1 Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto
University, Kyoto 606-8501, Japan
2 Department of Molecular Genetics Graduate School of Medicine, Kyoto
University, Kyoto 606-8501, Japan
3 RIKEN, Bioresource Center, Ibaraki 305-0074, Japan
4 Experimental Research Center for Infectious Diseases, Institute for Virus
Research, Kyoto University, Kyoto 606-8507, Japan
5 Medical Research Institute, Tokyo Medical and Dental University, Tokyo
101-0062, Japan
6 Department of Molecular and Cell Genetics, School of Life Sciences, Faculty of
Medicine, Tottori University, Yonago, Tottori 683-8503, Japan
7 Department of Pathology and Biology of Diseases, Graduate School of Medicine,
Kyoto University, Kyoto 606-8501, Japan
* Author for correspondence (e-mail: tshinoha{at}virus.kyoto-u.ac.jp)
Accepted 18 July 2005
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SUMMARY |
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Key words: Culture, Genomic imprinting, Karyotype, Spermatogenesis, Stem cell, Telomere
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Introduction |
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Spermatogonial stem cells are the founder cell population for
spermatogenesis (Meistrich, 1993; de Rooij
and Russell, 2002). Although spermatogonial stem cells are
infrequent and divide slowly in vivo, the addition of GDNF to in vitro
cultures of these cells induces self-renewing division of spermatogonial stem
cells: the cells increase logarithmically in vitro without losing the capacity
to produce spermatogenesis when transferred into the seminiferous tubules of
infertile mouse testes (Kanatsu-Shinohara
et al., 2003b
; Ogawa et al.,
2004
; Kubota et al.,
2004
). Recipient mice that received grafts of transfected cultured
spermatogonial stem cells sired transgenic offspring at a frequency
approaching 50%, which is five- to 10-fold higher than the frequencies
achieved using traditional methods based on oocytes or embryos
(Kanatsu-Shinohara et al.,
2005a
). Owing to their unique characteristics, we designated these
cells `germline stem' (GS) cells to distinguish them from ES cells or EG cells
(Evans and Kaufman, 1981
;
Martin, 1981
;
Matsui et al., 1992
;
Resnick et al., 1992
;
Kanatsu-Shinohara et al.,
2003b
). Thus, GS cells created a new possibility to study
spermatogonial stem cells.
In this study, we examined the replicative potential and stability of spermatogonial stem cells during long-term culture. Two independent GS cell cultures were maintained for 2 years, and the cultured cells were analyzed for their phenotypic and functional characteristics, including the germline potential.
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Materials and methods |
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Transplantation
The cultured cells were collected by trypsinization and were filtered
through 30 µm nylon mesh before transplantation. Approximately 3 µl of
the cell suspension were microinjected into the seminiferous tubules of the
testes of 4- to 8-week-old WBB6F1-W/Wv recipients (W mice; Japan
SLC, Shizuoka, Japan) via the efferent duct
(Ogawa et al., 1997). The
injections filled 75 to 85% of the tubules in each recipient testis. The
recipient W mice received anti-CD4 antibody injections to induce tolerance to
the allogeneic germ cells
(Kanatsu-Shinohara et al.,
2003a
). For the testing of tumor-forming potential, approximately
3x106 cells were injected into KSN nude mice (Japan SLC). The
Institutional Animal Care and Use Committee of Kyoto University approved all
of the animal experimentation protocols.
Analysis of testis
Donor cell colonization was analyzed by observation of fluorescence under
UV light (Kanatsu-Shinohara et al.,
2003a). This method allows the specific identification of
transplanted cells, because the host testis does not fluoresce. The recipient
testes were also fixed in 10% neutral buffered formalin, and processed for
paraffin sectioning. All sections were stained with Hematoxylin and Eosin for
histological analysis.
