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RC1-00136-1: Human Embryonic Stem Cell Survival and Transformation

Recommendation: Not recommended for funding 

Public Abstract (provided by applicant)

This proposal addresses fundamental questions in human embryonic stem cell (hESC) biology. The main goals of the research are to understand how hESCs remain as stem cells versus undergoing differentiation into different cell lineages such as heart muscle or neuronal brain cells. In addition to these choices, the proposal will examine how these choices are regulated and how we can improve the safety of the hESCs. One concern of hESC research is that prior to providing differentiated cells to patients for treatment, we need to understand how to control their decisions. If these choices are left unchecked, then hESCs have the potential to form tumor-like cells. Research in this proposal will provide new diagnostic tools to determine when hESCs are normal stem cells, differentiated derivatives that are stable, or abnormal cells that may form tumors. Therefore, this proposal will help develop new markers of cell choices and help to better identify the correct type of cell that may be usefull clinically.

Statement of Benefit to California (provided by applicant)

Human Embryonic Stem Cell (hESC) research has the potential to improve knowledge of disease progression, human development, the onset of tumor formation, and cellular therapeutics or what cell types may best be used for patient treatment. There is currently a great need to develop an understanding of the potential of hESCs prior to being used in clinical applications. Research in this proposal will examine how hESCs can be used clinically, only after understanding how to make these cells safe for patients. In this regard, funding this type of research will allow California and its citizens to become a leader in this area. This will benefit California and its citizens greatly by providing first hand knowledge of the clinical safety and relevance of hESC biology to the patients, citizens, clinicians and researchers. Information gained in this proposal could also help develop new partnerships with industry and academia, further promoting the growth of both this research and also promote financial growth for the state of California.

Review

SYNOPSIS: The goal of this proposal is to characterize the pathways important in cell fate by examining factors that determine genetic instability and tumor formation in human embryonic stem cells (hESCs). The first aim is to test the hypothesis that survival signals are propagated through a receptor/signaling pathway, which can be modulated, by additional survival or self-renewal signals in hESCs. The second aim is to test the hypothesis that alterations of survival or self-renewal signals in hESCs will lead to one of the first steps in transformation of stem cells and deregulation of differentiation from hESCs. Conversely, understanding these signals may improve hESC derivation. The third aim is to test the hypothesis that disrupting hESC growth environments will improve our understanding of hESC pluripotency, stability, and reveal potential therapeutic targets for both hESCs and tumor cells.

IMPACT AND SIGNIFICANCE: The questions being addressed in this proposal are vital to our understanding of the growth and sustainability of hESCs: what pathways influence their choice of fate, of self-renewal, differentiation or cell death, which genes influence these choices without inducing genetic transformation. Each aim contains a great idea that deserves study, and is fundamental to progress in hESC biology.

The experiments are innovative and deal with important issues in hESC biology. The general ideas put forward in this proposal have been explored previously with mouse embryonic stem cells and in the cancer field. If some of the experiments succeed, this work will move the field forward and could have some medical implications especially on regulation of uncontrolled growth of grafted hESCs in different pathological conditions.

QUALITY OF THE RESEARCH PLAN: The overall quality of this proposal is good and some of the experiments will produce meaningful results. However, each of the aims, especially as described, could constitute an entire proposal. While the hypotheses are straightforward and the aims reasonable; the Principal Investigator (PI) has packed so many experiments into this proposal that there was not room to elaborate on the experiments in detail. As a result, the outlines of the experiments are confusing at times and hard to follow and connect to the hypotheses. Without a description of specific steps and experimental details, it is hard to evaluate whether a particular experiment is scientifically sound. The reviewers questioned whether the PI will be able to conclude so many experiments on time. One reviewer suggested that perhaps, there should be a focus on aim 1 and 2 or 1 and a modified 3.

