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.
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.