The Cluster-Forming Activity Assay: A Short-Term In Vitro Method to Analyze the Activity of Mouse Spermatogonial Stem Cells - Male Germline Stem Cells Developmental and Regenerative Potential - page 132

6.3.5 Is Cluster Number the Only Readout of the CFA Assay?

The readout of the CFA assay discussed thus far was only cluster numbers, but the
constituents of clusters (SSCs and non-stem spermatogonia) can be another assay
outcome. It is conceivable that each cluster may represent a heterogeneous cell
community, as 2% or less of cluster cells have been estimated to be SSCs (Yeh et al.
2007 ; Kanatsu-Shinohara et al. 2005 ; de Rooij 2006 ). This suggests that as SSCs
divide and form clusters in culture, they not only self-renew but also generate cells
that are committed to differentiation. Such committed cells may also proliferate in
response to added growth factors, composing the vast majority of cluster cells.
Based on the detection and quantification of committed cells in clusters, therefore,
the CFA assay may potentially provide parameters to analyze the degree of sper-
matogonial differentiation. Since early spermatogenesis has been difficult to study
experimentally in vivo , the CFA assay, which provides a defined environment
in vitro , can be an ideal research tool to understand the regulatory mechanism of
SSC/spermatogonial differentiation.
One approach to accomplish this may be to measure the size of clusters. Since the
majority of cluster cells are apparently committed cells, the cluster size can be indica-
tive of the differentiation activity and the production level of differentiating cells.
To evaluate differentiation events in a more definitive manner, we need to deter-
mine differentiation markers. The most common marker thus far used is KIT (c-kit
proto-oncogene), since SSCs cannot be enriched in the KIT-positive cell population
(Shinohara et al. 1999 ; Ebata et al. 2005 ); a recent study disputed this notion
(Barroca et al. 2009 ), and further studies are required to clarify this issue. Thus,
spermatogonial differentiation can be monitored by detection of differentiation
marker expression. Alternatively, the loss of SSC markers can also allow for assess-
ing differentiation. In both cases, immunohistochemical analyses will give endpoint
readout, while the use of SSCs carrying fluorescent markers driven by a promoter
of a marker molecule will support a real-time observation of differentiation events.
Likewise, changes in cell cycle and apoptosis in clusters can be additional assay
readouts. The CFA assay can thus be an important tool to analyze spermatogonial
behavior and early spermatogenesis in vitro in a quantitative manner.

6.3.6 Other Considerations

The CFA assay is perhaps most effective when used in ex vivo studies, as shown in the
above examples. When this assay is applied for in vivo studies (to measure SSC activity
after in vivo manipulation), one issue that needs to be considered is the fact that cluster
induction requires enrichment of testis cells for SSCs (see Chap. 5). The degree of SSC
enrichment may vary, for example, if the expression of selection markers is affected by
in vivo manipulation. Thus, the requirement of an additional procedure before the CFA
assay represents a weakness of this technique when applied for in vivo studies.