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RC1-00137-1: Human oocyte development for genetic, pharmacological and reprogramming applications

Recommendation: Recommended for funding
Scientific Score: 79

First Year Funds Requested: $596,068.00
Total Funds Requested: $2,469,104.00

Public Abstract (provided by applicant)

There is much excitement over the possibility of new and fundamentally-different therapeutic applications for human embryonic stem cells and the cells to which they differentiate. In addition, there is equally great excitement in the scientific community regarding the potential of human embryonic stem cells for biological studies of how different human cell types differentiate into normal and diseased tissues. To date, there are more than 400 human embryonic stem cell lines that have been reported to have been derived from human embryos that are donated for research due to lack of suitability for transfer for reproduction or being in excess of the needs for reproductive purposes. The methodology to derive new human embryonic stem cell lines was largely derived from years of work in the clinic that resulted in optimal conditions to grow human embryos and from years of work in animal species, mostly the mouse. In spite of this great progress, we have not, however, yet derived chromosomally-normal (genetically-normal) human embryonic stem cell lines via reprogramming techniques such as somatic cell nuclear transfer. In part this is due to the inaccessibility of human oocytes or eggs for potential therapeutic and basic science applications. In this proposal, we seek to differentiate human oocytes/eggs from multiple hESC lines and use them to reprogram somatic cell DNA. Specifically, we will: 1) Assess and compare the ability of multiple nonfederal hESC lines to contribute to the germ cell lineage (form immature oocytes). 2) Differentiate hESCs to mature oocytes/eggs. 3) Assay the ability of oocytes derived in vitro, relative to immature oocytes donated for research, to reprogram a somatic nucleus. We expect to successfully complete this research with the end results contributing to the improvement of current assisted reproductive practices such as IVF, and improved ability to detect errors in oocytes that lead to birth defects. In addition, since oocytes are particularly susceptible to genetic and environmental perturbation, we expect to develop a system useful for others who study oocyte biology and potential impacts that may cause their demise. Finally, we hope to generate a novel cell source for reprogramming of human somatic cells. This research is clearly aimed at a central issue of women’s health as the formation, maintenance and demise of oocytes significantly impacts female physiology and health. The research is not eligible for federal funding as it relies heavily upon hESC lines that are excluded from federal funding and involves somatic cell nuclear transfer.

Statement of Benefit to California (provided by applicant)

Human embryonic stem cells are generally derived from human embryos that are not suitable for, or are in excess of, the reproductive needs of infertile men and women who present to assisted reproductive clinics. Evidence suggests that human embryonic stem cells can differentiate to many different cell types in the body and in fact, perhaps all the different cell types present in the adult. This is a remarkable fact especially in light of the tremendous burden of chronic diseases and injuries to the citizens of California. Many diseases and injuries, from birth, to childhood and adulthood, have a cellular basis and indeed may arise in the germ cells, the egg and sperm, or early embryo. A particular cell type or process within a group of cells that form a tissue may be specifically defective in disorders that range from cardiac disorders to neurodegenerative disorders and cancers. Thus, there is great hope that perhaps the diverse cell types that can be differentiated from human embryonic stem cells may be differentiated to cells with potential for novel therapeutics; however, immunological rejection after transplantation will be an obvious hurdle unless we can make cell lines that are compatible to individual genetic makeup. In addition, there is great hope that human embryonic stem cells will provide a system for the study of human genetic disorders and/or pharmacological properties. In this application, we propose to systematically examine the ability of hESC lines to form oocytes (eggs). This will allow us to characterize the potential of different lines more precisely and also provide us with a novel source of oocytes for basic studies and ultimately, it is hoped for somatic cell nuclear transfer in order to match the specific genetic makeup of individual patients. We compare results to those from donated immature oocytes. This research is of great promise to the citizens of California. We can develop new methods to diagnose normal oocyte development and potential that can be translated to diagnostic tests for patients who present for infertility. Indeed, infertility afflicts 10-15% of couples and it is these individuals who provide many of the resources necessary for human embryonic stem cell research. In addition, we will be able to develop new tests for diagnosis of common birth defects that arise in oocytes (such as imprinting and/or chromosomal abnormalities). Finally, it is hoped that we will provide a new source of oocytes without the ethical issues surrounding donation for potential therapeutics for chronic diseases and injuries that plague many in our state.

