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This Thesis Has Been Submitted in Fulfilment of the Requirements for a Postgraduate Degree (E.G This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. Isolation, Characterisation and In Vitro Potential of Oogonial Stem Cells Cheryl Elizabeth Dunlop BSc Med Sci (Hons), University of Edinburgh MBChB (Hons), University of Edinburgh Doctor of Philosophy (PhD) – The University of Edinburgh – 2016 Declaration This thesis has been composed by myself and the research described herein is my own, except where work by others has been duly acknowledged. The work described in this thesis has not been submitted for any other degree or professional qualification. Cheryl Dunlop 2016 i Abstract The longstanding belief that women are born with a finite ovarian reserve has been debated for over a decade, ever since the discovery, and subsequent isolation, of purported oogonial stem cells (OSCs) from adult mammalian ovaries. This rare cell population has now been reported in the mouse, rat, pig, rhesus macaque monkey and humans and, although a physiological role for the cells has not been proven, they do appear to generate oocytes when cultured in specific environments, resulting in live offspring in rodents. The primary aim of this thesis was to verify independently the existence of OSCs in human ovary and determine whether they could be isolated from a large animal model, the cow. The secondary aim was to investigate the cells’ in vitro potential, both to undergo neo-oogenesis and as a model for germ cell development. Putative bovine and human OSCs were isolated from disaggregated adult ovarian cortex using a previously validated fluorescence-activated cell sorting (FACS)-based technique, with cells sorted for externally expressed DDX4 (VASA). Freshly isolated and cultured cells were characterised by analysing their expression of pluripotency and germline markers, using RT-PCR, immunocytochemistry and Western blotting. The in vitro neo-oogenesis potential of the cells was explored by injecting fluorescently labelled cells into fragments of adult ovarian cortex and by forming aggregated artificial “ovaries” with putative OSCs and fetal ovarian somatic cells. Germ cell model experiments comprised treatment of cultured cells with BMP4 and/or retinoic acid (RA), with subsequent quantitative RT-PCR and immunocytochemistry analysis for downstream BMP4- and RA-response genes, and liposomal-mediated transfection of cells with a DAZL overexpression plasmid to assess their meiosis-related gene response. Scarce populations of putative OSCs were retrieved from 5 human samples (aged 13- 40 years) and 6 bovine samples. The cells were cultured long-term for up to 7 months and demonstrated consistent expression of several pluripotency-associated and germline markers at the mRNA and protein level, including LIN28, NANOG, POU5F1 (OCT4), IFITM3 (fragilis), STELLA, PRDM1 (BLIMP1), and C-KIT, indicating their early germline nature. Investigation of neo-oogenesis potential revealed that putative human OSCs were associated rarely with fetal somatic cells in ii primordial follicle-like structures, but could not be confirmed to have undergone oogenesis. However, like early germ cells, putative bovine and human OSCs were BMP4 and RA responsive, with both species demonstrating significant upregulation of expression of ID1 and bovine cells exhibiting a significant increase in MSX1, MSX2 and the meiotic marker SYCP3 in response to BMP4 and/or RA treatment. Cells could be successfully transfected to overexpress DAZL; however, no significant downstream gene expression changes were observed. This is the first report of putative bovine OSC isolation and corroborates a previous report showing putative human OSC isolation. Although the expression of both stem cell and germline markers indicates the cells have characteristics of OSCs, their capacity to enter meiosis and form functional oocytes has yet to be determined. Putative bovine OSCs, however, show promise as a novel model for investigating germ cell development. If their potential can be harnessed, then OSCs may have a role in clinical applications, for example in fertility preservation, in the future. Future experiments will examine the neo-oogenesis capabilities of the cells further and explore novel cell delivery systems for clinical use. iii Lay Summary It has long been believed that women are born with all the eggs they will ever have. However, since 2004, unique cells have been detected in the ovary that seemingly make new eggs when grown in the right conditions. These cells, called oogonial stem cells (OSCs), have been isolated from mice, rats, pigs, monkeys and humans. When mouse and