In humans and most other mammals, there are two copies, or alleles, of each gene, one from the mother and one from the father. For certain genes, the two copies are not used equally, and this process is highly regulated. This unequal use of the two gene alleles, or allele-specific gene expression, is normal and very important for normal development and health. Disturbances in this process can lead to major developmental problems, as are seen in Prader-Willi, Angelman’s Syndrome, and Beckwith-Wiedeman, and are implicated in many cancers.
An estimated 10% of all genes show allele-specific gene expression. Due to the lack of technology to identify these genes, there are many basic properties of these genes that remain unknown. For example, we do not know precisely how many of these genes there are, whether the genes are the same in every cell type, and what causes the two copies to be used unequally.
There are now several methods for reprogramming adult cells into pluripotent embryonic stem cell-like cells. These reprogrammed cells do not carry the ethical burden that embryonic stem cells do, and therefore are attractive sources of cells for preclinical and clinical applications. However, it has been observed that the cloned animals produced from cells generated by reprogramming of adult cells into embryonic stem-cell like cells (the iPS cells), or by somatic cell nuclear transfer (like Dolly the sheep) tend to have developmental abnormalities and lower life expectancies. Based on previous studies, we and others believe these outcomes are likely to be due to faulty gene regulation, and may be caused in part by the loss of normal allele-specific gene expression. These observations raise the concern that some apparently pluripotent cell lines may not be appropriate for clinical use due to disregulated gene expression. This is particularly worrisome given that the disregulated gene expression seen in many cancer cells may be caused by a similar mechanism.
We propose to develop two tools, one that measures allele-specific gene expression, and one that examines the possible contribution of allele-specific protein binding to allele-specific gene expression. These two tools are designed so that the same cell samples can be run on them in parallel, and the readout for the two tools is on the same microarray. This means that the resulting datasets can be directly compared.
We will demonstrate the application of these tools in the assessment of the stability of gene regulation in a range of cell lines, including human embryonic stem cells (hESCs), hESC-derived differentiated cells, and differentiated cells. In the future, these tools can be used as quality-control measures to measure stability of gene regulation in stem cell-derived cell preparations for applications such as toxicology testing or cell therapy.
Since two of the three hESC lines we will be studying is non-NIH approved, this study is not eligible for NIH funding.
Statement of Benefit to California:
The State of California faces immense challenges to its health care system, with soaring medical costs and an aging population.At the same time, investors are becoming more wary of funding the high-risk technology development that has fueled California’s high-tech and biotech booms. The taxpayers of California have made a substantial investment in scientists who are dedicated to development of stem cell therapies thatmay revolutionize medicine and health care by providing new treatments for incurable conditions such as diabetes, Parkinson's disease, and spinal cord injuries.
Stem cell therapies, however, are in an early stage, and it is critical that research over the next few years be focused on development of cells that will be both safe and effective. A significant roadblock to progress toward the clinic is our currently inadequate understanding of stem cells and their derivatives. The technology and data developed for this proposal will help to ensure that stem cells used for therapy are normal and free of a particular type of abnormality that can cause devastating effects in the cells, including the possibility of making them cancerous.
The technology we will be developing is an assay to detect “allele-specific” expression, which means that one copy of a gene, of the two copies in the chromosomes (one inherited the mother and the other from the father) is used in cells, while the other is not. We need to know this information about embryonic stem cells and induced pluripotent cells, because a failure for genes to be properly regulated appears to be responsible for developmental abnormalities and lower life expectancies in the cloned animals produced from adult cells (like Dolly the sheep and cloned mice). We propose to develop this technology so that it can be integrated into the repertory of methods that can be used to ensure quality control and safety of cell therapies. We will make the new test available to stem cell researchers and clinicians throughout California. Ultimately, this technology will benefit California by attracting highly skilled jobs and tax revenues, and by making the State a leader in a field that is poised to be the economic engine of the future.