Human embryonic stem (ES) cells have the capacity to self-renew but also give rise to other cell types. How this capacity is regulated and what factors determine one fate over another is an active area of research. This is because by understanding the decision making process the a stem cell goes through, we might be able to manipulate the process and make stem cells generate more of themselves or other cell types of interest. Preliminary studies indicate that one important determinant of stem cell fate is its ‘epigenetic’ information content.
In humans, DNA is tightly wrapped around a core of proteins called histones to form chromatin—the physiologically relevant form of the genome. The histones can be modified by small chemical molecules which can affect the structure of chromatin, allowing for a level of control on gene expression. The patterns of occurrences of the histone modifications throughout chromatin is highly regulated and can increase the capacity of the genome to store and process biological information beyond the DNA sequence. This information which is heritable but not encoded in the sequence of DNA is referred to as ‘epigenetics.’ The modifications of histones, therefore, contribute to the epigenetic information content of a human ES cell.
We have found that individual ES cells from mouse have different patterns of histone modifications, and thus, different epigenetic information content. We believe these differences may affect the fate decisions of stem cells. If so, then the histone modifications may act as a natural indicator of the potential of ES cells to make certain fate decisions. The histone modifications may also provide a natural tool by which cell fate decisions can be influenced. In this proposal, we intend to determine the epigenetic information of content of several human ES cell lines and relate that information to the potential of cells to make self-renewal versus differentiation decisions. Our work will provide a fast and high-throughput measure by which appropriate ES cells can be chosen for a clinical application of interest.
The available human embryonic stem (ES) cell lines display different capacities to proliferate to either generate more of themselves or differentiate to other cell types. Epigenetic regulatory mechanisms play critical roles in these developmental decisions, such as how a given cell establishes and maintains its identity. The identity of a pluripotent stem cell, such as an ES cell, is defined as being able to differentiate to all tissues of the body, yet, being able to switch off this differentiation process, otherwise, its self-renewal ability, or “stemness”, is lost. There have also been observations that certain ES cell lines are better is resisting spontaneous differentiation, or more efficient in making particular cell types such as those of blood, than other lines. It is conceivable that this kind of variability in ES cell lines is likely linked to underlying epigenetic differences. Currently, there is no rapid or reliable way to scan epigenetic differences between different ES cell populations, as current methods are either too laborious to perform on daily basis, or contain very little biological information. Our work proposes to develop a natural, fast and high-throughput tool for measuring cellular epigenetic patterns, which we hypothesize to be linked not only to the identity of various differentiated cell types, as we have shown already, but also to define the quality or functional potential of various ES cell lines. It is important to understand and be able to measure the capacity of various ES cells so that appropriate cells are chosen for a clinical application of interest. This would be beneficial to the people of California because ES cells can be quality controlled before they are used in patients.
Furthermore, if epigenetic modifications that link to cellular identity and functional potential become useful diagnostic tools, the approaches discussed in this project may lead to innovative discoveries and patents that may be exploited by the biotech industry in California, and thereby improve the economy of 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.