Early Predictors of Cell Fate Commitment by Embryonic Stem Cells: Cell Identification through Signaling Network Analysis in hESC Differentiation

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

The embryonic stem cell (ES cell) holds tremendous potential as a therapeutic cell source for repairing or replacing deceased tissues or organs. The current methodology based on the quantification of changes in gene expression are not sensitive enough to detect cells undergoing cell type switching. Therefore, current technology does not allow early detection of changes in cell type identity. The proposed study will allow us to develop a new approach to predict future cell type identity before ES cells have taken on permanent cell identity. If the goal of the proposed study is accomplished, we will have a rapid screening method for staging and identifying ES cells that are in transition from stem cell to differentiated cell types.

Statement of Benefit to California (provided by applicant)

The proposed study will allow us to develop new methodology in characterizing embryonic stem cells and their derivatives with special metabolic functions. If successful, it will greatly enhance the way that we phenotype cells for transplantation in terms of time-saving and accuracy. Patients who receive stem cell therapy will have a better assurance of purity and differentiation stages of transplanted cells. Future commercialization of the methodology will potentially create tax revenue for the State and employment opportunities for its citizens.

Review Summary

This proposal focuses on the development of a novel technique for phenotypic identification of human embryonic stem cells (hESCs) and embryoid bodies in various stages of differentiation. The applicant proposes to stimulate cells with ligands and monitor their responses with a panel of antibodies to specific epitopes in signaling cascades. The goal is to design tailored antibody arrays to recognize discrete response signatures in specific cell types. The applicant also proposes to identify important signaling nodes in these signatures that could be targeted to induce stem cell differentiation. Finally, the applicant plans to specifically profile retinal pigment epithelial cells and osteoblasts and compare their response signatures in vitro and in vivo.

The reviewers agreed that while the overall goal of this proposal is an important one, the rationale for this particular approach is poor. They commented that the proposal is poorly written, badly organized, and lacks sufficient experimental detail. The applicant is well qualified but has not published stem cell research and may be overcommitted. In addition, the budget is excessive for the scope of the experiments.

Methods for rapidly identifying hESCs that are in transition to differentiated cell types could be important for the stem cell field. However, reviewers questioned whether this proposal’s approach, using a panel of antibodies to comparing signaling responses to ligands, would lead to this endpoint. Understanding the early molecular signals that occur in response to differentiation signals in hESCs could potentially allow directed manipulation of these cells down a desired lineage, but again using antibodies to examine protein extracts at different stages of maturation would not be sufficient to reach this level of understanding of the molecular mechanisms underlying differentiation.

Reviewers questioned the feasibility of this proposal and raised a number of questions about the research design. The reviewers agreed that the antibody profiling described in the first aim would generate a large amount of data, but that it wasn’t at all clear how these data would lead to meaningful conclusions. They would have liked more detail about how results would be evaluated and interpreted. One reviewer directly questioned the feasibility of the approach and was not confident that response signatures found in heterogeneous cell populations would be distinct enough to allow for identification of individual cell types. Another reviewer asked why the applicant chose to examine the activity of the signaling node kinases in differentiated cells, instead of using established cell phenotype-specific markers.

While reviewers felt that the principal investigator (PI) is generally qualified to perform this type of research, one raised concern about the PI’s lack of stem cell publications. Another reviewer noted that 95% of the PI’s time is already committed to other projects, and there is only one other research scientist and a to-be-determined postdoctoral fellow devoted to this project. Reviewers commented that the budget costs seem excessive, given the scope of the project and the small number of staff.

Overall, reviewers found this proposal to be poorly organized and strongly questioned the impact and feasibility of its approach.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• None