A novel system for understanding the effects of natural genetic variation and epigenetics of stem cells.
New Faculty I
Understanding stem cell regulation holds the potential to treat many diseases. However, some experiments that would aid this understanding cannot be performed with humans due to ethics or practicality. One concern is whether the DNA marks (i.e. epigenetics) regulating stem cells and other genes are stable in cells grown in culture over long term. This is particularly important in that it is clear that cancers are also caused by stem cells. The effects of genetic variation on stem cells are also little known. Studies of house mouse stem cells have partly filled this gap. There are significant differences between house mouse stem and human stem cells. It is not clear whether these are simply species differences, or may partly reflect the artificial genetic make-up of laboratory mouse strains. For example, it is becoming clear that the complete inbreeding of the lab strains has affected their genome, and that the combination of genetic variants seen in these animals does not exist in nature. Further, these animals have been domesticated and selected for traits that do not exist in the wild (analogous to differences between poodles and wolves). Our system is unique in using animals with natural genetic variation. We use the most common Native American mammals, rodents of the genus Peromyscus. While these animals are commonly called deer or field mice, they are only distantly related to house mice and rats. One of many differences is that these animals live over twice as long as house mice. Captive strains derived from actual populations are available. Severe prenatal defects occur in hybrids between several of these populations are known. Many of these defects resemble human diseases. A common outcome is a growth with placental like tissues but no fetus. This is similar to a disease relatively common in California termed molar pregnancy. We have evidence that abnormal regulation of stem cells is involved in the hybrid defects. The research proposed here proposes to use these animals to study: 1. the stem cell basis of the hybrid defects, 2. the natural genetic variants that affect stem cells, 3. the stability of stem cell epigenetic marks of over long periods of time.
Statement of Benefit to California:
Understanding of stem cell regulation has the potential to treat or prevent many diseases. These diseases are not limited to those requiring regenerative capacity, but include cancers and developmental defects. Human studies on some aspects of stem cell biology are unethical or impractical. Current animal models are highly genetically modified relative to their ancestors. Our proposal suggests a new system that may be used to understand the role of natural genetic variation on stem cell regulation, and the stability of cultured stem cells. The proposed system also utilizes native California wildlife. We therefore expect several types of benefit to the Citizens of California. First, our studies will likely suggest genetic variants and epigenetic profiles that will lead to improved human studies or therapies. Second, these animals may provide an alternative system for use bybiotech and pharmaceutical companies. Third, the research will provide graduate and undergraduate student research opportunities, and brings together several disparate areas of biological study. Finally, better understanding of native species, and the role of environment on epigenetics may ultimately benefit our care of the ecosystem.
SYNOPSIS: This proposal explores the relationship between epigenetic specification of stem cell pluripotency and genetic imprinting. The PI believes that embryonic stem (ES) cells from either the human or the mouse system have limitations that complicate the study of this phenomenon. Human ES cell systems suffer from ethical concerns and impracticality, and the mouse ES cells are not totipotent and do not represent any naturally occurring combination of alleles. Because of these shortcomings, the PI proposes to use two species of field mice: P. maniculatus and P. polionotus. Crosses between these two species lead to parent of origin effects on development that are due to disruption of genomic imprinting. In Aim 1, the PI will create ES cell lines from each of the two species of field mice as well as from interspecific hybrids. The expression of genes associated with stem cell regulation will be measured in the hybrid ES cells, as will the ability of these cells to contribute to different lineages in chimeric embryos. In Aim 2, the PI will assess the integrity of multiple epigenetic marks at loci important for stem cell regulation in interspecies hybrid ES cells compared to parental ES cells. Furthermore, he will perform a gene expression analysis of early hybrid development, and culture and assess the potential of late gestation pluripotent cell types. In Aim 3, the PI will genetically map the locus that controls imprinting in interspecies field mouse hybrids and also test two possible candidate loci. He will further assess epigenetic/stem cell regulatory variants in other populations of mice and evaluate long-term stability of stem cells and derivatives. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The model proposed in this application is interesting, however the level of enthusiasm of all 3 reviewers is low. This grant proposal has many problems. The first problem is that the PI presents no support for the basic hypothesis that the observed defects in genomic imprinting also cause defects in the epigenetic regulation of stem cell regulatory genes. The background provided by the PI is limited to saying that because both imprinting and stem cell biology involve epigenetic changes, then both must be subject to regulation by the same gene (called Mei1). Furthermore, the assumption that the observed phenomena are due to epigenetics is also not supported by any data. A second problem is that even if stem cell regulatory genes are abnormally expressed in interspecies hybrids, there is no discussion as to how this would increase our understanding of stem cell biology. The PI is simply looking at genes already known to be important, and none of the experiments would provide any new information about their biological functions. The third specific aim is to map the locus responsible for abnormal imprinting in interspecies mouse hybrids. Not enough information is provided to know whether this has any reasonable chance of success. Finally, there are no preliminary data provided on whether ES cell lines can be derived from these field mice or from the hybrids. If this cannot be achieved, the rest of the aim--and most of the entire project—cannot proceed. One reviewer also pointed out that the same type of studies as the ones proposed here can potentially be performed using monoparental (parthenogenetic or androgenetic) ES cells in laboratory mouse where all the reagents and know-how are available. It is difficult to be enthusiastic about this project because instead of tackling emerging and high priority questions in the ES cell field, it adds an additional variable, in esoteric systems, not necessarily helpful for the understanding of ES cell fate specification in general, or for regenerative medicine in particular. QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Dr. Vrana is a talented investigator with many important contributions to his field, as shown by his list of publications. He received his PhD from Columbia, working in the lab of Ward Wheeler at the American Museum of Natural History. He was then a postdoc for 7 years with Shirley Tilghman, before becoming an Assistant Professor in the Biochemistry Department at UC Irvine in 2001. He began his work on interspecies mouse hybrids in the Tilghman lab, publishing two interesting papers which demonstrated the importance of abnormal imprinting to the resulting developmental anomalies. In the six years since he started his own lab he has continued to map genetically the locus responsible for controlling imprinting in these crosses, and has published three senior author papers in genetics specialty journals. He has a grant from the NSF to support that work, which ends in 2008. The PI has limited experience with ES cell work, he is more familiar with the animal model. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: UC Irvine is the best institution for these studies, encompassing all of the necessary resources and centers to successfully execute the proposed specific aims. The PI occupies 1200 square feet of independent lab space at the UCI School of Medicine campus. The Institutional letter of support does not indicate that the school is providing any ongoing support to the PI. DISCUSSION: During a brief discussion, it was reiterated that there is no precedent for the derivation of ES cell lines from the field mice, and no preliminary data are presented in the proposal to support the feasibility of such derivation. If not successful, many aims of this proposal cannot be pursued.