Review

SYNOPSIS: One of the natural and unique functions of the human oocyte is the ability to reprogram somatic cell nuclear DNA. This application targets a basic understanding of human oocyte development. To accomplish this significant goal, the Principal Investigator (PI) proposes three aims for the research.

The first aim is to assess and compare the potential of multiple human embryonic stem cell (hESC) lines to contribute to the germ cell lineage (form immature oocytes). In this aim, the PI proposes to quantitatively examine the ability of multiple non-federal lines relative to several NIH registry lines for their ability to contribute to the germ cell lineage as well as somatic cell differentiation. Successful accomplishment of this aim will provide lines of superior potential and stability.

The second proposed aim is to differentiate hESCs to mature oocytes. In this aim the PI seeks to differentiate hESCs to primoridial germ cells (PGCs)—and then, specifically, to oocytes. The success of this aim will generate a platform in which human oocytes can be obtained directly from hESC cultures.

The third aim is to assay the ability of these in vitro derived oocytes to reprogram a somatic nucleus. In this aim, the PI will assess the ability of the oocytes obtained from hESC differentiation for their ability to reprogram somatic nuclei during the oocyte to embryo transition. The successful execution of aim three will provide a means to reprogram human nuclei without direct oocyte harvest from donors.

IMPACT AND SIGNIFICANCE: All the reviewers noted that the proposed research, if successful, could have significant benefit for reproductive (assisted reproduction technology, ART) research and for hESC research and derivative cell therapies.

Perhaps mostly because of ethical and financial hurdles inherent to hESC biology over the last several years, very little progress has been made in understanding the potential of hESCs to contribute to the germ lineage. This proposal tackles this issue head on, with a focus on oocyte derivation that is relatively new. It is proposed by a leader in the field, who has made a name contributing to our fundamental knowledge of human oocyte development. In this sense alone, this grant is of the highest significance. In addition to filling our deplorable gap in knowledge of germ cell specification in humans, which on its own deserves full support, this project also has a direct impact on the clinical interface of hESCs. In that area, there are at least two independent major contributions that will result from this work. One is at the level of ART as it would provide an inexhaustable source of oocytes for womens’ reproductive health. Second is on the reprogramming aspects essential for cell based therapy. If hESCs could be used to generate oocytes, then it would we would no longer need to recruit women as egg donors for somatic cell nuclear transfer (SCNT) experiments. Women, serving as egg donors for SCNT research raises logistical, ethical, societal and medical questions and finding a way around their participation in this research would be desirable. Thus, this proposal impacts strongly the basic research of human development as well as a direct impact on the application in the clinic.

QUALITY OF THE RESEARCH PLAN: Most of the reviewers found this to be a very well written application with a reasonable research plan and well defined aims. The scientific and technical approaches reflect the talent of the investigator. In each individual experiment the outcome analysis is done to satisfaction. In addition, the review of the sometimes contradictory work done in this subject is a testament to the unbiased nature of the PI.

There is increasing evidence that the different hESC lines have rather different abilities to differentiate into a particular cell type and surveying the various lines for their ability to make germ cells seems like the logical place to begin. One reviewer stated that successful accomplishment of this aim alone would justify funding for this project, and thus this investment guarantees the best return for the money. Likewise, this group can capitalize on their substantial experience with working on human oocytes and preimplantation embryos to try and advance towards producing bona fide oocytes from germ cells. Finally, the idea of maturing human oocytes for SCNT is a very good one and might provide some progress in the field. Although this group has no experience in SCNT themselves, they have aligned themselves with those that do and this reviewer felt that this is a legitimate team.

