Basic Biology III
$1 371 600
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.
Project Synopsis: The goal of the proposed research is to elucidate the role of a specific member of the Forkhead family of transcription factors (FOX) in the establishment and maintenance of the pluripotent state. To achieve these ends, a series of three aims has been described. In the first, the applicant will employ knockdown approaches to determine whether certain core factors directly regulate the expression of the FOX protein in human embryonic stem cells (hESC). Aim 2 will focus on the identification of transcriptional targets for this FOX protein in hESCs using high throughput methodologies. In Aim 3, the applicant will explore a potential role for the FOX factor in regulating autophagy. Significance and Innovation: - The project is focused on elucidating the molecular basis of pluripotency. If successful, the insights obtained could lead towards improved methods for expanding and maintaining hESC, a prerequisite for securing the large numbers of cells that would be required for transplantation or other therapeutic applications. - The project is of limited innovation and is unlikely to have major impact. While the focus of Aim 3 represents a novel and potentially interesting avenue of investigation, the rationale for the importance of autophagy to pluripotent stem cell biology has not been convincingly established. Other project goals make use of standard methodologies and are unlikely to reveal insights that would be transformative. Feasibility and Experimental Design: - The research plan is extremely well written, logically constructed and based on strong preliminary data. Timelines are realistic, and potential pitfalls and alternative solutions have been described. - The proposed experiments are technically achievable, making use of approaches that are standard in the field and well within the capabilities of the applicant team. - Results from Aim 1 might be difficult to interpret. Knockdown of core regulatory factors will lead to differentiation and concomitant decrease in expression of the FOX factor. In addition, it is not clear that the FOX promoter would be fully included in the DNA fragment to be used in the reporter construct. Preliminary data showing unequivocal evidence of promoter identity would have been helpful. - The efficiency of siRNA transfection and knockdown can vary tremendously and has the potential to confound resulting data and their interpretation, particularly given the transient state of many early fate decisions. Use of internal controls and stably transfected loss-of-function constructs is highly recommended. - The experimental plans lacked details on the time frames to be analyzed for several experiments. Reviewers suggested that analyses on the scale of hours, as opposed to days, would be more informative. - Aim 3 does not fully consider the complexity of autophagy and its various forms, and it is not clear that the proposed approach will be informative. Nonetheless, reviewers considered the use of electron microscopy to be a strength, although proper fixing techniques have not been demonstrated. Principal Investigator (PI) and Research Team: - The PI is an acknowledged leader in the field of pluripotent stem cell biology and has an extensive track record with numerous publications in top tier journals. - The research team has all of the appropriate expertise to conduct the proposed research. - The PI has a 20% commitment and the budget is appropriate. Responsiveness to the RFA: - The proposed research utilizes hESC and directly addresses molecular mechanisms of pluripotency and stem cell identity. The proposal adequately and appropriately addresses the goals and objectives of the RFA.
- This application scored below the initial scientific merit funding line, no programmatic reason to fund the application was proposed, and the GWG voted to place the application in Tier 3, Not Recommended for Funding.