Embryonic stem cell-based therapies hold great promise for the treatment of many human diseases. These therapeutic strategies involve the culture and manipulation of embryonic stem cells grown outside the human body. Culture conditions outside the human body can encourage the development of changes to the cells that facilitate rapid and sustained cell growth. Some of these changes can resemble abnormal changes that occur in cancer cells. These include “epigenetic” changes, which are changes in the structure of the packaging of the DNA, as opposed to “genetic” changes, which are changes in the DNA sequence.
Cancer cells frequently have abnormalities in one type of epigenetic change, called “DNA methylation”. We have found that cultured embryonic stem cells may be particularly prone to develop the type of DNA methylation abnormalities seen in cancer cells. A single rogue cell with DNA methylation abnormalities predisposing the cell to malignancy can jeopardize the life of the recipient of stem cell therapy. We have developed highly sensitive and accurate technology to detect DNA methylation abnormalities in a single cell hidden among 10,000 normal cells.
In this seed grant, we propose to screen DNA methylation abnormalities at a large number of genes in different embryonic stem cells and compare their DNA methylation profiles to normal and cancer cells. This will allows us to identify the dangerous DNA methylation abnormalities most likely to occur in cultured embryonic stem cells. We will then develop highly sensitive assays to detect these DNA methylation abnormalities, using our technology. We will then use these assays to determine ES cell culture conditions and differentiation protocols most likely to cause these DNA methylation abnormalities to arise in cultured ES cells.
The long-term benefits of this project include 1) an increased understanding of the epigenetics of human embryonic stem cells, 2) insight into culture conditions to avoid the occurrence of epigenetic abnormalities, and 3) a technology to monitor for epigenetic abnormalities in ES cells intended for introduction into stem cell therapy patients.
The successful implementation of human embryonic stem cell therapy will require rigorous quality control measures to assure the safety of these therapies. Cells cultured outside the human body are known to be at risk of developing abnormalities similar to those found in cancer cells. Since a single rogue cell hidden among thousands of normal cells could cause cancer in an embryonic stem cell therapy recipient, it will be essential to have highly sensitive and accurate assays to detect these abnormalities in cultured embryonic stem cells before they are introduced into the patient. The goal of this proposal is to develop such sensitive and accurate assays. The citizens of the State of California will benefit from the availability of such assay technology to help assure the safety of human embryonic stem cell therapies.
SYNOPSIS: The goal of this project is to comprehensively characterize DNA methylation patterns at a large number of promoters, focused mostly on polycomb (PcG) target genes, to determine if stem cells are subject to aberrant irreversible gene silencing. The PI describes currently unpublished data to support the logic. PcG is known to target repressive marks (e.g. H3K27me3). The PI looked at 177 PcG target genes in primary colorectal tumors and found that 77 had cancer-associated hypermethylation marks compared to normal. Based on this result, they suggest the interesting hypothesis that repression of differentiation genes in cancer cells contributes to their stem cell properties such as unlimited proliferation. This supports the concept of the stem cell basis for tumors and suggests that it may be very important to monitor the epigenetic state of ES-derived cell populations prior to use in therapies. The goal here is to demonstrate feasibility of screening human embryonic stem cell (hESC) lines or derivatives.
In aim1, the applicant will use a high throughput DNA methylation Illumina-based platform to screen a number of cell lines. He will attempt to analyze over 1000 PRC2 target promoters and classify hESC lines based on the epigenetic profile. Aim 2 will monitor epigenetic abnormalities of selected hESC lines under different culture and/or differentiation conditions.
INNOVATION AND SIGNIFICANCE: The potential danger of introducing irreversibly self-renewing stem cells in hESC-based therapies is an important issue that is well recognized, and the development of assays to screen for this potential could be very valuable. This project uses innovative approaches, developed largely by the PI, to evaluate promoter methylation state, and although descriptive, the comparison of different lines will be interesting and provide valuable information.
STRENGTHS: Strengths involve the application of high-throughput, sensitve technology to profile DNA methylation patterns in several embryonic stem cell lines. The technology will then be applied to monitor DNA methylation abnormalities that might occur under various culture and differentiation conditions in stem cells. While the details of the two approaches were not well explained in the brief application, they are bisulfite-based PCR protocols that have been used in previous publications. The big advantage of Aim 1 experiments is that the PI does not even need to culture cells, but only needs to obtain DNA samples for the analysis (although it will be important to know the state of the cells when harvested). Even in the absence of DNA methylation abnormalities, a strength of the proposal is that a large amount of valuable comparative data should be generated that may provide a unique database for the community and that could serve as a benchmark for stem cell quality. The proposal applies powerful technology that could help scrutinize stem cells and material derived from them before they are transferred to patients.
The PI is a leader in the field of DNA methylation and cancer. The degree of confidence for successful execution of the experiments is therefore high. A backup plan for obtaining ESCs is in place in case ESC lines from the international stem cell consortium cannot be obtained.
WEAKNESSES: The research plan could be much more focused and well described. While the rationale for aim 1 was well described, it is less clear how the experiments will proceed in aim 2. There is not a clear plan or set of priorities in terms of differentiation protocols. Will cell subsets be sorted for comparison?
Overall, the research plan is presented without well-defined benchmarks or priorities. There is some concern that abnormal methylation patterns may not be found in hESC lines. If rare methylation marks are detected it is not clear how this could be used to determine meaning or relevance. What will determine success of the project? There is a strong hypothesis presented but beyond the admittedly interesting characterization, how will these experiments really test the hypothesis?
While the expertise of the PI in this field is appreciated, there is some concern regarding available effort. Also. depite being supported by 3 RO1 grants over the past 4-5 years, the PI's published productivity has been modest.
DISCUSSION: The applicant proposes to profile DNA methylation patterns of hESC lines with the Illumina DNA methylation analysis platform; this methylation technology is not real high throughput but is sensitive, it can detect 1 aberrantly methylated promoter in a background of 10000 cells. One reviewer questioned how would know in aim 2 whether epigenetic abnormalities detected under different differentiation and/or culture conditions were positive or negative.