Recommended if funds allow
Statement of Benefit to California:
SYNOPSIS: This proposal aims to test the ability of a variety of hESC lines to form germ cells in vitro. Building on previous observations which suggested that when cultured as embryoid bodies (EB), hESC can differentiate into cells that look a lot like oocytes, albeit in very few numbers and few cases, the PI proposes to develop new technology to systematically obtain good quality human oocytes from these cultures. Toward this goal the author proposes 3 specific aims. Aim 1 investigates the relative ability of 2 NIH-approved and 7 non-NIH approved hES cell lines to form germ cells and oocyte-containing follicles in vitro. Aim 2 will analyze the effect of co-culture of hES cell derived EBs - that are exposed to different BMP growth factors followed by re-aggregation on 3D polymer scaffolds - on the incidence of formation of germ cells in EBs. Aim 3 will investigate the ability of neurotrophins to facilitate targeted modification of the genome via homogulous recombination at two independent loci in hESCs. INNOVATION AND SIGNIFICANCE: Previous studies have reported that both in the context of mouse and humans, ESCs can differentiate into germ cells or germ cell progenitors. While some of these findings remain controversial and have not been independently reproduced, the ability to generate germ cells, especially oocytes, from hESCs remains one of the highest priorities for this bourgeoning field. The successful accomplishment of this goal has great significance for both basic and clinical research, with a direct impact on SCNT. Obtainment of human oocytes is clearly the limiting factor in SCNT, which has forced some investigators to desperately perform SCNT across species, using human somatic nuclei in rabbit oocytes. Obtainment of healthy, fertilizable human oocytes from hESCs would be the most effective way to overcome this important limiting step. STRENGTHS: The principal strengths of this proposal are its collaborative nature and a well-designed experimental approach. The PI is using overwhelming power in three specific aims to tackle the issue, using 9 hESC lines from both registry and non-registry collections, comparatively using EB, and VASA as a germ cell specific marker. The use of what has been published for the mouse, namely the TGFb ligands BMP2 and BMP4, to address signaling pathways and perhaps thresholds required for the optimization of germ cell specification and finally the generation of genetically marked hESCs for Alkalin Phosphatase and VASA as reporters of optimization in specific aim 3 all represent the tremendous strength of the approach. WEAKNESSES: The main weakness of the proposal is the lack of preliminary results, starting with the reproducibility of what has been published. The simple reproduction of the mouse work on oocyte induction is not shown. One might assume if these investigators are serious about this research they would have at least attempted some experiments with mouse or human cells prior to applying for funding. In addition, Aim 2 concentrates entirely on the use of BMPs despite a clear lack of evidence that these molecules are important in humans for the induction of PGCs. A minor weakness of specific aim 2 is that a single concentration of BMP2 or 4 is proposed while it is appreciated by the TGFb signaling field that these ligands operate as morphogens eliciting different outcomes based on their threshold of activity. Exposure to different concentrations of the same ligands or the variation on timing of exposure to the ligands might optimize the desired outcome. DISCUSSION: This is a high-priority, high-risk application to optimize the formation of germ cells in-vitro. Generation of germ cells from mouse ES cells has been previously reported but not reproduced and therefore a concern of the reviewers. A key strength of the proposal is in the use of several lines to find out which are most able to form germ cells, and the testing of different BMP growth factors. Reviewers raised a minor question about using only a single concentration of BMP2/4. One reviewer commented that Aim 3 seems to be a little irrelevant to the rest of the story (seems to be tacked on), but the homologous recombination will be important in genetically marking the newly-formed germ cells. These markers (Alkaline Phosphatase and VASA) might also be useful in subsequent small molecule screens. Another reviewer noted that these reporters are expressed at high concentrations in undifferentiated cells, and said that a discussion of other useful markers would have helped.