Although some hESC lines have been reported to be stable in culture, we and others have found that the stability is variable for different lines. Furthermore, small DNA rearrangements that cannot be detected by chromosome analysis, which has been used as a key measure of hESC stability, exist in many cell lines. It is of crucial importance to detect any potential genetic abnormalities in hESC, and hESC genomes must be stable if they are to become useful clinical reagents. Our long-term goal is to generate genetically stable hESC lines that can be eventually used safely for therapeutic purposes. However, knowledge of long-term genetic stability of these cell lines is necessary to ensure the safe therapeutic use of these cells, and it is essential for the generation of new cell lines in the future. We believe that the groundwork for improving the genetic stability of the existing cell lines should be done before we derive new cell lines. Therefore, we would like to use the existing hESC lines to explore culture conditions for improving genomic stability of hESC upon long-term culture. We propose to follow the molecular features of existing cell lines, both federally- and non-federally-approved lines, over time to identify differences potentially caused by culturing methods and conditions. There is very little information on the non-federally approved cell lines due to the lack of funding. It is important to compare these cell lines with the federally approved cell lines because they are derived and cultured under different conditions and methods. The common genetic and epigenetic features of these cells may also provide insights into the characteristics and plasticity of embryonic stem cells. We also propose to study how genetic changes such as permanent removal or addition of a gene to the hESC will impact these cells, especially the genes involved in DNA repair and modification. We have already begun to design new ways to manipulate these cells for both the understanding of basic biology and the future therapeutic application.
Statement of Benefit to California:
Similar to many other proposals, the work proposed here would help California in general ways such as biotechnology development and human health improvement. The work proposed here would also provide several specific benefits for the California community. The strength of the research team is genetics and functional analysis, and the approach is focused on fundamental understanding of stem cell genetics and is hypothesis-driven. Therefore, the work proposed will not only serve as a stepping stone for clinical application of embryonic cells, it will also provide a solid understanding of the biological function of these cells. Much work on embryonic stem cells has been focused on the potential clinical applications of these cells and not as much on genetic stability and fundamental biology. Also, most of the work has been done using the federally-approved cell lines due to funding constraints. The stability of these cells is of key importance for their clinical application, so improving genetic stability in culture and increasing the genetic adaptability of these cells are essential. Comparing federally-approved and non-federally-approved cell lines is also important since they were derived and cultured under different conditions. Therefore, we have begun work in these two directions with cell lines from both sources. Experiments designed to manipulate these cells based on critical genetic information may provide important insight into the function and genetic adaptability of human embryonic stem cells. The research reagents generated in this proposal will make many other experiments in normal human cells possible. The unique aspect of this proposal may move basic scientific understanding of normal cells forward and give California a leading edge in both clinical application and basic biology of human embryonic stem cells.
SYNOPSIS: In this broad approach, the author proposes to tackle three independent objectives. First is to measure the differences of culturing hESCs in low and high oxygen. The second objective is to optimize conditions for genetic knock-outs in hESCs, and to develop a stable episomal system to study the impact of DNA methylation in hESCs. The third aim is to establish a stable episomal system to study how overexpression of genes impacts hESCs without positional effects and with minimal alteration of the endogenous genome. SIGNIFICANCE AND INNOVATION: The most significant impact of this project is specific aim 2. For reasons that are not well-understood, knock-outs in hESCs have been tremendously difficult to create. The resolution of this important technology will provide the community with genetic tools to manipulate the human genome and address the functionality of individual genes in hESCs. There are no innovations. Each of the 3 aims have merit and successful results would have a significant impact. The most important aims are the second and third. The development of efficient homologous recombination technology for hES cells would have a large impact on the field. Similarly, the development of efficient episomal vector systems for these cells would have impact. The technologies that will be pursued and developed are not particularly innovative; however, they are appropriate to the goals of the proposal. STRENGTHS: The proposed studies are all fairly strong, and each individual goal in the 3 aims has considerable merit. The technologies to be applied are clearly within the expertise of the P.I. Particularly important would be to develop effective tools to genetically manipulate hES cells. This has been difficult for reasons that are not clear. A strength of the proposal is the selection of the priority problems to be solved. The author courageously wants to tackle all of them. If just aim 2 works, it will justify the investment. WEAKNESSES: This is an overly ambitious grant that will certainly not fulfill all of its objectives. Each one of the aims could have constituted a separate proposal, and would have provided the opportunity to develop the arguments, rationale, and outcome analysis in a more comprehensive fashion. Two years of funding is not enough to accomplish these goals, especially in the view of the fact that the PI does not have a senior authorship paper since 2002. The broad scale of the proposal increases the likelihood that the proposed studies will result in a significant dilution of effort, and may compromise the success of each of the 3 aims. A greater degree of focus, and limited set of goals for a 2-year proposal would be appropriate. This proposal would have been more highly regarded if it had been focused solely on aim 2. DISCUSSION: There was no further discussion following the reviewers' comments.