Translating Spermatogonial Stem Cell Transplantation to the Clinic - Male Germline Stem Cells Developmental and Regenerative Potential - page 245

(Kanatsu-Shinohara et al. 2003 ; Kubota et al. 2004a, b ; Ryu et al. 2005 ; Hamra
et al. 2005 ). These methods allow for rodent SSCs to be maintained for a very long
time (perhaps indefinitely) with a significant amplification in numbers [doubling
time: mouse, 5.6 days (Kubota et al. 2004a ); rat, 3-4 days (Hamra et al. 2005 ) or
11 days (Ryu et al. 2005 )]. Progress was also reported recently in two studies that
established conditions for culturing human spermatogonia, potentially including
SSCs (Sadri-Ardekani et al. 2009 ; He et al. 2010 ). In the first study, testicular tissue
from six adult men who underwent orchiectomy for prostate cancer treatment was
used for cell isolation and culture (Sadri-Ardekani et al. 2009 ). Testicular cell sus-
pensions from these donors were initially depleted of somatic cells by differential
plating on plastic overnight and subsequent maintenance on uncoated plastic in
StemPro-34 medium containing EGF, LIF, GDNF, and soluble GFRa1. Cultures of
testicular cells established in this way contained cells expressing spermatogonial
markers (e.g., PLZF) and could be maintained in short-term culture in the absence
of feeder cells for up to 15 weeks with up to 53-fold amplification in colonization
activity as determined by xenotransplantation (Sadri-Ardekani et al. 2009 ).
Subsequent subculture of these cells onto laminin-coated dishes allowed for longer
maintenance of up to 28 weeks and significant amplification of xenotransplant
colonization activity (up to 18,450-fold) (Sadri-Ardekani et al. 2009 ). The latter
study by He et al. used testicular tissue from five organ donors to isolate cells for
culture (He et al. 2010 ). Testicular cell suspensions from organ donors were
depleted of somatic cells by differential plating on plastic for 3 h (He et al. 2010 ).
Subsequently, GPR125-positive spermatogonia were enriched by MACS and posi-
tive cells were maintained on gelatin-coated plates in StemPro-34 medium supple-
mented with bFGF (NUDT6), EGF, LIF, GDNF, soluble GFRa1, TGFbeta, and
Nodal. Cultures containing cells expressing spermatogonial markers (e.g., GPR125,
ITGA6, THY1, and GFRA1) could be maintained for 2 weeks with a fivefold
increase in numbers (He et al. 2010 ). It is important to note that the testicular tissue
for both studies was initially cryopreserved and thawed prior to cell isolation in a
way that might be suitable for future translation to the clinic. If these methods are
broadly applicable to human donor testis tissue and reproducible, it could be pos-
sible to isolate a small testicular biopsy from a patient and produce enough thera-
peutic SSCs in culture for future transplantation.

11.4.4 Risk of Reintroducing Malignant Cells During SSCs

Transplants

Transplantation of cryopreserved testis cells isolated from patients with malignant
diseases carries an inherent risk of reintroducing contaminating malignant cells
back into patients. Caution is certainly warranted as Jahnukainen and co-workers
have demonstrated that as few as 20 leukemic cells transplanted to a rat testis can
result in the development of terminal leukemia within 21 days (Jahnukainen et al.
2001 ). A recent report indicated that MACS sorting for CD4 was ineffective for