The explanations in the “analysis of the results” are not sufficient in some of the aims. Also, the investigator’s intentions if specific ideas or experiments don’t work as planned are not spelled out. Rather, the investigator has listed a series of alternative, lengthy experiments. Also, a few of the experiments and tools proposed in specific aims are not suited to addressing the hypotheses.

STRENGTHS: The PI is qualified and has published interesting work in the field of the hESCs. The PI’s work showing that, in the presence of specific factors, hESCs can be expanded clonally and more efficiently, will help address some of the ideas put forward in this proposal. The collaborators have the tools necessary to do the proposed experiments.

The questions being asked are important. The proposal contains many good ideas, but deserves focus. Also, in aim 3, the aim’s hypothesis and the plans don’t match. Similar issues of focus would help all three aims, which have the kernels of great ideas.

The initial studies proposed for the first aim examine how specific gene products regulate survival and self-renewal of hESCs. This is quite important for the basic and applicative biology of these cells. Subsequent studies on understanding how hESCs differentiate along lineages of different embryonic layers will provide a rational framework for differentiation and expansion of specific cell types. Addressing how hESCs change and acquire malignant characteristics in the second aim is of the outmost importance. The discovery type approaches to research proposed in the third aim are always interesting though lacking mechanistic approaches to experiments.

WEAKNESSES: The research plan is overly ambitious; each of the three aims could be developed into separate grants with greater detail given and more interpretations provided. While the individual experiments are reasonable and feasible, the total package here is not, it is completely too ambitious and currently in the beginning stages. The PI has several years experience with hESCs but has few publications as first/senior author. The project is very immature at this point; the PI is a newly independent investigator.

A major criticism is that there are no times given for the culture experiments. It is possible that monitoring for genetic instability and transformation will take many passages (months and years of continuous culture). Given that no time frames are presented, it is hard to judge the feasibility of the experiments. This is combination with the over-ambitious nature of the proposal (any of the aims alone could constitute a full proposal) tempers enthusiasm for an application with a lot of great ideas.

In the proposed studies for aim 1, the investigator gives a list of genes to be manipulated without explaining why several of these genes were selected. Furthermore, the types of analysis to be performed and the exact end-points of analysis don’t seem to be decided. The experimental procedures including type of culture conditions, timing of specific steps, etc. are missing, thus making difficult to evaluate whether a specific experiment will yield results that can be interpreted clearly. The investigator needs to spell out some of these details. In aim 1, “design part” 2, again no details are given.

Survival factor signaling is complicated and controversial. The tools used in the preliminary result are not sufficient, nor are eliminating or overexpressing receptors or some of the putative down stream signaling molecules. There are some tools out there that could be quite useful in elucidating the role of the different receptors.

In aim 2, expressing one gene at a time might not yield the desired results. Several mutations in different genes have to occur for a cell to be transformed. So, expressing one gene at a time might not work. Detailing alternative strategies to the single gene approach would improve the application.

One experiment or idea for consideration that is not developed in this proposal and probably relevant to this aim is the role of epigenetic changes in the hESC; especially considering the propensity of these cells to have abnormal epigenetic phenotypes after have been cultivated for a long time. Exploration of this idea might be more relevant to how these cells acquire malignant phenotypes.

As stated earlier, experimental details for aim 3 like timing steps and other conditions are not spelled out—there is just a mix of experiments. The whole basis of this aim is the design of appropriate assay end-points, and they are not presented.

Approaches to analysis of complex datasets are poorly described or absent. The application is hard to read. Figure legends are sometimes unintelligible (they should stand alone) and important sentences are nonsensical or confusing. Important information is left out.

DISCUSSION: This proposal is from a newly independent investigator and suffered from direct comparison with a comparable competing application. Although the questions to be addressed by the proposal are really important, the content of the proposal underscored the inexperienced nature of the thinking behind the proposal. The hypothesis was “poorly posed and vague” and lacked focus. The applicant has no idea how long the experiments would take (despite experience in the area) and endpoints seemed not decided. This is not a mature project but the investigator is to be encouraged along this line of inquiry.

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:

• Lansing, Sherry