Antibodies and staining
The primary antibodies used were: rat anti-mouse EpCAM (G8.8), mouse
anti-mouse SSEA-1 (MC-480; Developmental Studies Hybridoma Bank, University of
Iowa), rat anti-human 6-integrin (CD49f) (GoH3), biotinylated hamster
anti-rat ß1-integrin (CD29) (Ha2/5), biotinylated rat anti-mouse CD9
(KMC8) and allophycocyanin (APC)-conjugated rat anti-mouse Kit (2B8; all from
BD Biosciences, Franklin Lakes, NJ). APC-conjugated goat anti-rat-IgG
(Cedarlane Laboratories, Ontario, Canada), APC-conjugated streptavidin (BD
Biosciences) and Alexa Fluor 633-conjugated goat anti-mouse IgM (Molecular
Probes, Eugene, OR) were used as secondary antibodies. The cell staining
technique used for flow cytometry was as previously described
(Shinohara et al., 1999
). The
stained cells were analyzed using a FACS-Calibur system (BD Biosciences), and
only EGFP-positive cells were gated for analysis. At least 10,000 events were
acquired for each sample. Alkaline phosphatase staining was carried out using
a Vector Blue substrate kit (Vector Laboratories, Burlingame, CA) according to
manufacturer's protocol.
Analysis of marker gene expression
Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA). For
reverse transcriptase-polymerase chain reaction (RT-PCR), first-strand cDNA
was synthesized using SuperscriptTM II (RNase H- reverse
transcriptase, Invitrogen). PCR was carried out using appropriate primer sets,
as described previously (Kanatsu-Shinohara
et al., 2004b;
Kanatsu-Shinohara et al.,
2005b
; Falender et al.,
2005
).
Pulsed-field gel electrophoresis, terminal restriction fragments (TRF) analysis, and measurement of telomerase activity
The DNA was harvested from 2x106 cells by salting out
method, using a buffer containing 20 mM Tris-HCl pH 8.0, 10 mM EDTA, 400 mM
NaCl, 0.5% SDS and 100 µg/ml of proteinase K. The DNA was digested with
HinfI overnight and separated by electrophoresis on a 1.1% agarose
gel at 14°C, using a pulsed-field apparatus (BioRad, Hercules, CA).
Pulsed-field electrophoresis was performed with 6 V/cm constant for 12 hours
and a ramped pulse from 1 to 10 seconds. To examine the TRFs containing
telomeric sequences, the gel was dried at 60°C for 3 hours, soaked in
denaturing solution (1.5 M NaCl-0.5 M NaOH solution for 30 minutes,
neutralized in 1.5 M NaCl, 0.5 M Tris-HCl (pH 8.0) buffer for 30 minutes, and
probed with (C3TA2)4 telomeric DNA
oligonucleotides at 37°C overnight. Telomerase activity was measured using
the TeloChaser detection kit (Toyobo, Osaka, Japan) as previously described
(Tatematsu et al., 1996).
Microinsemination
Microinsemination was performed by intracytoplasmic injection (Kimura et
al., 1995) of round spermatids from EGFP-positive spermatogenic colonies from
donor testes into C57BL/6xDBA/2 F1 (BDF1) oocytes, collected from
superovulated females. The embryos were transferred into the oviducts of
pseudopregnant ICR females, 24 hours after in vitro culture. Live fetuses were
retrieved on day 19.5 and were raised by lactating foster mothers.
Combined bisulfite restriction analysis (COBRA)
Genomic DNA was treated with sodium bisulfite, which deaminates
unmethylated cytosines to uracils but does not affect 5-methylated cytosines.
PCR amplification of differentially methylated regions (DMRs) from the
bisulfite-treated genomic DNA was carried out using specific primers as
previously described (Xiong and Laird,
1997; Kanatsu-Shinohara et
al., 2004b
). The amplified PCR products were digested with the
indicated restriction enzymes, which have recognition sequences containing CpG
in the original unconverted DNA. The intensity of the digested DNA bands was
quantified using ImageGauge software (Fuji Photo Film, Tokyo, Japan).
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Results |
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To test the effect of long-term in vitro culture, we maintained two
independent GS cells for 2 years (Fig.
