Pluripotent Stem Cells from the Postnatal Testis: Unlocking the Potential of Spermatogonial Stem Cells - Male Germline Stem Cells Developmental and Regenerative Potential - page 27

growth factors, including glial cell line-derived neurotrophic factor (GDNF), basic
fibroblast growth factor (bFGF), epidermal growth factor (EGF), and leukemia
inhibitory factor (LIF) (Kanatsu-Shinohara et al. 2003 ). As opposed to immunose-
lection, only a negative selection was required to remove the majority of somatic
cells via binding to gelatin. However, the efficiency of deriving long-term SSC lines
from adult mouse testis was only 20-50% in these culture conditions (Kanatsu-
Shinohara et al. 2004 ; Ogawa et al. 2004 ). This could be due to a relative decline
in the number of functional stem cells in older animals or in the self-renewal capacity
of such cells. Nonetheless, multiple studies have subsequently confirmed that the
SSCs could be passaged over many generations and retain the ability to restore
fertility in animals with deficient spermatogenesis (Kanatsu-Shinohara et al. 2003,
2005a, b ; Ryu et al. 2005 ; Kubota et al. 2004a, b ). However, it has been estimated
that only 1-2% of cultured SSCs exhibit testicular repopulation capacity (Kanatsu-
Shinohara et al. 2005b ). Conversely, recent data suggest that differentiating germ
cells can display plasticity, potentially reverting back to the stem cell phenotype
in vitro or in vivo (Nakagawa et al. 2007 ; Barroca et al. 2009 ). As our understanding
of the nature of SSCs has evolved, the tools to study them have become increasingly
sophisticated, revealing a number of unique properties as discussed below.

2.3 Molecular Features that Could Predispose

SSCs to Pluripotency

It is reasonable to suppose that some of the same characteristics of male germ cells
that facilitate initiation of embryogenesis at the time of fertilization could also play
a role in spontaneous cellular reprogramming that would lead to formation of pluri-
potent stem cells in vitro . But what are these special molecular characteristics?
Both in terms of gene expression and chromatin structure, SSCs have been found
to share certain features (but also notable differences) with pluripotent stem cells.
In the sections below, we first review the current understanding of the normal
expression levels of the core pluripotency genes (particularly Oct4, Nanog , and
Sox2 ) in the testis and in cultured SSCs then examine data describing the unique
state of chromatin and its modifications in the germ lineage (see Fig. 2.1 ).
Oct4 is a homeobox transcription factor that is crucial for pluripotency in embry-
onic stem cells (Nichols et al. 1998 ; Niwa et al. 2000 ). Oct4 is part of a core network
of molecules, including Sox2 and Nanog, that both autoregulate and co-regulate
downstream factors that maintain self-renewal and block differentiation (Boyer et al.
2005 ). Studies revealing the expression of Oct4 in the postnatal testis have relied
both on immunological methods and genetic reporter systems with varying results,
though no study has documented levels in postnatal germ cells comparable to those
observed in ES cells. Pesce et al. ( 1998 ) found diffuse Oct4 protein by immunohis-
tochemistry in spermatogonia up to 7 days postnatally but in adult animals only a
subset of spermatogonia (type A) were positive (Pesce et al. 1998 ). However, in a
report using transgenic mice that expressed GFP under control of an 18 kilobase