Studying Chromosomal Aneuploidy During Human Embryogenesis Using Human Embryonic Stem Cells Derived From Embryos Characterized by Preimplantation Genetic Diagnosis
$2 000 000
Stem cells are the building blocks of the human body. They play a major role in the regeneration of tissues, and in the development of the human embryo. Stem cells are now at the center of world attention, since it has become evident that they possess the potential to change the face of medical research and human health. In our own research we have studied human embryonic stem cells for many years, and we have made seminal discoveries in the field. We were pioneers in the demonstration of both spontaneous and directed differentiation of human embryonic stem cells in culture into a dozen different cell types. In addition, we were the first to demonstrate genetic manipulation of the cells, to analyze their immunogenicity, and to suggest ways to overcome their tumorigenicity. In addition to our research on the potential of human embryonic stem cells for transplantation, we have been pioneers in establishing models for human diseases using human embryonic stem cells. Derivation of human embryonic stem cell lines which harbor specific genetic defects has great value for modeling human inherited disorders and for finding new drugs for many of these diseases, especially in cases where no good animal model exists. We have already created models for two genetic disorders. By creating a specific mutation in human embryonic stem cells, we have generated a model for Lesch-Nyhan syndrome, a fatal kidney disease resulting from accumulation of uric acid. The mouse model failed to recapitulate the symptoms of this devastating disorder, but our human embryonic stem cells did accumulate uric acid. Moreover, we showed that a specific drug can reduce the levels of uric acid produced by the cells. In addition, we have created a model for the most common cause of hereditary mental retardation, namely fragile X syndrome. The molecular basis of this disease could not be demonstrated in animal models. We have isolated a cell line carrying the fragile X syndrome after pre-implantation genetic diagnosis for this disease, and characterized its molecular basis. We aim to continue creating such models for various genetic disorders, and will now concentrate on chromosomal aberrations, the number one cause of spontaneous miscarriages. Our research will yield a repository of many different cell types for our own research and for the research of other investigators in California and the rest of the world. This proposed research will enable us to define the earliest stages that lead to embryonic death during the first trimester of pregnancy.
Statement of Benefit to California:
California in now in a special position to lead research on stem cells for years to come. Research with human embryonic stem cells has the capacity to change the face of medical research. Our own study will focus on the number one cause of spontaneous abortions, namely an abnormal number of chromosomes in the embryo. If the embryo survives an abnormal number of chromosomes it will develop abnormalities of the body and brain, dependent on the specific chromosomal excess or deficiency . The most known such disorders are Down Syndrome which results from an extra chromosome, and Turner Syndrome which results from a lack of a chromosome. Our research will be beneficial to the state of California and its citizens in various ways. First, in our study we will create a repository of human embryonic stem cells that are missing a chromosome or that have an extra chromosome. Such a repository will be extremely important for our research, but also will serve other investigators in California. We have vast experience in research with human embryonic stem cells and their derivation, so we expect to have a substantial repository in the near future. Second, our research is aimed at the very important clinical issue of spontaneous abortions. Most early abortions occur during the first trimester, and most of them result from chromosomal aberrations. The reasons for the developmental arrest are not fully understood, but now we have a unique opportunity to study developmental arrest using materials that were not previously available. In this regard, this research proposal is aimed at the study and potential methods of prevention of spontaneous abortions. The third level of benefit is basic understanding of human embryogenesis. Our research will touch the most crucial aspects of early human development and the molecular events that are involved in this process. Thus, our research will serve as a unique source of materials and knowledge for stem cell research in California.
