Translating Spermatogonial Stem Cell Transplantation to the Clinic - Male Germline Stem Cells Developmental and Regenerative Potential - page 236

11.3.4 Recipient Models for SSC Transplantation

The best way to study the regenerative capacity of SSCs is using a transplantation
paradigm. To facilitate SSC engraftment, donor cells are typically transplanted into
recipient seminiferous tubules that lack endogenous spermatogenesis and, there-
fore, have niches available for occupancy by donor cells. Several approaches for
depleting endogenous spermatogenesis in recipients have been described, including
cytotoxic treatment (de Rooij and Kramer 1970 ; Brinster and Avarbock 1994 ;
Brinster and Zimmermann 1994 ; Ogawa et al. 1997, 2000 ; Nagano and Brinster
1998 ; Zhang et al. 2006a ) and genetic mutation (Ohta et al. 2001 ; Shinohara et al.
2001 ). In rodents, the cytotoxic treatments used to deplete endogenous spermato-
genesis in recipients involve anti-mitotic treatment with chemotherapy or local
irradiation (de Rooij and Kramer 1970 ; Brinster et al. 2003 ; Zhang et al. 2006a ).
The gonadotoxicity of various chemotherapy and radiation treatment regimens is
reviewed extensively in Chap. 9.

11.3.5 Busulfan Treatment

To evaluate the full regenerative potential of rhesus SSCs and study stem cell/niche
interactions, we developed a similar germ cell depleted (infertile) rhesus testis
model. Experiments were conducted to identify a busulfan treatment dose that is
compatible with long-term survival and which results in germ cell and SSC ablation
(Hermann et al. 2007 ). Adult rhesus macaques were treated with high doses of the
alkylating chemotherapeutic agent busulfan (Busulfex IV, ISP Pharma), which led
to long-term loss of sperm in the ejaculate beginning ~10 weeks after treatment and
lasting for more than 1 year [Fig. 11.2a , (Hermann et al. 2007 )]. Loss of sperm
production after high-dose busulfan treatment also correlated with a complete
depletion of spermatogenesis in histological sections of the testis (Fig. 11.2b ).
VASA (DDX4) and DAZL (Ruggiu et al. 1997 ; Castrillon et al. 2000 ; Reijo et al.
2000 ; Toyooka et al. 2000 ) are germ cell markers, and both were absent following
busulfan treatment (Hermann et al. 2007 ). Furthermore, GFRa1 (GDNF receptor)
and PLZF, consensus markers of stem and progenitor spermatogonia (Meng et al.
2000 ; Buaas et al. 2004 ; Ryu et al. 2004 ; Costoya et al. 2004 ; Buageaw et al. 2005 ;
Ryu et al. 2005 ; Naughton et al. 2006 ), were also lost coincident with spermato-
genic depletion (Hermann et al. 2007 ).
To augment the immunohistochemical evidence that busulfan depletes the pri-
mate SSC pool, the rhesus-to-nude mouse xenotransplantation assay was used to
measure the effects of busulfan on the putative stem cell pool (Hermann et al.
2007 ). As previously demonstrated, germ cells from primate species including
[rhesus macaques (Hermann et al. 2007, 2009 ), baboons (Nagano et al. 2001b ) and
humans (Nagano et al. 2002 )] produce chains and patches of spermatogonia that
resemble early rodent transplant colonies [reviewed in (Hermann et al. 2010 )].
While primate xenotransplant colonies do not produce complete spermatogenesis,