Exploration of the oogenic potential of putative germ line stem cells isolated from the ovaries of girls and adult women

For decades, scientists have believed that women were born with all the eggs they would ever have and that no new ones could ever be made. Recent work has shown that the ovaries of adult women have cells that are capable of forming what appears to be new eggs under certain conditions. The implications of this are potentially enormous. If we could make new eggs we could eventually help restore fertility in women who have become infertile for a variety of reasons, including chemotherapy treatment, early menopause or just normal aging. We need to learn more about these cells and determine the conditions that allow them to form eggs and to know whether the eggs are normal. We have recently isolated two populations of cells and we need to study them in more detail. This project will define the cells that can be isolated from ovarian material from a range of ages. Then using a system where we can grow eggs outside the body (culture system), we will determine whether these cells can form new eggs and whether they are normal. We will also find out the conditions that support this process, The ultimate end point of this work would be to determine whether these cells were capable of forming eggs that could be fertilised and form normal embryos.

Cells with germ line potential have been isolated and purified from ovaries of a range of mammalian species including adult women. The ability to develop these cells into mature oocytes would have many applications in assisted reproduction and fertility preservation as well as being of great value as a model for the study of human oogenesis. However, questions still remain whether these cells are bona fide oogonial stem cells (OSCs) or the result of an in vitro transformation. In our previous MRC funded study, we developed a tissue dissociation protocol yielding a high number of viable cells amenable to FACS and molecular analysis without the need for expansion in vitro. The germ line marker DDX4 (at both gene and protein level) has been demonstrated in these freshly isolated cells and a FACS technique incorporating dual-detection of DDX4 with aldehyde dehydrogenase 1 (ALDH1) (marker of progenitor and stem cells) demonstrates that the putative OSC population in human ovaries can be divided into two sub-populations distinguished by expression of differentially spliced DDX4 transcripts and of DAZL, a major regulator of germ cell differentiation. The aim of this proposal is to determine the optimal sorted cell population to form human oocytes (determined by morphology and expression of oocyte specific genes) and to determine the somatic cell support that will facilitate this process. The function and developmental potential of the sub-populations of human OSCs will be tested by combine them with somatic cells from fetal and adult ovarian sources as well as injection of cells into ovarian cortex that can be developed in vitro. Once oocytes/follicles have formed we will be able to explore robustly the full meiotic and developmental potential of these cells and determine whether they are functional oocytes. These studies will provide the basis for exploring the therapeutic potential of these cells in areas such as Fertility Preservation and Infertility treatment.

This research will inform how we view the human ovary and will have impact in several areas academic, clinical (including patient support) and commercial communities. We have established connections in all these areas. This work is dealing with a highly contentious area which has been mired in confusion and uncertainty. The results of this project will have a direct impact on our basic thinking of how the ovary works but could also have an impact on medical practice and society as a whole in terms of strategies for fertility preservation or even restoration, and in assisted reproduction. The pathways to impact outlines our approach to engaging with interested parties. This study will impact the wider scientific community as it may alter the way in which we view ovarian function. Because of our network of scientists and clinicians we are well placed to anticipate potential impacts and prepare for the next stage of implementation.