Towards 'in vitro' produced germline stem cells in the bovine

Abstract

Bovine spermatogonial stem cells (SSCs) have potential to be used in advanced reproductive technologies such as testis cell transplantation, where identification and purification of large numbers of SSCs is required. There are at least two possible sources of SSCs: isolation from the testis, or in vitro differentiation from pluripotent stem cells. The long-term goal of this thesis was to work towards the generation of SSCs from bovine somatic cells using induced pluripotent stem (iPS) cell technology. In order to do so, it was first important to characterise molecular markers expressed by bovine SSCs to allow for their identification in culture, and secondly to explore the feasibility of producing bovine iPS cells. In order to achieve the first goal, a screening platform was developed based on comparative analysis of gene expression levels in SSC enriched and depleted cell populations. Expression of established testis cell markers was used to confirm the validity of the screening platform. This method was then used to examine expression of candidate spermatogonial markers in the bovine testis. STRA8, KIT, GFRA1, CLDN8, DDX6 and NAP1L4 were shown to be putative markers for bovine spermatogonia. Further analysis of CLDN8 showed expression by both a subset of spermatogonia and a subset of Sertoli cells, leading to the hypothesis that CLDN8 plays a role in the maintenance of SSCs in the SSC niche. Reprogramming of bovine somatic cells was undertaken by introducing canonical reprogramming factors through a lentiviral vector. Initial experiments found that the reprogramming protocol was sufficient to produce cells exhibiting stem cell-like characteristics. Analysis of these cells indicated partial reprogramming had been achieved. A number of small molecules were then tested for their ability to enhance the success of cell reprogramming. A combination of three small molecules was found to accelerate the kinetics of the reprogramming process and also promoted further reprogramming where cells could differentiate to the three different germ layers. Further research is now required to define the optimal culture conditions for the maintenance and expansion of bovine pluripotent cells in long term culture, and to test whether they can be differentiated towards the germ line.