Cellular epigenetic diversity as a blueprint for defining the identity and functional potential of human embryonic stem cells
Statement of Benefit to California:
SYNOPSIS: In this proposal, the investigators wish to determine the histone modificiation pattern of hESCs and the changes in this epigenetic modification when ESCs are allowed to differentiate to hemangioblasts, HSC-like cells, and single lineage hematopoietic cells. They obtained evidence in several cell populations, including murine ESC, that histone modifications occur to different extents at the single cell level, with cell-to-cell differences in total levels of histone modifications. Therefore they propose to study the pattern of histone modification by FACS which has been optimized to identify 14 different histone modifications. This will also allow selection of cells with specific histone modification patterns. This will be used in a final aim, wherein Chip on Chip will be attempted on cells selected based on such histone modification to evaluate specific genes, transcriptional programs and functional status of such cell. INNOVATION AND SIGNIFICANCE: This is an innovative proposal that will examine changes in epigenetic states in populations of stem cells at the both the cellular and genomic levels. It has become clear that major changes in the differentiated state of stem cells and in particular ESCs are regulated epigentically wherein histone modifiactions allow certain parts of the genome as a whole to be transcribed or not; and the fate of cell is not determined one gene at the time. The investigators have shown initially in cancerous cell populations and more recently also in mouse ESCs that such epigenetic patterns are not controlled at the population level, but that individual cells within a population may have different modification patterns. It is therefore perhaps necessary to evaluate subpopulations of cells with similar epigenetic modifications to start to understand the influence of the epigentic changes on cell fate. In the aforementionned study on tumor cells (by the investigator and colleagues), tumor cells displayed differences in total levels of certain histone modifications in a cytological immunodetection assay. Importantly, the frequency of some modifications was a predicative measure of clinical outcome for prostate cancer. All together, the proposed experiments should provide important new insight about cellular epigenetic variations in the available ESCs and how these variations contribute to a specific differentiation event. This is critical not only for developing a knowledge base to guide stem cells along specific differentiation pathways, but also for evaluating the quality of different stem cell lines. The studies proposed are therefore innovative and of significant importance to further insights in ESC behavior. STRENGTHS: Strengths involve the application of a new, innovative approach to analysis of cellular epigenetic variation between different stem cell lines and in populations of cells derived from the same ESC line. The proposed experiments are very likely to define epigenetic difference between ESC lines and determine whether these differences play a role in the ability of ESCs to differentiate. The proposed experiments are very well described, possible problems are acknowledged, including that the studies proposed to define the molecular differences between epigenetically distinct sub-populations of hESC (aim 3) may pose significant technical problems. The PI has an outstanding track record in studies of histone modifications using the approaches described in the proposal and has established a number of collaborations to obtain the required ESC lines and other reagents. WEAKNESSES: The proposal is extremely ambitious. Although aim 1 and aim 2 should be relatively straightforward, the studies proposed in aim 3 may pose unresolvable technical problems. This is acknowledged by the investigators. The PI indicates, although does not show specific data, that all parts of this technique separately have been accomplished even though the whole procedure has not. Obviously, this aim will be required to interpret the implications for cell fate status of changes in histone modification. The molecular basis of cellular epigenetic variations as reflected by changes in total levels of certain histone modifications is currently not understood. However, both the published data by this group and the preliminary evidence is compelling and suggests that global changes in histone modifications, perhaps mediated by changes in the level or activity of modifying enzymes, contribute to cellular epigenetic variation that is likely to play a role in stem cell biology and cancer. DISCUSSION: The reviewers' were enthusiastic about the proposal - a novel and unusual way of looking at epigenetic modifications on a cell to cell basis. The PI is outstanding. Reviewers concerned about aim 3; but one cited work in other labs that suggests that the technical challenges posed by this aim can be addressed.