Biomedical Engineering Reference
In-Depth Information
Another issue is that the effectiveness of the CFA assay has not been evaluated
using adult SSC-derived clusters. Since clusters can be more readily induced with
pup than adult testis cells (Chap. 5), the CFA assay was established using pup SSCs.
However, adult SSC-derived clusters can also be maintained for a long time, and
SSCs amplified (Chap. 5). It is thus likely that the CFA assay is effective with adult
SSCs, but this may need to be confirmed.
6.4
Future Prospects
While the CFA assay can be used immediately as a short-term functional assay for
SSCs, the technique can be improved and extended. Here we discuss two
possibilities.
In a published report (Yeh et al.
2007
), we measured cluster numbers visually
under a microscope, but this is a tedious process that reduces the value of the short-
term assay. The use of SSCs or spermatogonia carrying a fluorescent marker (e.g.,
green fluorescent protein) should allow for a more facile detection of clusters. In
addition, combined with a computer-assisted, automated fluorescence detection
system, the assay can provide readouts more readily and in real-time.
Second, when the SSC culture and the CFA assay are used to analyze the effect
of a growth factor, the presence of feeder cells becomes a confounding factor. It is
possible that a factor affects SSC activity indirectly by changing the viability or
actions of feeder cells. This problem can be resolved by using a feeder-free SSC
culture. Since such a culture system has been reported (Kanatsu-Shinohara et al.
2005
), its applicability for quantitative SSC analyses needs to be evaluated.
In this regard, it is important to establish a human SSC culture system. Then, a
CFA assay for human SSCs will become possible and will be a powerful technique
to analyze their biology in a short-term and semi-quantitative manner. Since regen-
eration of human spermatogenesis in experimental animals through spermatogonial
transplantation has not been achieved, the CFA assay could become a critical
method to study human SSCs. We realize that such an assay has great potential to
evaluate the quality and quantity of human SSCs in the future, and expect that the
clinical impact of such a system will be immense.
Acknowledgments
We thank F. Clerk for editing this manuscript. The study conducted in our
laboratory was supported by Canadian Institutes of Health Research (CIHR) (MOP-49444 and
86532). MCN is a Fonds de la Recherche en Santé du Québec (FRSQ) scholar and JRY is a recipi-
ent of Research Institute of McGill University Health Centre (MUHC-RI) studentship.
References
Barroca V, La ssalle B, Coureuil M, Louis JP, Le PF, Testart J, Allemand I, Riou L & Fouchet P
2009 Mouse differentiating spermatogonia can generate germinal stem cells in vivo.
Nat. Cell
Biol.
11
190-196.
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