Establishment of a Short-Term In Vitro Assay for Mouse Spermatogonial Stem Cells1

Abstract

Spermatogonial stem cells (SSCs) are responsible for life-long, daily production of male gametes and for the transmission of genetic information to the next generation. Unequivocal detection of SSCs has relied on spermatogonial transplantation, in which functional SSCs are analyzed qualitatively and quantitatively based on their regenerative capacity. However, this technique has some significant limitations. For example, it is a time-consuming procedure, as data acquisition requires at least 8 weeks after transplantation. It is also laborious, requiring microinjection of target cells into the seminiferous tubules of individual testes. Donor-recipient immunocompatibility for successful transplantation and large variations in data obtained represent further limitations of this technique. In the present study, we provide evidence that a recently developed SSC culture system can be employed as a reliable, short-term in vitro assay for SSCs. In this system, donor cells generate three-dimensional structures of aggregated germ cells (clusters) in vitro within 6 days. We show that each cluster originates from a single cell. Thus, by counting the clusters, cluster-forming cells can be quantified. We observed a strong linear correlation between the numbers of clusters and SSCs over extended culture periods. Therefore, cluster numbers faithfully reflect SSC numbers. These results indicate that by simply counting the number of clusters, functional SSCs can be readily detected within 1 week in a semi-quantitative manner. The faithfulness of this in vitro assay to the transplantation assay was further confirmed under two experimental situations. This in vitro cluster formation assay provides a reliable short-term technique to detect SSCs.

Wnt5a is a cell-extrinsic factor that supports self-renewal of mouse spermatogonial stem cells

“…3B). Because cluster numbers correlate with SSC numbers (Yeh et al, 2007), these results suggest that inhibition of -catenin signaling did not affect SSC activity. However, sFRP1 dose-dependently reduced cluster numbers (Fig.…”

“…These mice carry the lacZ reporter gene linked to -catenin-TCF/LEF binding sites, allowing for faithful monitoring of -catenin signaling activation (Mohamed et al, 2004a). The cultured cells formed three-dimensional aggregates of spermatogonia (Kubota et al, 2004b;Yeh et al, 2007), termed 'clusters' hereafter. lacZ expression was observed in a subpopulation of cluster cells (Fig.…”

The effects of poly L-lactic acid nanofiber scaffold on mouse spermatogonial stem cell culture

“…44 The diameter and the number of colonies were determined every 7 days during the culture for 3 weeks. Cluster formation was assessed using the procedure described by Yeh et al 45 Identity confirmation of the spermatogonial cells…”

Spermatogonial stem cell biomarkers: improved outcomes of spermatogonial transplantation in male fertility restoration?

“…These cultures require the presence of GDNF and FGF2, and under these conditions, SSCs form distinct cell accumu lations, or 'clusters', of spermatogonia. Transplantation of cluster cells leads to reconstitution of spermatogenesis demonstrating that SSCs are found in the clusters [37]. Clusters can be passaged serially and maintained virtually indefinitely (for at least up to 2 years [38]), without losing SSC activity.…”

“…Clusters can be passaged serially and maintained virtually indefinitely (for at least up to 2 years [38]), without losing SSC activity. Using these culturing systems, therefore, SSCs can be robustly amplified (4000-to 5000-fold over 10 weeks [35,37]). GDNF is an indispensable growth factor to achieve in vitro expansion of mouse SSCs.…”