1A), during which the cells continued logarithmic growth. The
established GS cells were weakly positive for alkaline phosphatase
(Fig. 1A), and they were
regularly passaged every 5 to 6 days at a dilution of 1:3 to 1:6. Every 3
months during the 24-month experimental period, representative cell samples
were cryopreserved. Flow cytometric analysis after 24 months in culture showed
that the GS cells expressed the spermatogonial markers ß1- and
6-integrins, CD9 and EpCAM
(Shinohara et al., 1999
;
Kanatsu-Shinohara et al.,
2004a
; Ryu et al.,
2004
); the cells weakly expressed Kit, a marker of differentiated
spermatogonia (Shinohara et al.,
2000
), and did not express SSEA-1, an ES cell marker
(Solter and Knowles, 1978
)
(Fig. 1B). Similarly, RT-PCR
analysis after 24 months of culture showed that, although the GS cells
expressed the ES cell markers Oct4, Rex1 and ERas, they did not express other
ES-specific markers, such as Nanog (Pesce
and Schöler, 2001
; Mitsui
et al., 2003
; Takahashi et
al., 2003
; Chambers et al.,
2003
). By contrast, many spermatogonia or germ cell markers,
including neurogenin 3, Ret, Stra8 (spermatogonia markers)
(Meng et al., 2000
;
Giuili et al., 2002
;
Yoshida et al., 2004
), Mvh and
Stella (germ cell markers) (Fujiwara et
al., 1994
; Saitou et al.,
2002
), were expressed in the GS cells. Both PLZF and TAF4b,
transcription factors involved in spermatogonial stem cell renewal
(Buaas et al., 2004
,
Costoya et al., 2004
;
Falender et al., 2005
), were
also expressed in the GS cells (Fig.
1C). Thus, the overall morphology and marker expression profiles
of the GS cells, as examined by flow cytometry and RT-PCR, did not change
after an approximately 1085-fold expansion by 139 passages over a
24-month period (Fig. 1E).
Although the GS cells continued to proliferate without noticeable changes
in either of the two independent cultures over the 24-month experimental
period, ES-like cells (mGS cells) developed in one case after freeze-thaw
treatment (Kanatsu-Shinohara et al.,
2004b) (Fig. 1A).
In this case, the cultured cells were divided into two fractions at 51 days
after the initiation of the culture. Some of the cells were maintained for the
2-year period without cryopreservation, whereas the remaining cells were
cryopreserved. In one of the cultures derived from the frozen cell stocks, mGS
cells appeared 46 days after thawing, a total of 91 days from the initiation
of culture. However, we did not find ES-like cells in any other of the more
than 30 freeze-thaw-treated GS cell cultures, suggesting that the freeze-thaw
procedure per se does not always induce the production of mGS cells.
Consistent with our previous study
(Kanatsu-Shinohara et al.,
2004b
), the mGS cells exhibited ES cell markers
(Fig. 1C) and produced
teratomas by subcutaneous injection into nude mice (data not shown).
|
We also determined the genomic imprinting pattern in the GS cells, because
the methylation patterns of germline cells tend to change during culture. The
methylation patterns of the DMRs of three paternally methylated regions
[H19, Meg3 IG (Gtl2 Mouse Genome Informatics) and
Rasgrf1] and two maternally methylated regions (Igf2r and
Peg10) were examined in GS cells harvested after 3, 12, 18 and 24
months of continuous culture (Fig.
3). Consistent with the findings of our previous study
(Kanatsu-Shinohara et al.,
2004b), COBRA analysis of the GS cells demonstrated androgenetic
imprinting: methylation of the paternal DMRs and demethylation of the maternal
DMRs. This androgenetic pattern was not altered in the two cultures at 24
months, indicating that GS cells are epigenetically stable. By contrast, the
mGS cells that developed in a 3-month-old culture after freeze-thaw treatment
had a different methylation pattern. Although paternally methylated regions in
H19 and Rasgrf1 were highly methylated, DMRs in the Meg3
IG region were slightly undermethylated compared with those in the GS
cells. Furthermore, while DMRs in Igf2r are unmethylated in the GS
cells, they are hypermethylated in the mGS cells. These results support our
previous observation that genomic imprinting is variable in mGS cells
(Kanatsu-Shinohara et al., 2004).
