Spermatogonial Stem Cell Transplantation and Culture - Male Germline Stem Cells Developmental and Regenerative Potential - page 117

In subsequent experiments, a positive role of glial cell line-derived neurotrophic
factor (GDNF) for SSC maintenance in vitro was demonstrated (Nagano et al. 2003 ).
Several groups have now reported the development of long-term SSC culture
systems using techniques to enrich for germ cells, thereby removing contaminating
cells, and including cocktails of growth factors in the growth media. In 2003, it was
demonstrated that gonocytes isolated from day 0 mouse pups could be maintained
and proliferate over a 5-month period (Kanatsu-Shinohara et al. 2003 ). These
experiments resulted in a long-term culture system that supported SSC self-renewal
in DBA-derived strains of mice, but not other mouse strains. However, the culture
medium included serum and proprietary components; therefore, it was difficult to
determine the critical growth factors required for SSC self-renewal. In 2004, a
procedure to maintain the SSCs of many mouse strains in vitro in a defined culture
medium containing GDNF, GDNF family receptor alpha 1 (GFRa1), and basic
fibroblast growth factor (bFGF) was reported (Kubota et al. 2004a ). This work
unequivocally proved that GDNF was the essential growth factor responsible for
self-renewal of mouse SSCs. Subsequently, others reported a more complex serum-
free medium containing proprietary ingredients was also able to maintain SSC
function and support SSC self-renewal of DBA-derived strains of mice (Kanatsu-
Shinohara et al. 2005a ). In these culture systems, germ cells, including the SSC,
form clump-like structures in vitro that resemble colonies of ES cells in culture
(Fig. 5.4 ). Mouse SSC clumps appear more three-dimensional than rat clumps.
Since the discovery of GDNF as the main regulator of SSC self-renewal, many
experiments have been conducted evaluating mechanisms of SSC function using
long-term culture systems. For example, gene expression in these cultured cells
using stable or transient transfection of transgenes or siRNA has been evaluated
(Ogawa et al. 2003 ; Oatley et al. 2006 ; Dann et al. 2008 ; Schmidt et al. 2008 ,
unpublished). Using SSC cultures treated with siRNA in combination with SSC
transplantation, it was demonstrated that Bcl6b , Etv5 , and Lhx1 are important for
mouse SSC self-renewal (Oatley et al. 2006 ). Recently, both Bcl6b and Etv5 were
found important for rat SSC self-renewal (Schmidt et al. 2008, unpublished). With
a similar experimental approach and treatment of cultured cells with inhibitors of
Fig. 5.4 Microscopic images of cultured germ cells. ( a ) EpCam positive rat germ cells enriched
for SSCs growing as clumps. ( b ) Thy1 positive mouse germ cells enriched for SSCs growing as
clumps. ( c ) The clumps in ( B ) were isolated from a GFP-positive individual and fluoresce when
stimulated with UV light. Scale bars = 100 mm