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.
The original goal of this project was to generate oocytes (eggs) from human embryonic stem (hES) cells in cell culture dishes in the laboratory. Such oocytes could be of use as vehicles to reprogram the DNA from cells of patients with life-threatening or debilitating conditions, thereby allowing generation of new lines of hES cells that are immune matched to the patient. The paucity of donated human oocytes precludes research using such material, and production of human oocytes from hES cells in the laboratory would in theory provide a limitless source of material.
Since our last progress report another CIRM-funded group, Dr. Renee Reijo Pera’s lab at Stanford University, has published exciting results demonstrating successful production of primordial oocytes from mouse ES (mES) and human ES (hES) cells. Consequently, during the remaining period of the award we propose to use the Reijo Pera methods for production of female germ line cells in our lab using H9 and HUES-9 female hES cells. After accomplishing this, we will introduce human mtDNA containing mutations that cause either a severe or mild reduction in oxidative phosphorylation (energy) production into H9 and HUES-9 hES cells and investigate the impact of the different mtDNA mutations on the ability of hES cells to form cells with characteristics of PGCs, then primordial oocytes in vitro and in vivo. An important related goal of this research is to investigate whether development of oocytes from ES cells could be used as a method to remove deleterious mtDNA mutations from the hES cell population, thereby improving the utility and possibly safety of derived cell types for therapeutic purposes.
During this reporting period we have focused on our approved revised research plan. We continued our efforts to generate embryonic female germ cells from human embryonic stem (hES) cells in vitro using methods reported in the literature at the end of 2009. Our revised plan included the new goal of using in vitro developed female embryonic germ cells (oocytes) as a resource to investigate how mitochondrial genomic DNA containing deleterious mutations is segregated during female germ cell development. As well as providing novel information about the biology of germ cell development, this research may provide important information relevant to development of safe methods for therapeutic cloning. We began by using two different female hES cell lines (HUES-6 and H9) to investigate whether we could use the reported methods to develop female germ cells from hES in our lab. A third female hES cell line (HUES-9) we originally intended to use was found to have a high propensity to gain an extra chromosome (become aneuploid) and was therefore not used. Despite following the reported methods that demonstrated in vitro differentiation of hES cells into female germ cells, we were unable to reproduce the previously reported results in our own lab. The reasons for this are currently unclear but may involve subtle, but important, differences in the methodology or materials we used.