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testes is defined at least in part by contributions from cells of the interstitium. More
recent evidence has strengthened this concept, showing that the cytokine colony
stimulating factor 1 (CSF1) influences self-renewal of mouse SSCs in vitro and its
production in vivo is localized to interstitial Leydig cells and peritubular myoid cells
(Oatley et al. 2009 ).
Studying SSC fate decisions in mammals is challenging due to their scarcity, and
lack of known specific morphological, phenotypical, or molecular markers to
specifically identify SSCs (Oatley and Brinster 2008 ). Previous studies showed that
nearly all SSCs (>90%) in both pre-pubertal and adult mouse testes are contained
within the THY1+ (CD90) germ cell fraction (Kubota et al. 2004a, b ). To date, the
greatest enrichment of SSCs from mouse testes is achieved by isolation of THY1+
cells (Oatley and Brinster 2008 ). Using functional germ cell transplantation,
Kubota et al. ( 2004a ) showed that SSC concentration of the THY1+ cell fraction is
5- to 30-fold greater compared to the unselected total cell population of pre-pubertal
and adult mouse testes, respectively (Kubota et al. 2004b ). Other cell surface
molecules reported to be expressed by rodent SSCs include a6-integrin (Shinohara
et al. 1999 ) and GPR125 (Seandel et al. 2007 ). Examination of cell populations
enriched for SSCs, such as the THY1+ germ cell population, compared to the total
testis provides insights into characteristics of SSCs. Unfortunately, no isolated
testis cell fraction based on any surface marker reported to date, including the
THY1+, a6-integrin+, or GPR125+ cell population, is composed purely of SSCs
and likely also contain A pr and A al spermatogonia that are produced upon SSC
differentiation. For this reason, examination of SSCs should not rely on cell surface
markers and must include functional transplantation analysis to draw unequivocal
conclusions about the biology of these cells (Oatley and Brinster 2008 ).
Impairment of SSC function in vivo results in formation of seminiferous tubules
devoid of germ cells, a phenotype referred to as Sertoli-cell-only (Fig. 7.1 ), which
leads to sub-fertility or infertility. Progressive increase in the percentage of seminif-
erous tubules with this phenotype is regarded as a hallmark of impaired SSC self-
renewal (Buaas et al. 2004 ; Costoya et al. 2004 ; Oatley et al. 2006 ). However, this
interpretation can be misleading because disruption of SSC differentiation to A pr
spermatogonia, SSC death, or quiescence could cause an identical phenotype
(Fig. 7.2 ). Because stem cell is a functional definition, the truest measure of an SSC
is the ability to reestablish spermatogenesis following transplantation into a recipient
testis (Brinster and Avarbock 1994 ; Brinster and Zimmermann 1994 ). However, this
assay cannot distinguish between defects in SSC self-renewal, differentiation, or
survival because disruption of any of these fates will result in impaired reestablish-
ment of spermatogenesis. Thus, distinguishing between defective SSC self-renewal
and differentiation by in vivo examination is challenging. Another means to evaluate
SSC functions is use of in vitro culture systems that support their self-renewal and
differentiation.
Methods to maintain mouse SSCs in vitro for extended periods of time have
been devised, providing a tool to critically study their fate decisions (Kanatsu-
Shinohara et al. 2003, 2005 ; Kubota et al. 2004a ). Currently, techniques for isolation
of SSC-enriched testis fractions and long-tem culture of SSCs are only available for
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