Tools and Technologies I
Embryonic stem (ES) cells not only hold considerable promise for the treatment of a number of devastating diseases (e.g. cardiovascular diseases, neurodegenerative diseases, diabetes and cancers), they also provide an ideal tractable culture system for studying early embryonic development of all cell types and modeling human diseases. Harnessing these potentials of ESCs will require an improved ability to manipulate their self-renewal and differentiation, and a better understanding of the signaling pathways that control their fate. To address the critical challenges in hESC culture (i.e. robust self-renewal and single cell survival in chemically defined conditions), we propose to (1) optimize two previously identified and functionally characterized novel small molecules (from high throughput screens in hESCs) via medicinal chemistry for much improved self-renewal and survival of hESCs, and screen additional 85,000 diverse compounds to identify new small molecules with different mechanisms of action that can maintain self-renewal of hESCs in the absence of growth factors, or promote single cell survival of hESCs under the chemically defined media. We will further confirm and characterize their effects and activities via various in-depth cellular/biochemical assays, and carry out structure-activity-relationship (SAR) studies of the selected hit compounds (that especially act on different targets/mechanisms as compared to the two molecules in Aim 1) to optimize their potency and specificity. Collectively, the studies described in this proposal will provide novel chemical tools for better understanding and controlling hESC, and may ultimately allow development of therapeutics employing hESCs for treating diseases.
Statement of Benefit to California:
The proposed studies will provide new chemical tools and technologies and substantial knowledge for better understanding and controlling hESC self-renewal and differentiation (e.g. development of robust self-renewal and clonal expansion conditions for hESCs) , and may ultimately allow development of therapeutics employing hESCs for treating diseases.
There is a need to identify defined conditions in which human embryonic stem cells (hESC) can survive, self-renew and clonally expand. The applicant describes defined conditions as culture conditions not requiring feeder layers, serum, and growth factors. The purpose of this study is to further optimize two small molecules, previously discovered by the principal investigator (PI), that promote hESC self-renewal under chemically defined conditions or single cell survival, respectively (aim 1). In addition, the PI seeks to identify new small molecules with similar activities by high throughput screening (HTS) of 85,000 compounds (aim 2). The newly identified compounds will then be further optimized utilizing structure-function relationships to potentially enhance their biological activity and further characterized to discover their mechanism of action (aim 3). The goal is to focus on molecules that act on different targets than the two molecules already identified. The development of chemically defined media components that support the self-renewal and survival of hESCs is an important area of research and will be crucial for clinical development of stem cell technology. If successful, the applicant will identify novel small synthetic molecules that support hESC proliferation in a chemically defined medium. However, a reviewer noted that much of the activity seems to duplicate previous efforts towards a similar goal. Thus, the impact of this particular series of studies does not stand out significantly from previous work, but could aid in the development of hESC culture media reagents. Reviewers differed in their overall assessment of this application. Some felt that it presents a well-articulated research plan, proposing a series of well-crafted experiments based on solid preliminary data. The lack of discussion of potential limitations or alternative approaches was noted as a weakness of the proposal. Reviewers raised specific concerns regarding each aim. Although optimization of the two already identified compounds in aim 1 was considered feasible, it remained unclear how the experimental design will permit reaching the specified target objective, a 10-fold improvement in potency. Furthermore, a reviewer would have liked to see an analysis of in vitro properties of the compounds such as solubility and metabolic stability. In light of already identified compounds, some reviewers questioned the need for pursuing additional molecules with similar activities in aim 2, and others suggested that combinations of compounds, rather than single molecules, will likely be most effective in supporting hESC cultures. The proposed screen was judged to be feasible given that the assays have already been run previously, but it remained unclear what criteria will be used to prioritize compounds. Although reviewers appreciated that the PI intends to thoroughly characterize the identified compounds in aim 3, they expressed concern that this aim is too ambitious and its feasibility would have benefited by focusing on more specific key characterization metrics. Reviewers observed that the applicant proposes to use a non-federally approved hESC line because it has better properties and is easier to work with than the federally approved lines. The PI is a recognized leader in the field of chemical biology with an outstanding publication record in top journals, and has discovered and characterized numerous small molecules capable of influencing stem cell self-renewal and differentiation. The applicant is well funded and has significant laboratory space and appropriate equipment at his/her disposal, and has committed sufficient amount of his/her own time and lab personnel to this project. The budget was considered appropriate except for seemingly excessive travel costs. In conclusion, reviewers felt that this proposal addresses an important area of stem cell research with relevance to clinical development. In support of the feasibility of the project, reviewers cited prior work and preliminary data, and the PI’s status as an accomplished chemical biologist. However, concerns raised about experimental details of each aim reduced enthusiasm for this proposal, culminating in its Tier 2 placement. PROGRAMMATIC REVIEW A motion was made to recommend that this application be moved to Tier 1 – Recommended for Funding. Panelists emphasized that the applicant is one of the best academic chemists working in the stem cell field, and that serious high throughput screening, as proposed here, is of strategic value for CIRM. Panelists expressed unease about moving an application with this relatively low score into the funding range, and reiterated that the concerns raised about this application included the overly ambitious scope of aim 3, an aim critical for the potential impact of this proposal, and the dearth of combinatorial chemistry approaches. Furthermore, questions were raised regarding the PI’s workload. The applicant is relatively junior and may be over-committed. The motion to move this application to Tier 1 carried.