Pluripotent adult stem cells from the mouse testis

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Master of Science to the School of Molecular Sciences, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

Spermatogonial Stem Cells (SSCs) obtained from the testis can give rise to cells in vitro with pluripotent characteristics similar to those of Embryonic Stem (ES) cells. The pluripotency transcription factor Nanog is expressed in these pluripotent ES-like cells and may have a role in their derivation. We have found, using a LacZ reporter allele in the pluripotency gene Nanog, that a unique population of Nanog expressing cells exist in the testis throughout development and in the adult. Nanog may therefore be a unique marker of pluripotent stem cells in the testis. We have tested multiple culture conditions in an effort to optimise the culture of SSCs and the generation and propagation of ES-like cells from the testis. These methods, though proven to be successful for other research groups, did not improve the efficiency of our SSC and ES-like cell culture. Despite difficulties encountered, we obtained ES-like cells, as supported by long term self-renewal, morphology and immunocytochemistry of pluripotency markers. However, these cells proliferated too slowly to be useful in further analysis and attempts to accelerate cell expansion failed. We investigated Nanog expression in the stem cell compartment of adult mouse testis by flow cytometric analysis. Although, we were able to successfully purify Side Population stem cells, spermatocytes and spermatids, correlation with Nanog gene expression was not achieved due to technical difficulties. Testis derived pluripotent stem cells may hold great promise for future biomedical research and application in human regenerative and therapeutic treatments; however, our understanding of their biology is still limited. Our investigation has shown how crucial the culture environment is and how difficult and unpredictable working with these cells can be without efficient isolation and culture protocols.