rat OSCs are transplanted back into ovaries, live mice and rats have been born from eggs apparently created by the stem cells. However, there is no proof that these cells make new eggs under normal circumstances within the ovary and the idea of egg-producing stem cells has been controversial, with many scientists challenging the findings. The aim of this research was to investigate whether these cells exist in cow and human ovaries and, if so, to explore whether they could make new eggs when grown in a dish. By cutting up pieces of ovary and using an antibody to select cells containing a marker called DDX4, a rare population of cells was isolated from both bovine and human adult ovaries. These cells exhibited markers of a stem cell with egg-like qualities, implying the cells may be able to divide repeatedly over a long period of time and also develop into new eggs. The cells multiplied successfully for up to 7 months in the laboratory and continued to show the stem cell and egg markers. Attempts were made to create eggs from the cells by injecting them back into ovaries and also by creating “artificial ovaries” by combining them with another type of cell that supports egg growth, but it could not be proven that the cells could form new eggs. However, when the cells were treated with two substances (BMP4 and retinoic acid) that encourage the precursors of eggs to mature into eggs before females are born, they behaved in the same way as the egg precursors. This suggested that these cells may provide a great opportunity to look more closely at how eggs grow and develop as they could be used in experiments to simulate what happens to these egg precursors in real life. This is of great scientific value as there is a very limited availability of human eggs to perform experiments on. This is the first time in the world that these cells have been discovered in the cow and confirms previous research in humans. If these cells can form new, healthy eggs, then they could provide a new treatment option for girls and women at risk of infertility due to cancer treatment who, due to age or time constraints, cannot have eggs collected iv prior to starting treatment. Future experiments will explore further the ability of the cells to form new eggs in a dish and also investigate new ways of delivering the cells into ovaries so they can be used in the hospital setting. v Publications relating to this thesis Published papers Dunlop CE, Telfer EE, Anderson RA. Ovarian stem cells - potential roles in infertility treatment and fertility preservation. Maturitas. 2013 Nov;76(3):279-83. Dunlop CE, Telfer EE, Anderson RA. Ovarian germline stem cells. Stem Cell Res Ther. 2014 Aug 18;5(4):98. Dunlop CE, Anderson RA. The regulation and assessment of follicular growth. Scand J Clin Lab Invest Suppl. 2014;244:13-7; discussion 17. Grieve KM, McLaughlin M, Dunlop CE, Telfer EE, Anderson RA. The controversial existence and functional potential of oogonial stem cells. Maturitas. 2015 Nov;82(3):278-81. Full text of first author publications can be found in Appendix 2. vi Presentations relating to this thesis Oral presentations Human and bovine ovarian cortex contain oogonial stem cells with potential for modelling germ cell development and entry to meiosis. Dunlop CE, McLaughlin M, Bayne RA, Rosario R, Telfer EE, Anderson RA. British Fertility Society Birmingham, UK 2016 The isolation, characterisation and in vitro culture of ovarian stem cells. Dunlop CE, McLaughlin M, Bayne RA, Telfer EE, Anderson RA. Edinburgh Obstetrical Society Edinburgh, UK 2013 Poster presentations Isolation, propagation and characterisation of oogonial stem cells from human and bovine ovarian cortex. Dunlop CE, McLaughlin M, Bayne RAL, Anderson RA, Telfer EE. Society for Reproduction and Fertility Edinburgh, UK 2014 Isolation, propagation and characterisation of oogonial stem cells from human and bovine ovarian cortex. Telfer EE, Dunlop CE, McLaughlin M, Bayne RAL, Anderson RA. ESHRE Annual Meeting Munich, Germany 2014 vii Isolation, purification, and culture of oogonial stem cells from adult human and bovine ovarian cortex. [Abstract published in Lancet. 2014 Feb; 383(S45)]. Dunlop CE, Bayne RA, McLaughlin M, Telfer EE, Anderson RA. Academy of Medical Sciences Spring Meeting London, UK 2014 Isolation, characterisation and in vitro culture of oogonial stem cells from adult human and bovine ovarian cortex. Dunlop CE, Bayne RA, McLaughlin M, Telfer EE, Anderson RA. RCOG Annual Academic Meeting London, UK 2013 Isolation, purification and culture of oogonial stem cells from adult human and bovine ovarian cortex. Dunlop CE, Bayne RA, McLaughlin M, Telfer EE, Anderson RA.
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