers agreed that the principal investigator (PI) is an established, highly regarded cancer biologist with a strong track record in studying the Wnt/TCF pathway. While his/her experience with iPSCs/pluripotency is limited, the comprehensive and complementary expertise of the collaborating investigators spoke strongly to the capabilities of this team. The inclusion of a Ph.D. student who was instrumental in generating preliminary data for effort was a notable asset. One reviewer felt that the budget did not adequately justify the purchase of generic lab equipment that appears to already be available to these investigators. In summary, this proposal addresses a significant and fundamental question in stem cell biology. Despite some minor technical concerns, the strong research plan and excellent qualifications of the team convinced the reviewers of its potential to succeed.