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Discussion |
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Our results contrast with previous reports that demonstrated instability of
germline cells in vitro. Although ES cells have a spontaneous mutation
frequency that is 100-fold below that of somatic cells
(Cervantes et al., 2002
),
culture of germline cells, such as preimplantation embryo, ES cells and EG
cells, generally induces genetic and epigenetic changes. For example, previous
studies have shown that ES cells progressively lose euploidy with increasing
numbers of culture passages. More than 70% of ES cells became aneuploid by
passages 25 (equivalent to more than 2 months of continuous culture), and
these cells could no longer contribute to the germline by blastocyst injection
(Longo et al., 1997
). The
aneuploid abnormalities induced by long-term culture appear to occur at
specific chromosome loci: whereas mouse ES cells commonly develop trisomy
eight after long-term culture (Liu et al.,
1997
), human ES cells have trisomy 17q and 12
(Draper et al., 2004
). These
karyotypic changes probably confer a growth advantage in vitro and hence
represent a common cause of the loss of germline potential. In addition to
their chromosomal instability, in vitro culture of preimplantation embryo, ES
cells and EG cells induces aberrant genomic imprinting, which can lead to
morphological or functional abnormalities in embryo or offspring
(Sasaki et al., 1995
;
Dean et al., 1998
;
Humpherys et al., 2001
).
Surprisingly, composition of the medium can also influence the imprint pattern
(Doherty et al., 2000
; Khosla
et al., 2002; Ecker et al.,
2004
). The instability of these embryonic germline cells probably
reflects their embryonic origin, which is susceptible to subtle changes in the
maternal environment. However, our results strongly suggest that
spermatogonial stem cells have sophisticated repair mechanisms to prevent
transmission of genetic or epigenetic damage to the progenitors, which may be
similar to the those found in postnatal stem cells in other self-renewing
tissues (Cairns, 2002
;
Potten et al., 2002
;
Gilbert, 2005
).
|
|
Although our results show the remarkable stability of spermatogonial stem
cells, our previous study has suggested that GS cells had multipotential
characteristics (Kanatsu-Shinohara et al.,
2004b). We reported that ES-like cells (mGS cells) appeared in the
cultures during the initiation of GS cell cultures from neonatal testis cells.
The origin of these mGS cells in neonatal testis cell culture is currently
unknown (Kanatsu-Shinohara et al.,
2004b
); it is possible that they arise from a population of
distinct undifferentiated pluripotent cells that persist in the testis from
fetal stage. However, we previously demonstrated the direct conversion of GS
cells into mGS cells in experiments using GS cells derived from p53 mutant
mice that have a high frequency of teratoma
(Lam and Nadeau, 2003
;
Kanatsu-Shinohara et al.,
2004b
). Based on this observation, we speculated that
spermatogonial stem cells retain the ability to become multipotent cells
during the early passages in vitro, an ability that may be lost during
long-term cell culture (Kanatsu-Shinohara
et al., 2004b
). In this study, mGS cell colonies developed in
3-month-old cultures after a freeze-thaw treatment, but thereafter no other GS
cells converted to mGS cells during the 2-year experimental period. This
result confirms our previous observation that mGS cells appear in the early
phase of neonatal testis cell culture, and also suggests that established GS
cells are resistant to mGS cell conversion. Although current culture
conditions support the germline potential in spermatogonial stem cells, they
do not fully support the multilineage potential attributed to spermatogonial
stem cells. Further studies will be necessary to determine whether mGS cells
are derived from GS cells, and a special effort should be made to establish
improved culture conditions that maintain the full developmental potential of
spermatogonial stem cells.
