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:
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.
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 (hESC). The first aim is to test the hypothesis that survival signals are propagated through the TRK receptor family through AKT activation, 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 hESC will lead to one of the first steps in transformation of stem cells and deregulation of differentiation from hESC. 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 or 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 by addressing: 1) the way survival factors mediate survival versus other fate options; 2) the role of genes located in chromosomal areas that are amplified in culture in transformation (vs. other cell fates); and 3) the role of the environment in genetic instability and/or tumorigenesis of hESC. 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 hESC cells 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 investigator has packed so many experiments in this proposal that he/she did not have time 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 investigators 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 hESC. The PI’s work showing that, in the presence of BDNF or NT-3, hESC can be expanded clonally and more efficiently, will help address some of the ideas put forward in this proposal. The PI is a new assistant professor at UCLA and director of a hESC core facility that is part of a PO1. 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. For example, in studying the environment of cells that contributes to tumorigenesis: is O2 or the location in the body the priority? In the description of experimental plans for aim 3 only high-throughput screens are described. 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 hESC. This is quite important for the basic and applicative biology of these cells. Subsequent studies on understanding how hESC differentiate along lineages of different embryonic layers will provide a rational framework for differentiation and expansion of specific cell types. Addressing how hESC change and acquire malignant characteristics in the second aim is of the outmost importance. The author’s idea of focusing on genes found in chromosomes that are often amplified or changed in hESC is a good start. Coincidentally, specific genes involved in their survival and maintenance are located on these chromosomes. Overexpressing candidate genes and then following the formation of teratomas in immuno-compromised animals is a simple approach and perhaps will yield results. 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 hESC but has few publications as first/senior author. The project is very immature at this point; the PI is a newly independent investigator with just starting a lab at UCLA as of July 2006. 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. Low O2 is certainly a good idea, but the PI seems unaware of several papers on this subject in hESC cultivation. (3% O2 is the standard in the core facility known to this reviewer.) An important consideration in O2 manipulations is reperfusion of room air (21% O2) with changing medium or cell splitting, unless a specialized facility is developed. These factors can contribute to considerable variability and deserve consideration. What is “RT O2”? 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. For example, why use FOXO1, mTOR and REST and not others genes like SOX2, FGF4, UTF1, ZFP42, FOXD3, and TERT that are essential for the multipotentiality? Furthermore, the exact end-points of analysis don’t seem to be decided. On page 2 of the Research Design section, survival assays will be “annexin V staining…or additional markers.” On the same page, transformation will be monitored by “Karyotype or SNP analysis and changes in expression of transformation markers such at CD30”… The controls in this important paragraph are also confusing. How will the CD30-positive cells be used as a control? Later, other possible analyses are listed “array CGH, DNA methylation status, histone modifications by ChIP, and global expression profiles.” 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. For example, take the use of CD30 as a marker for transformation. This gene is up-regulated after several passages in suboptimal conditions of hESC growth and not in the presence of feeders and serum. Therefore, knowing the conditions and timing of these experiments is important. Also, experiments like survival and differentiation will be analyzed at different time points than those analyzed for CD30. The investigator needs to spell out some of these details. In aim 1, “design part” 2, again no details are given. For example, what markers will be used to distinguish between trophectoderm versus primitive ectoderm? Once micro-arrays are done, how will the author use these data to verify and explain the findings? Page 3 of Research Design states they will use “multiple NIH and non-NIH approved cell lines as well as obtaining blastocysts”. Again, how many and which ones? How many blastocysts? Neurotrophin signaling is complicated and controversial. The tools used in the preliminary result such as using selective inhibitors are not sufficient, nor are eliminating or overexpressing receptors like p75 or some of the putative down stream signaling molecules. There are some tools out there that could help. For example, the use of neurotrophins that bind only trk receptors and not p75 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. Furthermore, what is the justification for performing tumor analysis in different locations in the body (3 are mentioned)? The experiments to determine “minimal number of cells required for tumor formation” could be incredibly time-consuming and are not described at all. 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 methylation 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 high throughput screening end-points, and they are not presented. If ‘survival’ of cells plated at clonal density is the screening end-point then it should be clearly stated. Instead there is a discussion of who will be involved in picking out the screening end-points. Using phosphorylated histone 3 will not reveal chromosomal abnormalities even if it is used with high numerical aperture objectives. Phospho-H3 to Ser10 is a good marker to reveal cells in late G2 and M phase. Beyond that, it is a not a good marker for abnormalities in chromosome structure. The only reliable way is to look for karyotypic abnormalities or other tools that cannot be used in high content screenings. Approaches to analysis of complex datasets are poorly described or absent. The grant 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. How long were the cells cultured in Figure 3? Just above Figure 7, “In addition we have performed initial screens with hESC in different environments to validate Aim 3.” What environments? DISCUSSION: This proposal is from a new assistant professor at UCLA and suffered from direct comparison with competing application submitted by post-doctoral mentor. 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 post-doctoral experience in the area) and endpoints seemed not decided. This is not a mature project but investigator is to be encouraged along this line of inquiry.