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A new microarray tool for discovering DNA methylation stem cell biomarkers

Funding Type: 
Tools and Technologies I
Grant Number: 
Funds requested: 
$828 296
Funding Recommendations: 
Not recommended
Grant approved: 
Public Abstract: 
A significant barrier to progress in development of safe and effective stem cell therapies is the lack of reliable inexpensive tools that can be used for quality control and characterization of transplantable cells. This is particularly challenging for analysis of the epigenetic state of cells, which is critical to their normal function. We propose to develop a high-resolution tool that can be used routinely to monitor cells to be used for therapy, and to ensure that they have normal epigenetic profiles. DNA methylation is an important epigenetic mechanism that helps to control the orderly expression of genes. Inappropriate methylation patterns are linked to genetic disease and to the uncontrolled growth characteristics of cancer cells. Given the consequences of abnormal DNA methylation it is critical that cells used for clinical therapy maintain a stable and normal DNA methylation pattern. Existing DNA methylation detection methods are either limited in scope or extremely expensive and technically challenging. These factors limit the feasibility of using these methods for routine, large-scale profiling of DNA methylation status. We propose to develop a high-density DNA methylation microarray, which will test 100,000 individual sites in promoter regions of all known coding genes and known microRNAs, as well as a sampling of CpG sites located in intergenic regions. The platform will be a cost-effective and accessible tool for routine analysis of cell cultures prior to their qualification for cell therapy.
Statement of Benefit to California: 
The heath care system of California will greatly benefit from the taxpayer’s investment in stem cell research. But a significant barrier to progress in development of safe effective stem cell therapies is the lack of reliable inexpensive tools that can be used for quality control and to assure the safely of transplantable cells. This is particularly challenging for analysis of the epigenetic state of cells. The exquisite control of gene activity in normal cells is regulated through regulatory molecules collectively known as “epigenetic” factors because they determine what genes are active in the DNA sequence without altering the sequence itself. Changes in the epigenetic state of cells can lead to uncontrolled growth, as in cancer cells, or to abnormal differentiation of stem cells. Our research has indicated that human embryonic stem cells have a very precise epigenetic profile that distinguishes them from all other cells, and that this profile changes in very predicable ways as the cells differentiate. Our current technology, however, lacks the resolution to monitor the epigenetic state of all the genes in human DNA. We propose to develop a greatly improved high-resolution tool that can be used routinely to monitor stem cells that will be used for therapy, to ensure that they have normal epigenetic profiles.This technology will be developed specifically for human embryonic stem cells and induced pluripotent stem cells in the proposed research project, but it will have wide applicability to cancer research and studies of other human disease.
Review Summary: 
The applicants propose to develop a robust, cost effective tool for determining the epigenetic profiles of stem cells prior to consideration for therapy. Specifically, a high density DNA methylation array will be devised to assess 100,000 epigenetic modifications in concert, including promoters of all known coding genes, miRNAs and a sampling of intergenic regions. In the first aim, the applicants will design, select and test potential probes for subsequent incorporation into methylation arrays. Next, the resulting technology will be evaluated for performance and utility using a comprehensive panel of human cells, including multiple stem cell lineages. For the final aim, the applicants propose to develop bioinformatics tools to facilitate data acquisition and analysis. The reviewers agreed that the proposed technology could be of tremendous value to stem cell science. While current methods for profiling DNA methylation are useful, they are technically challenging, labor intensive, of limited sensitivity, and very expensive. By addressing these issues, the proposed technology could enable such studies to be widely implemented, thereby facilitating stem cell characterization both for basic research purposes as well as for quality control of stem cell-based therapeutics. Despite their enthusiasm of the proposal in theory, the reviewers were uncertain of its impact in practice. Although it is a stated goal to develop inexpensive technology, the applicants did not address how this would be achieved. For instance, a concern was raised about the number of methylation sites to be included in the arrays. One reviewer indicated that it should be possible to differentiate epigenetic states amongst cell types using a smaller, more select set of sites and questioned the inclusion of such a wide range of targets, if the goal is to produce inexpensive tools. The reviewers agreed that the proposal was well written, rationally designed, and the milestones were clearly defined. The preliminary studies were very encouraging and spoke well to the overall feasibility. The reviewers were also impressed with the comprehensive selection of cell lines and controls to be used for validation. Despite these strengths, the reviewers were apprehensive that insufficient consideration was given to the biology behind the technology. For example, it was unclear what specific cell types would be distinguished by the proposed technology, whether the goal is to detect undifferentiated human embryonic stem cells (hESCs), or to verify the identity of a specific lineage of cells to be transplanted. If it were the latter, then the proposed validation criteria would be insufficient and more samples per lineage would have to be analyzed to obtain significant results. The research team was considered to be outstanding. The principal investigator has excellent publications in the area of this proposal, and the consultants have substantial expertise in hESC biology. The research environment was considered to be ideal. One reviewer commented that no team member contributed more than 75% effort to this project and was concerned that this project will not get sufficient attention it. The budget was largely considered appropriate and justified. Overall, while the proposed technology could be useful for a broad range of studies, the reviewers were not convinced of its potential to advance stem cell biology.

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