Another reviewer considered the research plan lacking in strategic context and mechanistic insight. Aim 1, to assess and compare the potential of hESC lines to contribute to germ cell lineages, is a straightforward test of multiple lines, using established conditions and markers of germ cell lineage. The aim is more a listing of methods than a strategic analysis of the experiments. It is largely descriptive and will provide some understanding of the heterogeneity without much insight into mechanism. Aim 2, to differentiate hESCs to oocytes, is an area of great need but unfortunately, again the aim is largely a list of methods, without much discussion of ways the investigators will improve oocyte development. They also do not build on the observations of aim 1. The imprinting studies are a bit confusing. The proposed measure of germ cell commitment is by a measure of epigenetic modification. Yet imprinting and epigenetic modification can be disrupted in ES or EG cells in culture, as shown by several groups. Furthermore, the epigenetic modification is not an obligate feature of imprinting and may arise late. Allele-specific expression would be more reliable. The epigenetic changes might be difficult to see as well. The method proposed to distinguish alleles is not a quantitative method and requires a linked polymorphism to distinguish alleles. The third aim is to assay the ability of differentiated germ cells to reprogram a somatic nucleus. The investigator and colleagues will use a large number of suitable oocytes per year from the clinic. Given that this is a huge experimental barrier at present, it is not clear how they will improve the technology. This aim could be a grant in and of itself in the hands of an expert SCNT group. The aim is not well developed in the application, and the main issue, how to improve the technology, is not substantially explored.

STRENGTHS OF PROPOSAL: The strongest aspect of this grant is the quality of the PI and the group undertaking the task. The PI is a world-renowned scientist and a well respected embryologist. The group has the pertinent experience required to be successful. The institution represents the perfect environment to undertake this type of work. The application addresses an important problem. Contingency plans seem to be well laid out. The generation of oocytes from hESC would be a valuable resource if the research were successful. Because of this application’s scope and topic, this would never be funded by the NIH. The proposed research is directly online with the aims of the CIRM.

WEAKNESSES OF PROPOSAL: Most of the reviewers found this to be a strong proposal, the only shortcoming being that it might be considered overly ambitious, as the PI is proposing a series of very tough experiments to be accomplished in four years. This, however, should not be held against the PI, as his/her track record clearly shows that an ability to accomplish goals proposed. Another reviewer expressed concern about the lack of strategic depth; methodological problems in the imprinting analysis and a weak third aim as well as noting the lack of much detail in the types of experimental manipulation proposed.

DISCUSSION: This application is from a strong investigator, a world expert in embryology. The PI proposes to study human oocyte development including comparing different lines for ability to produce oogonial stem cells; differentiating hESC to oocytes and then investigating hESC derived oocytes for their ability to reprogram somatic nuclei. The significance of the proposed research is high. The work would provide a basic understanding of human oocyte differentiation and if successful could address a major stumbling block in hESC research.

The proposal is very well-written, the aims are well-defined and flowed well; it was a pleasure to read. The scope of the proposed research is of a volume and breadth that is amazing and from anyone else would question feasibility but the PI has an excellent track record. There is solid preliminary data. The PI is one of few persons in the world who has (or will shortly have) permission to perform SCNT and is the only group in the world with expertise in in vitro maturation of germinal vesicle oocytes.

One reviewer, while agreeing that the preliminary data was very solid and that the proposal was for significant and important work, was concerned by the lack of strategic context for the work which was unexpected from a senior investigator. This reviewer found a lack of integration among the aims.

There was discussion about the epigenetic analysis of imprinted genes to determine if reprogramming occurred . There was concern that epigeneitic instability in stem cells may influence the ability to follow this epigenetic modification as a marker for reprogramming by the methods proposed.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• Feit, Marcy
• Lansing, Sherry
• Sheehy, Jeff