Biomedical Engineering Reference
In-Depth Information
Seandel et al. (
2007
) developed a means to derive adult SSC lines from mice up
to 11 months of age with overall >90% efficiency, using feeders comprised of mitoti-
cally inactivated primary testicular stromal cells. We found that the novel G-protein
coupled receptor Gpr125 was expressed in a population of cultured germ cells that
contained SSC activity based on transplantation assays. These experiments were
performed using engineered mice (and SSCs derived from such mice) in which
lacZ
was placed within the endogenous
Gpr125
locus (
Gpr125-lacZ
), representing an
extremely sensitive and specific reporter system. After about 3 months following
initiation of SSC cultures even without preselection of cells from the testes of
Gpr125-lacZ mice (or from other strains), morphologically distinct colonies com-
prised of ES-like cells [referred to as multipotent adult spermatogonial-derived stem
cells (MASCs)] appeared spontaneously (Fig.
2.3
). These colonies were selected
and transferred into ES culture conditions for long-term propagation, establishing
new colonies that closely resembled mouse ES cells when plated upon inactivated
MEFs. These adult-derived ES-like cells expressed Oct4 and Nanog protein and
readily differentiated into derivatives of all three germ layers
in vitro
, including
contractile cardiogenic tissue. Similarly, the ES-like cells produced tri-lineage tera-
tomas in immunodeficient mice (Seandel et al
.
2007
), including foci of de novo
germ cell differentiation (Seandel et al. unpublished data).
As more stringent evidence of pluripotency, the ES-like cells that had been
cloned at the single cell level were found to be competent at forming chimeric
embryos, with contributions to multiple organ systems. Of note, despite obtaining
live-born chimeric mice from ES-like cells, we have observed generally low contri-
butions of the adult ES-like cells in chimeras and a reproducible congenital abnor-
mality comprised of hyperplasia and abnormal chondrogenesis of the anterior rib
cage (Falciatori et al. unpublished data). This phenotype, reminiscent of androge-
netic embryos, would be consistent with the partial androgenetic imprinting profile
previously described for ES-like cells (Kanatsu-Shinohara et al.
2004
; Mann et al.
1990
). Also of great interest, the gene expression profile of the adult-derived
ES-like cells was not identical to that of ES cells. Among the pluripotency genes
markedly lower in the ES-like cells were
Rex1, Esg1,
and
Gdf3
, while
Nanog
was
also lower but still expressed at absolutely high levels. Certain lineage commitment
markers were present at substantially higher levels in ES-like cells than in bona fide
ES cells, including the mesodermal gene
brachyury
.
More recently, the ability of adult SSCs in culture to produce pluripotent stem
cells was confirmed by Ko et al. (
2009
). These authors not only demonstrated the
ability of authentic SSC clones to acquire pluripotency (including a germline con-
tribution) but also showed that the initial plating density of SSCs was crucial for
efficient conversion (estimated at about 0.01% of cells). Furthermore, this study
also compared methylation patterns at imprinted genes to demonstrate the origin of
the pluripotent stem cells from spermatogonia. The latter analysis showed that the
ES-like cells exhibited an androgenetic imprinting pattern at the differentially
methylated region of the H19 gene, similar to what was seen in spermatogonia but
dissimilar to the somatic pattern. These data argued against a possible origin from a
somatic stem cell. As with the prior studies, the authors demonstrated that derivatives
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