Biomedical Engineering Reference
In-Depth Information
after a 1-week culture of mixed testicular cells followed by a manual selection and
transfer of putative stem cell colonies onto MEF feeders. While these ES-like cells
lacked
NANOG
expression, they did express
OCT4
and
SOX2
. In addition, the
ES-like cells exhibited high levels of telomerase and normal karyotype. The ES-like
cells exhibit an intermediate pattern of methylation at both imprinted and non-
imprinted loci, similar, in general, to that described by (Kossack et al.
2009
; Conrad
et al. (
2008
)). Although functional during
in vitro
differentiation assays, the ES-like
cells did not form teratomas in immunocompromised mice. This contrasts to a third
report in which ES-like cells derived from biopsy-sized quantities of human testicu-
lar tissue from organ donors were able to give rise to small teratomas (Golestaneh
et al.
2009
). In this study, total testicular cell suspensions were plated in defined
human ES cell culture medium in order to generate ES-like colonies within 1 week
after testicular cell isolation. Another report also describes the generation of pluri-
potent cells from testicular biopsies but with slower kinetics (3-8 weeks) (Mizrak
et al.
2010
). When transplanted into immunodeficient mice, these cultured testicu-
lar cells were also able to give rise to tissues containing some differentiated human
cells but not complex teratomas (which were produced, in contrast, using either
hESC or iPS cells). Since neither of these latter studies used preselection of germ
cells, the exact origin of the pluripotent cells derived is unclear.
Thus, the aforementioned pluripotent cell lines derived from postnatal testis
share certain important properties, including expression of pluripotency genes, tri-
lineage differentiation ability, and variations on the expected androgenetic methyla-
tion profile at imprinted genes, in conjunction with intermediate levels of
methylation at other important loci (Table
2.1
). The functionality of human pluri-
potent cell lines is generally somewhat more difficult to assess due to technical and
ethical limitations. Nonetheless, data initially generated using the murine system
appear to be relevant for the human testis. Unfortunately, different approaches were
used to generate the pluripotent lines in each study, precluding their direct compari-
son (see Fig.
2.2
). For example, multiple different culture media were used for
induction conditions, in the presence or absence of fetal bovine serum, GDNF,
bFGF, EGF, and LIF, with somewhat conflicting results about their respective
effects (de Rooij and Mizrak
2008
). Furthermore, the timeframe for conversion of
unipotent germ cells into pluripotent stem cells is also quite variable. These obser-
vations suggest that more than one mechanism could theoretically be responsible.
It should be noted, however, that spontaneous teratomas occur only very rarely
in males, with an incidence of <1 in ~11,000 in wild-type laboratory mice and
<1 in ~16,000 in the human testis (Krausz and Looijenga
2008
; Stevens and
Mackensen
1961
). In comparison, the experimental rate of emergence of pluripo-
tent cells observed in the aforementioned studies is much higher in aggregate and
no such similar process is known to occur in somatic cells without experimental
delivery of pluripotency factors. Nonetheless, it is difficult to formally rule out the
possibility that a spontaneous genetic change, as opposed to an epigenetic or cul-
ture-induced phenotypic change, contributed the observed results. While the
mechanism of conversion will be extremely important to determine and may have
relevance for acquisition of pluripotency by other cell types, the crucial criteria in
Search WWH ::
Custom Search