Objective: 

We have recently identified a small population of solitary Nanog-expressing spermatogonial stem cells (SSC) that are located specifically in stage XII of the mouse seminiferous epithelial cycle. Besides expressing pluripotency-associated factors Nanog and Oct4, these cells are regulated in an embryonic stem (ES) cell fashion (Ventelä et al. 2012). Due to the complex architecture of the seminiferous tubules the interaction of these cells with their stem cell niche could be studied more thoroughly in vitro. Many different groups have succeeded in establishing long-term SSC cultures. However, none of these systems have been reported to support Nanog-expressing SSCs. This challenged us to develop a method that would allow in vitro studies of these cells. Methods Mouse seminiferous tubules were dissected and cut to short segments, which were moved onto cultivation flasks and cultured in ES cell conditions. The migration and proliferation of the seminiferous tubule cells were monitored up to seven weeks and samples were collected weekly for biochemical analysis.

Results and Conclusions: 

The culture is composed of the constituents of testis stem cell niche: Sertoli, peritubular myoid and spermatogonial stem cells. Nanog is detectable at both protein and mRNA level. Our data demonstrate that one-week-old co-culture offers a stem cell niche for auto- and allogeneic spermatogonia. Glial cell line-derived neurotrophic factor (Gdnf) mRNA levels can be elevated by follicle-stimulating hormone (FSH) which shows that the Sertoli cells respond to physiological stimuli. Nutlin-3 inhibits Nanog and CIP2A (cancerous inhibitor of PP2A) and stimulated p21 mRNA expression, and induced a proliferation and differentiation arrest of spermatogonia.

References: 

Ventelä, S., Mäkelä, J.-A., Kulmala, J., Westermarck, J. & Toppari, J. 2012. Stem Cells 30, 1008–1020.