Basic Biology I
Human embryonic stem cells (hESCs) are capable of unlimited reproduction and retain the ability to differentiate into all cell types in the human body. Therefore, hESCs hold great promise for human cell and tissue replacement therapy. However, our knowledge on how to grow hESCs and how to differentiate them into desired cell types for therapy remains limited. The overall goal of this proposal is to address this lack of knowledge to improve the feasibility of large production of hESCs and routine derivation of therapeutically valuable cells from hESCs. We propose to establish a systems biology approach, which will be continuously optimized with our experimental data, to provide intelligent guidance on how to enable the robust growth of hESCs without inducing differentiation as well as on how to differentiate hESCs into various cell lineages for therapy. The combination of the proposed bioinformatics and experimental approaches will provide a unique opportunity to address the needs for hESC-based replacement therapy.
Statement of Benefit to California:
Human embryonic stem cells (hESCs) are capable of unlimited self-renewal and retain the ability to differentiate into all cell types in the human body. Therefore, hESCs hold great promise for human cell and tissue replacement therapy. However, due to our limited knowledge of the mechanism underlying the self-renewal and lineage-specific differentiation, it becomes increasingly urgent that more effort must be made to address these knowledge bottlenecks. Our overall goal is to establish a systems biology approach to provide intelligent guidance for our experimental effort to elucidate the mechanisms underlying the self-renewal and lineage-specific differentiation. Achieving this goal will significantly improve our capacity for large scale production of hESCs and reliable differentiation of these cells into therapeutically useful cell types. Therefore, the proposed research will benefit California citizens by contributing to the eventual realization of the therapeutic potential of hESCs.
This proposal has two main goals 1) to understand transcriptional pluripotency networks and 2) to develop a computational model that will ultimately enable rational design of methods to drive pluripotent cells to specific fates. Aim 1 will attempt to identify targets of key pluripotency factors Oct4, Nanog and FoxD3, and determine their roles in pluripotency maintenance by modulating their expression. Aim 2 seeks to identify cofactors that bind proximal promoters the above targets and investigate their roles by modulating expression. Aim 3 will attempt to develop a computational model to predict the impact of perturbing hESC transcriptional networks. Reviewers universally agreed that a systems understanding of how the currently identified network of transcription factors maintains self-renewal and pluripotency of hESCs would have a major impact on the stem cell field. The approach to investigate FoxD3 and other factors in the proposed screen should yield interesting data. Preliminary data demonstrating the model’s prediction of Oct4 knockdown mediated transcriptional changes were intriguing and would benefit from peer review. However, the proposal was overly ambitious and its rationale contained serious logical flaws. Specific Aims 1 and 2 are extremely time consuming and the initiation of key experiments depends upon the generation of new data, rendering the timeline vulnerable to substantial delays. The proposal lacks discussion of how feedback between pluripotency regulators would be discovered and modeled. Extensive concern was expressed regarding Specific Aim 3, the generation of the predictive model. A more complete description of preliminary results and mathematics related to the model would benefit the proposal. Reviewers were skeptical that a model built using data derived solely from manipulation of pluripotency regulators would achieve the proposal’s goal of developing rational methods to direct stem cells toward specific fates. This left reviewers uncertain that the applicants have considered carefully whether the proposed experiments address the questions they seek to answer. The selection of methods also raised concern. Disruption of pluripotency will result in heterogeneous responses such as differentiation to multiple cell types over time. Interpretation of the resultant data will be difficult using population based, rather than single cell based, assays. Finally, one reviewer felt that some of the model’s predictions presented in the preliminary data were not sufficiently accurate to provide meaningful results. The investigators should substantiate feasibility of their approach by performing a simulation to see if the level of error observed would impact the model’s ability to predict differentiation. The PI is talented, has a solid publication record and the team is well qualified to perform the described experiments. The host institution and environment are excellent. In summary, this is an ambitious proposal addressing a complex topic. Preliminary data requires further development, and methods should be better explained. Most critical though, reviewers were not convinced that completion of the proposed studies would achieve the goals of the project.