The ability of human embryonic stem (hES) cells to form a wide variety of adult human cell types offers hope for development of novel therapies to treat human degenerative diseases such as Alzheimer’s, diabetes, and muscular dystrophy. However, to prevent rejection of the transplanted cells by a patient’s immune system it will be important to use hES cell derived tissues that are immunologically matched to the patient. One way to do this involves somatic cell nuclear transplantation (SCNT) where the nucleus containing the genetic information is transferred from a patient’s cell into a human oocyte (egg) from which the nucleus has been removed. The oocyte is then stimulated to divide into a small group of cells from which new hES cells are derived. As cells derived from these hES cells contain the patient’s DNA they will be immune-matched to the patient, thereby preventing tissue-rejection.While SCNT has been performed using cells and eggs from mice, it is not yet possible to do this on a routine basis using human cells and eggs. One reason for lack of progress concerns the scarce supply of human oocytes available for research. Oocytes are usually obtained from women undergoing hormonal treatment for infertility, when permission is given for unused and unwanted oocytes to be used for research. However, a severe shortage and great demand for such material has stimulated efforts to recruit suitable donors from the general public, often with financial incentive. There is significant health and ethical concern about such policies and the potential negative impacts of such procedures on long-term health of women are unclear.Remarkably, recent research with mice suggests that it may be possible to produce oocytes from hES cells. If so, the oocytes produced may be of use for SCNT to produce new lines of personalized-hES cells for treatment of patients. If successful, this would be expected to have at least two major benefits to the public. First, women would no longer be required as a source of eggs for research, which would reduce the risk of such treatments on women’s long-term health. Second, this would generate a theoretically infinite increase in the quantity of oocytes available for research, which would in increase the rate at which technical advances could be made in production of immune-matched hES via SCNT.Consequently, the proposed research will investigate methods for production of germ cells and oocytes from female hES cells. Specifically, we will test our prediction that it is possible to coax hES cells in culture to form germ cells and ultimately mature oocytes by exposing hES cells to different cell proteins and hormones that are normally used by the body to generate eggs. If successful, these protocols will help accelerate research on development of therapeutic cloning for a wide range of diseases. This would also obviate ethical concerns regarding egg-donation by women and would help protect women’s health.
The proposed research involves investigating how human embryonic stem (hES) cells form germ cells and oocytes, in a cell culture dish in the laboratory. The long-term goal of the research is to develop methods that enable production of such eggs in vitro. If successful, the research will benefit Californians in areas of healthcare and economy. Regarding healthcare, the ability to produce significant quantities of human oocytes in the laboratory will accelerate development of methods for reliable development of new hES cell lines from human oocytes following transfer of nuclei from a patient’s cell (i.e. therapeutic cloning). The ability to generate hES cells that are immunologically matched to a patient would also preclude the need for use of immunosuppressive drugs during treatment of patients with hES cell-derived material. Such drugs can have unwanted side effects on patients and could also have as yet unrecognized negative effects on ability of hES cell-derived tissues to engraft into a patient’s organs. Immunosuppressive therapy also increases the overall expense of treatment, due to the treatment itself as well as treating any side effects from its use.
Success of the proposed research would significantly reduce any requirement for donation of unused human oocytes. This would benefit California women by protecting them from undergoing unwanted and unnecessary hormonal stimulation for which the long-term health effects are currently unclear. If successful, the research could also have significant economic benefit to the State of California. First, successful development of this technology could be transferred to biomedical industry, and the potential worldwide market for such methods is considerable. This could stimulate employment in a variety of sectors as well as increasing state tax revenue. Equally important, a successful research outcome could also reduce the overall cost of healthcare to the State. It is anticipated that low-income individuals are more likely to be willing to donate eggs for research. While this may provide them with short term financial benefit, it is possible that such individuals could develop significant age-related health problems involving hormonal imbalance, such as infertility, polycystic ovarian syndrome, bone-loss, cognitive decline and obesity. In this event, the burden of providing healthcare to such individuals would likely fall to the State. Thus, reducing the incidence of egg-donation by in-state residents is likely to minimize any costs associated with treatment of such donors in the future.
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.