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of the pluripotent stem cells were functional in various assays. Together, these
results highlight the conclusion that the adult spermatogonial-derived ES-like cells
should not be considered equivalent to ES cells, despite their common ability to
form functional tissues, since differences were found in both gene expression by
Seandel et al. (
2007
) and in imprinting status by Ko et al. (
2009
), respectively.
2.6
Culture-Induced Pluripotency in Humans
Recent provocative studies have found evidence for pluripotent stem cells derived
from the adult human testis (Conrad et al.
2008
; Kossack et al.
2009
; Golestaneh
et al.
2009
; Mizrak et al.
2010
). Conrad et al. (
2008
) employed the following steps
to obtain highly enriched germ cells from fresh tissue: 4 days of culture of mixed
enzymatically dispersed testicular cells, followed by immunoselection using alpha6
integrin, and differential matrix selection for collagen-non-binding and laminin-
binding cells. It is quite remarkable that within 4 days of culture the selected germ
cells activated expression of OCT4 protein in the nucleus and cytoplasm, even
though no OCT4 is found subsequent to embryonic stages in the human testis
(Rajpert-De et al.
2004
; Conrad et al.
2008
). After several additional weeks of
incubation, cultures were obtained containing fibroblast-like monolayers that sur-
rounded discrete multilayered colonies of cells with ES-like properties. Subsequent
culture in the presence of LIF resulted in generation of new colonies of such stem
cells, although it is not clear whether these represented de novo conversion from the
precursor spermatogonial cells.
The ES-like cells expressed
OCT4, SOX2,
and
NANOG
, demonstrated by
RT-PCR, while both OCT4 and NANOG protein were present by immunofluores-
cence, flow cytometry, and Western blot analysis. Microarray expression profiling
detected expression of all three of these pluripotency genes not only in the ES-like
stem cells but interestingly also in the precursor spermatogonial cells, implying that
transcriptional activation also of
SOX2
and
NANOG
(in addition to
OCT4
and other
pluripotency associated genes such as
REX1
) must take place during the initial
four-day culture period. While this study actually detected
SOX2
by RT-PCR in normal
adult testis, neither
SOX2
nor
NANOG
were found expressed after embryonic stages
by other investigators (Perrett et al.
2008
; de Jong et al.
2008
; Hoei-Hansen et al.
2005
; Conrad et al.
2008
). The reason for these discrepancies between laboratories
is unclear but may be due to technical differences. While the global expression
profile of the human ES-like cells was generally similar to ES cells, the retention
of a germline signature could be seen in high levels of expression of
DAZL
and
POU6F1
. To demonstrate pluripotency, the authors formed teratomas from inde-
pendent ES-like cultures from eight normal samples in 23 of 32 attempts, using
cells with normal karyotype. These data portray a relatively robust system for
obtaining pluripotent stem cells.
Kossack et al. (
2009
) reported the derivation of two ES-like cell lines from
testicular biopsy-derived cells (Kossack et al.
2009
). These lines were obtained
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