Spermatogonial Stem Cell Transplantation and Culture - Male Germline Stem Cells Developmental and Regenerative Potential - page 119

The authors concluded that the technique supported the expansion of SSCs and the
development of postmeiotic germ cells over a 15-day culture period. However,
transplantation assays were not conducted to determine the level of SSC prolifera-
tion and no functional spermatozoa were produced.

5.3.4 Species

5.3.4.1 Rodent

The first long-term self-renewing SSC cultures that were validated with SSC trans-
plantation were generated from mice in 2003 and 2004 (Kubota et al. 2004a ;
Nagano et al. 2003 ; Kanatsu-Shinohara et al. 2005a ). These experiments demon-
strated that GDNF was the essential molecule for SSC self renewal and that SSCs
were maintained for extended periods of time. In some systems of rodent SSC
culture, the starting cells come from newborn pups of day 0, 1, or 2 after birth
(Kanatsu-Shinohara et al. 2003 ), which contain gonocytes that reside in the center
of the seminiferous tubules. In other systems, the cells are recovered from pups at
days 5-8 or from adults (Nagano et al. 2003 ); in both instances, the gonocytes have
converted to spermatogonial stem cells and reside on the basement membrane of
the seminiferous tubule. Shortly after birth the gonocytes migrate to the basement
membrane of the seminiferous tubules and between day 0 and 6 in mice have begun
to initiate the first wave of spermatogenesis and to differentiate into SSCs (Huckins
and Clermont 1968 ; de Rooij and Russell 2000 ; Yoshida et al. 2007 ; McLean et al.
2003 ). For these reasons, is has been suggested that results from germ cells isolated
at 6 days of age or older may differ from results obtained using cells isolated at day
0, 1, or 2 after birth. Identification of differences in these two populations could be
informative regarding the transition from true gonocytes to stem cell. The defined
culture system that was reported in 2004 (Kubota et al. 2004b ) readily supports the
maintenance of SSCs from both pup and adult testes from a variety of different
mouse donor strains. In addition to the mouse, systems for the long-term culture of
rat SSCs have been reported (Ryu et al. 2005 ; Hamra et al. 2005 ). Experiments
using the rat SSC culture system, which is based on a defined medium very similar
to that of the mouse system, have identified several important characteristics of
rodent SSCs in vitro (Ryu et al. 2005 ). First, cultured mouse SSCs double approxi-
mately every 5 days; whereas, rat SSCs double every 11 days (Kubota et al. 2004a ;
Ryu et al. 2005 ), which may result from intrinsic differences in SSC proliferation
between these two species. Second, different cell surface markers are utilized to
enrich for mouse and rat SSCs. Mouse SSCs are readily enriched using MACS for
the cell surface marker Thy1; whereas, isolation of EpCam is more effective for
isolating SSCs from the rat testis. In the rat SSC culture system reported by Hamra
et al. ( 2005 ) an undefined culture medium and different feeders are used, but long-
term growth of rat SSCs is obtained. Additional work from this laboratory also
describes detailed methodologies for rat SSC isolation and enrichment using