Screening for Oncogenic Epigenetic Alterations in Human ES Cells
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.
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, DNA methylation. In this grant, we screened for 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 allowed us to identify potentially dangerous DNA methylation abnormalities, which occur in cultured embryonic stem cells. In the first year of this seed grant, we have developed a custom microarray to screen for DNA methylation changes at predisposed genes. In addition, we have analyzed DNA methylation in embryonic stem cells at more than 14,000 genes on a generic platform. This has allowed us to identify hundreds of genes that are abnormally methylated in various types of human cancers, and that show some evidence of this alteration in ES cells.
In the last phase of our study, we have screened the DNA methylation level of 1,536 genes in 142 different human embryonic stem cell pairs. Each member of the pair differed in the length of time it was in culture. Thus, our sample set was comprised of 284 paired specimens, one derived from an early passage and one derived from a late passage.
Our results indicate that the levels of DNA methylation varied considerably at a significant portion of the screened genes, some of which gained and some of which lost DNA methylation. These results indicate that DNA methylation in human embryonic stem cells seems to be susceptible to change over, at least in the genes examined in this study. Overall, our results suggest that the monitoring of DNA methylation changes in human embryonic stem cells may have to be incorporated as a routine protocol in stem cell manipulation.
During the past 12 months we have made significant progress on the data analysis of 141 paired (early passage-late passage) human embryonic stem cell lines (HESCs). The data in question was generated using a custom Illumina GoldenGate array of known Polycomb targets in HESCs, as described by Lee et al 2006. Briefly, we profiled the DNA methylation status of 1,536 loci on 282 specimens. This profiling was used to determine whether DNA methylation changes in HESCs arise as a result of time in culture at the examined loci. This determination was made by comparing the DNA methylation status of a sample of an early passage line with a late passage sample of the same line.
Interestingly, we found that DNA methylation in Polycomb target genes is highly affected by time in culture in a cell line-specific manner. That is, in some cell lines few DNA methylation changes were observed, while in the majority of them a large number of loci showed either an increase or decrease in DNA methylation. Via collaboration with the University of Sheffield, we were able to determine that DNA methylation instability seems to be independent of genetic instability. Furthermore, genetic instability seems to be a function of passage time in culture.