Spermatogonial Stem Cell Transplantation and Culture - Male Germline Stem Cells Developmental and Regenerative Potential - page 111

Transplantation of SSCs between individuals in non-rodent species is complicated
because of the difficulty in generating recipients devoid of endogenous spermato-
genesis, technical limitations on the delivery of SSCs to the seminiferous tubules,
and absence of inbred lines of animals that provide for immunologically compatible
donor-recipient combinations. Nevertheless, complete donor-derived spermatogen-
esis following transplantation in species such as goats (Honaramooz et al. 2003 ),
dogs (Kim et al. 2008 ), and pigs (Honaramooz et al. 2002 ; Mikkola et al. 2006 ) has
been reported. Furthermore, it has been demonstrated that ultrasound guided intrat-
esticular transfer is the most appropriate method for transplantation into bovine,
monkey, and human testes (Schlatt et al. 1999 ). Recently, reports have described
successful SSC colonization in sheep (Rodriguez-Sosa et al. 2006, 2009 ) and cattle
(Herrid et al. 2006 ).
SSCs from human and primate testes colonize mouse seminiferous tubules, but do
not differentiate (Nagano et al. 2001, 2002 ). When primate testis cells are trans-
planted into primate recipient testes, it has been suggested that donor cell colonization
occurs based on an increase in testis weight in some recipients (Schlatt et al. 2002 ).

5.2.5 Procedures and Considerations

5.2.5.1 Cell Labeling and Enrichment

The ability to visually identify donor colonies after SSC transplantation is dramati-
cally improved by the use of transgenic donor cells with a visible reporter gene.
Common transgenic animals that have been used for SSC transplantation include
those carrying genes either for Lac Z or GFP. Additionally, animals that are trans-
genic for other fluorescent markers (such as DS red) can also be useful for SSC
identification post transplantation (Fig. 5.2 ). Donor-derived spermatogenesis can
also be identified using microsatellite markers, cell membrane dyes, or by genotyping
spermatozoa or offspring.
Isolation of populations of cells enriched for the SSC is important for efficient
generation of donor-derived spermatogenesis post-transplantation. Many methods
have been reported that enrich for the SSC. These methods include cell isolation
from immature or cryptorchid males (Shinohara et al. 2000a ), Percoll centrifuga-
tion, differential plating (Shinohara et al. 2000b ), and selection using fluorescence-
or magnetic-activated cell sorting (FACS or MACS) based on expressed specific
cell surface markers or fluorescence driven by germline-specific promoters
(Shinohara et al. 1999 ; Kubota et al. 2003, 2004a, b ). The most efficient methods
are those that employ selection using cell surface markers such as Thy-1 (5-30-fold)
and CD9 (7-fold) in the mouse (Kubota et al. 2004a ; Kanatsu-Shinohara et al.
2004 ) and Tacstd1/Epcam in the rat [11-fold; Schmidt et al. 2008 , unpublished; and
Ryu et al. ( 2004 )]. When deciding which marker should be used, care must be taken
to assure that putative markers do indeed enrich for the SSC. Furthermore, even
with selection, age of the donor and species can have a significant effect on degree