SYNOPSIS: This proposal is directed at establishing a repository of hESC lines with specific chromosomal aberrations, as well as studying changes in gene expression in monosomic hESC lines, and analyzing imprinting on the X chromosome in Turner syndrome hESCs. Lines will be established from human blastocyts in which chromosome anomalies have been identified by preimplantation genetic diagnosis (PGD). IMPACT AND SIGNIFICANCE: This application proposes to produce a collection of karyotypically abnormal hESC lines derived from PGD embryos. Thus the application focus is on disease model hESCs rather than the conditions for normal ESC development. The key argument is that this approach might be more revealing than mouse models. However there is a substantial limitation to the value of the human model, in that one cannot study normal or aberrant organogenesis, or perform in vivo therapy trials as in a mouse model, because of the ethical limitation on human experimentation which is fortunately in place. Though the work could lead to a better understanding of spontaneous abortion and certain genetic diseases, the proposed research would utilize hESCs as a tool to study such topics, but would likely not greatly advance the search for new therapies based on stem cells. The work might lead to the identification of chromosomes that are critical for pluripotency and may lead to the discovery of new imprinted genes. QUALITY OF THE RESEARCH PLAN: The application is actually a combination of two separate experimental goals and approaches. The first is to develop a respository of hESCs carrying chromosomal aberrations. A collection of hESCs would be derived from preimplantation human embryos known to contain chromosomal abnormalities as judged by PGD. Dr. Rimoin is an expert geneticist and has a strong collaboration with an active ART center. He and his team have demonstrated ability to derive such lines. It is hoped that a nearly comprehensive set of hESC lines can be established that cover most possible monosomies, as well as some trisomies. A limitation is that aneuploidy may be heterogeneous, and the lines derived may not reflect the underlying monosomy. Further, aneuploidy may arise during hESC derivation itself, and it will be difficult to distinguish between this and preexisting aneuploidy. Also, if the dosage of a particular chromosome is especially important for hESC derivation or maintenance in culture, it is likely that the corresponding aneuploid lines could not be isolated. The lines will then be expanded by 20 passages under standard ES derivation conditions, then the phenotype determined. This strategy seems biased to recover only “ES-like” cell lines. Pluripotency will be assessed using appropriate markers and by embryoid body formation and karyotype will be determined. The number of lines of each type is not specified. The second aim involves the molecular characterization of these lines. The PI proposes to determine the transcriptional profiles of the cell lines, and from this, it is hoped that imprinted genes can be identified. The PI also describes a transcription profile-based strategy to identify the developmental potential of specific cell lines based on transcription profiles of EBs. The plan is to perform array-based gene expression analysis of undifferentiated and differentiated cells. A concern is that determining the developmental potential of specific cell lines based on transcription profiles of EBs may be quite difficult since EBs contain multiple cell types. Also, this aim underlies a fundamental weakness of the application, as this will provide a large amount of descriptive information. Furthermore, it is not clear how one will sort through gene expression differences. It has been shown in yeast and also in human aneuploidies that there are many changes in gene expression, naturally on the altered chromosome but genome-wide as well. But what does one then do with the information? The applicant also argues that one can use these hESC lines as models where mouse models are unsuccessful. But that approach is severely limited by the appropriate ethical restrictions that prevent most in vivo analysis. Part of the second aim, as well as the third aim, is addressed at identifying new imprinted genes through reduced expression depending on parent of origin. Mouse studies of androgenotes and parthenogenotes have been quite limited in identifying new imprinted genes, and in those cases the conditions are much better than this, with both parents and many known polymorphisms. However, this may be possible, especially if several maternal and paternal monosomic lines for each chromosome are identified. The third aim also addresses methylation of putative imprinted genes, but the methylation pattern can be quite complex and distant from the promoter, and imprinting can occur in the absence of methylation variation. The third aim also involves gene replacement to correct developmental abnormalities caused by loss of an imprinted gene. Likely most of the developmental changes will have nothing to do with imprinting but simply with gene dosage. Also, they do not explain how to restore a precisely balanced level of expression with a transgene, and as noted earlier, the assessment of development is quite limited in this cellular in vitro system. STRENGTHS: The PI has a strong track record and the researchers have the demonstrated expertise necessary to derive and study a large number of abnormal hESC lines. This is a good system for identifying and isolating monosomic hESC lines. The work would lead to a bank of mutant hESC lines, perhaps of use for investigation of disease mechanisms of selected human genetic disorders. These studies might also lead to the identification of new imprinted genes. WEAKNESSES: The approach is relatively unfocused. There is limited biological application as an in vivo model. If a monosomy has a great impact on pluripotency, the corresponding hESC line might well be impossible to derive. All expressions profiles of EBs are likely to suffer from complications arising from a mixture of cell types. To identify imprinted genes using expression profiling, probably 3 paternal and 3 maternal monosomic lines for each chromosome would be required, once experimental replication is considered. This could be a difficult task. The third aim is weak. DISCUSSION: This proposal is from a famous geneticist. It is thoughtfully written and there is no doubt that the investigators are capable of carrying out the proposed research but there were questions about the utility of the research and the impact on the field. The strongest part of the application would be to identify new imprinted genes but this would require derivation of multiple lines with monosomies which would be problematic.