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 proposes three aims for the research.
The first aim is to assess and compare the potential of multiple hESC lines to contribute to the germ cell lineage. In this aim, the PI proposes quantitatively examine the ability of 12 non-federal lines relative to 3 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 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 differentiated germ cells 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 reprogramming 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 (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 germ line. 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 assisted reproduction technology 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 human ES cells could be used to generate oocytes, then it would we would no longer need to recruit women as egg donors for SCNT experiments. Women, serving as egg donors for SCNT research, raises logistical, 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: Two 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 NT 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 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 15 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. They will use the level of methylation of DMRs in several imprinted genes as a measure of germ cell commitment. Yet imprinting and methylation can be disrupted in ES or EG cells in culture, as shown by several groups. Furthermore, the methylation is not an obligate feature of imprinting and may arise late. Allele-specific expression would be more reliable. The methylation changes might be difficult to see as well, since they might involve a change from ½ methylated to full or zero methylated, and back to ½. Cloning PCR products after bisulfite treatment 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 up to 200 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. Dr. Pera is a world-renowned scientist and a well respected embryologist. The group has the pertinent experience required to be successful. UCSF 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: Two 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 taken against the candidate, as her track record clearly shows that she is able to accomplish the goals that she sets out. 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, the feasibility of the proposed research would be questioned 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. This reviewer also had concerns with the generation of embryos after SCNT - specifically how far are they being grown. This did not enter into scoring but the reviewer was was concerned as this is a very charged ethical issue.
There was discussion about the methylation of imprinted genes to determine if reprogramming occured by quantifying the level of DNA methylation. There was concern that DNA methylation instability in stem cells may influence the ability to follow methylation as a marker for reprogramming by the methods proposed.