$2 509 438
Statement of Benefit to California:
SYNOPSIS OF PROPOSAL: The application, by a senior scientist, is focused on the molecular mechanisms regulating hES survival. The application has very specific targets of investigation: to determine the role of Oct-4, sox2 and nanog in regulation of TRKs, and to determine whether TRKs mediate hES survival via PTEN and forkhead pathways. In the latter area of investigation, a broader approach is taken to identify hES survival factors, and the approaches are accompanied by a pledge to make the resulting reagents generally available. Another area of investigation focuses on the use of siRNA screening to identify kinases that have survival functions in hES. Finally, the applicant proposes to use gene traps to identify genes that are activated by NTs and mediate NT survival function in hES. The grant is clearly written and the studies are feasible, as supported by preliminary data and the experience of the assembled research team. IMPACT AND SIGNIFICANCE: The PI proposes to study the effects of neurotrophins (NT) on hESC survival, and has recently identified particular neurotrophins as survival factors. The PI has long studied germ cells and other testicular cell growth. In recent years, he has studied the role of oncostatin in nociceptive function of ganglia. He claims to have shown that NT markedly inreases survival of isolated hESC cells, reducing apoptosis and doubling time, as well as chromosomal abnromalities. The PI has the ability and a plan to drill down on this observation to determine the molecular underpinnings of hES survival, and this information will be important to translational therapies based on hES. The PI also proposes to develop general tools that will help the community of hES biologists (particular lines of general utility, methods). QUALITY OF THE RESEARCH PLAN: The quality of the research plan is high; this is a very well written application addressing an important problem: the mechanism of NT effects on hESCs. Sophisticated and elegant methods will be used, including studies of single cells (to remove indirect effects from other cells) and an elegant gene trap method to identify transcriptional activation of genes required for survival of hESCs. The plans are outlined in a manner that makes it apparent that the timetable is feasible for producing the proposed work and results. STRENGTHS: A major strength is that the work is a natural continuation of recent important studies in the lab. The PI is addressing a fundamental issue (survival mechanisms) both through focused pathway studies and high throughput screens. The application is well written, the techniques proposed are elegant, and the PI has proposed rigorous, feasible experiments that are very likely to yield important insights into regulators of hESC survival and growth. WEAKNESSES: There are few weaknesses in this proposal. Since the method of passaging has more than practical consequences (trypsinization may be changing the fundamental biology of the cells) it would be a welcome addition to the proposal to look at the effects of trypsin on the pathways of interest. But this is a minor point. DISCUSSION: This proposal is head and shoulders above the others focused on this important and understudied topic. This is great research proposed by a great lab with great preliminary data, and they will generate fundamental data on ESC biology. Three of the four aims proposed are relevant to the observation the neurotrophins are important for survival. The high-throughput screening objective was regarded by some discussants as off the mark, but one reviewer felt this was appropriate since siRNA works well in hESCs. The cell lines proposed in aim 4 may be of great value to the research community, and would better be managed in a central core facility, rather than by relying on individual postdoctoral fellows to maintain and characterize. One reviewer suggested that this would be a significant financial burden. Large-scale gene targeting of hESC seems premature without some preliminary results suggesting feasibility, which is not supported by previously published studies. The PI did not really address the fact that the methods of passaging the cells may have an effect on neurotrophin pathways. The proposed gene trapping method was discussed and regarded as elegant but complex. Discussants agreed that the efficiency of this technique is likely to be low in hESCs. One of the reviewers was very supportive of the proposed single cell studies since they are likely to remove any consideration of paracrine effects. Another reviewer was not so enthused by this proposal. While it was agreed that new survival factors were needed, the reviewer felt that the work proposed deals with known components of the TRK pathway, including the receptors, ligands, kinases and transcription factors. Are these pathway components working similarly in hESC? If so, there is little new information to be gained. The functional genomics and gene trapping are good approaches, but were regarded as rather simplistic. These qualities make the proposal straightforward and somewhat less exciting, but if the pathway does function at a high level to support survival, then this is important work. Overall, the proposed studies are considered to be important, feasible and a nice extension of the previous work of the lab.