Basic Biology I
Infertility affects 6.1 million individuals and their partners in the United States. The underlying causes of many cases of infertility are not known. The germ cell lineage is central to fertility, as this cell lineage is ultimately responsible for creating eggs and sperm. Therefore, abnormal formation and function of germ cells would undoubtedly be a major cause of infertility. Formation of human germ cells begins very early in fetal life. During this time, fetal germ line cells undergo germ cell-specific reprogramming events to create the foundation upon which further germ cell differentiation into functional gametes can occur. Currently, we do not understand the basic mechanisms by which human fetal germ cells differentiate, or undergo germ cell reprogramming. This lack of understanding is due to the fact that up until recently, there has been no robust method for examining human germ cell formation during fetal life. Now, the use of human pluripotent stem cells has created the opportunity to generate fetal germ cells in the lab, and to use these in vitro derived germ cells to study the basic mechanisms that control germ cell differentiation. Using this method the experiments outlined in this proposal are directed at understanding how the supporting cells of the fetal gonad regulate differentiation of human fetal germ cells. Results from this work are directed towards establishing the enabling technology for understanding the basic mechanisms that regulate germ cell formation and therefore deciphering the underlying causes of human infertility, germ cell tumors or birth defects.
Statement of Benefit to California:
In the short term, the proposed research is designed to create jobs as a technical position will be created to undertake the proposed research program. In the long term, the goal of the proposed research is to create new technology which could be used by academic, research and development or pharmaceutical laboratories. Finally, this research program is ultimately designed to benefit human reproductive health. This problem is not restricted to Californians, however 6.1 million individuals and their partners are diagnosed as infertile in the United States, and results from this grant are designed to understand the underlying causes of this disease.
The overall goal of this proposal is to generate progenitor germ cells, called primordial germ cells (PGCs), in vitro from human pluripotent stem cells and then evaluate the role of gonadal somatic cells in PGC differentiation. In Aim 1, the applicant proposes to compare imprint erasure, a criticial step in germ cell differentiation, between PGCs derived from human embryonic stem cells (hESCs) and those derived from induced pluripotent stem cells (iPSCs). In Aim 2, the applicant will investigate whether the stage of co-cultured human gonadal somatic cells affects imprint erasure in pluripotent stem cell-derived PGCs. Finally, in Aim 3, the applicant proposes to test the hypothesis that human gonadal somatic cells suppress the transcription of pluripotency genes by analyzing their effect on gene expression in co-cultured human pluripotent stem cells. Reviewers agreed that this proposal addresses a major unsolved problem and could have a significant impact. The proposed in vitro model system could enable mechanistic study of an otherwise difficult to access cell type, human germ cells. These cells could also serve as tools for toxicological screening. However, without evidence that the in vitro specified PGCs could complete the first meiotic division in culture, one reviewer found the applicant’s suggestion that in vitro specified PGCs could be used as a source of gametes for the treatment of infertility to be premature. Reviewers did not find the proposal to be particularly innovative, as no novel approaches are being utilized or developed. While the proposal has the potential to provide new mechanistic insights, the experimental plan is limited in scope, overly descriptive and not focused on mechanism. Reviewers raised a number of issues with the research plan that they felt would limit its impact. They appreciated that the plan is straightforward and clearly outlined, with appropriate descriptions of potential pitfalls, alternative approaches and milestones. The proposal’s strengths include the applicant’s demonstrated ability to generate PGC from pluripotent cells and an established bank of human gonadal somatic cell lines. However, reviewers expressed concern whether statistical significance can be achieved in the proposed experiments. For example, Aim 1 will compare PGCs derived from six hESC and five hiPSC lines for imprint erasure at five imprinted loci. Given the variation of demethylation described for three loci in the preliminary data, the five loci proposed for the study of imprint erasure may be inadequate. One reviewer suggested a whole genome analysis of sex-specific CpG methylation profiles should be pursued instead. Similarly, concern was expressed that enough cell lines would be analyzed to achieve the statistical power necessary for the assessment of gonadal somatic cell developmental stage on imprint erasure in Aim 2. Furthermore, differences may occur in the kinetics of hESC lines’ imprint erasure, complicating the interpretation of hESC to hiPSC comparisons. Reviewers felt that the labor-intensive expression profiling approach would not prove informative, especially since the proposed studies will generate large quantities of data, but a detailed discussion of their interpretation is lacking. Reviewers praised the applicant’s track record and expertise in the differentiation of human germ cells from pluripotent stem cells, calling him/her a “leader in this field”. They noted that the applicant has built an excellent research team in an excellent research environment. However, considering the amount of work proposed, they expressed concern that the only team member assigned 100% effort is a research associate to-be-determined. Overall, reviewers appreciated the significance of the scientific question addressed in this proposal and the applicant’s expertise in this field. However, they found the research plan to be limited in scope, overly descriptive and limited in feasibility, thus questioning its potential for impact.