Spermatogonial stem cells represent an ideal model in which to investigate
the unique biology of stem cells. Spermatogonial stem cells can be
transplanted between animals, genetically manipulated and cultivated under
feeder-cell or serum-free conditions
(Brinster and Zimmermann, 1994;
Kubota et al., 2004
;
Kanatsu-Shinohara et al.,
2005a
, Kanatsu-Shinohara et
al., 2005b
). These advantages are not available for stem cells in
other self-renewing systems. Using these techniques, spermatogonial stem cells
can now be routinely expanded and subsequently examined for molecular and
genetic characteristics. Future studies will determine the mechanism by which
spermatogonial stem cells maintain the stable characteristics. In addition,
the long-term stability of GS cells will provide a strong advantage in
practical application, and may complement ES cell technology. Although the
shortening telomeres suggests that spermatogonial stem cells have limited
proliferative potential, it would not be a serious concern in practical
application of GS cells; assuming a constant rate of telomere loss, GS cells
should continue to proliferate for 34 months and achieve
10120-fold expansion before they undergo crisis. This should
allow a sufficient time for in vitro genetic modification of GS cells for
various purposes, including gene targeting. Thus, GS cells provide a new
possibility in the study of stem cell biology and will be an attractive target
of germline modification.
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ACKNOWLEDGMENTS |
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Allsopp, R. C., Morin, G. B., Horner, J. W., DePinho, R., Harley, C. B. and Weissman, I. L. (2003). Effect of TERT over-expression on the long-term transplantation capacity of hematopoietic stem cells. Nat. Med. 9,369 -371.[CrossRef][Medline]
Blackburn, E. H. (2005). Telomeres and telomerase: their mechanisms of action and the effects of altering their functions. FEBS Lett. 579,859 -862.[CrossRef][Medline]
Brinster, R. L. and Zimmermann, J. W. (1994).
Spermatogenesis following male germ-cell transplantation. Proc.
Natl. Acad. Sci. USA 91,11298
-11302.
Buaas, F. W., Kirsch, A. L., Sharma, M., McLean, D. J., Morris, J. L., Griswold, M. D., de Rooij, D. G. and Braun, R. E. (2004). Plzf is required in adult male germ cells for stem cell self-renewal. Nat. Genet. 36,647 -652.[CrossRef][Medline]
Cairns, J. (2002). Somatic stem cells and the
kinetics of mutagenesis and carcinogenesis. Proc. Natl. Acad. Sci.
USA 99,10567
-10570.
Cervantes, R. B., Stringer, J. R., Shao, C., Tischfield, J. A.
and Stambrook, P. J. (2002). Embryonic stem cells and somatic
cells differ in mutation frequency and type. Proc. Natl. Acad. Sci.
USA 99,3586
-3590.
Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S. and Smith, A. (2003). Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113,643 -655.[CrossRef][Medline]
Costoya, J. A., Hobbs, R. M., Barna, M., Cattoretti, G., Manova, K., Sukhwani, M., Orwig, K. E., Wolgemuth, D. J. and Pandolfi, P. P. (2004). Essential role of Plzf in maintenance of spermatogonial stem cells. Nat. Genet. 36,653 -659.[CrossRef][Medline]
de Rooij, D. G. and Russell, L. D. (2000). All
you wanted to know about spermatogonia but were afraid to ask. J.
Androl. 21,776
-798.
Dean, W., Bowden, L., Aitchison, A., Klose, J., Moore, T.,
Menesses, J. J., Reik, W. and Feil, R. (1998). Altered
imprinted gene methylation and expression in completely ES cell-derived mouse
fetuses: association with aberrant phenotypes.
Development 125,2273
-2282.
Doherty, A. S., Mann, M. R. W., Tremblay, K. D., Bartolomei, M.
S. and Schultz, R. M. (2000). Differential effects of culture
on imprinted H19 expression in the preimplantation embryo. Biol.
Reprod. 62,1526
-1535.
Draper, J. S., Smiths, K., Gokhale, P., Moore, H. D., Maltby, E., Johnson, J., Meisner, L., Zwaka, T. P., Thomson, J. A. and Andrews, P. W. (2004). Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat. Biotechnol. 22, 53-54.[CrossRef][Medline]
Ecker, D. J., Stein, P., Xu, Z., Williams, C. J., Kopf, G. S.,
Bilker, W. B., Abel, T. and Schultz, R. M. (2004). Long-term
effects of culture of preimplantation mouse embryos on behavior.
Proc. Natl. Acad. Sci. USA
101,1595
-1600.
Evans, M. J. and Kaufman, M. H. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature 292,154 -156.[CrossRef][Medline]
Falender, A. E., Freiman, R. N., Geles, K. G., Lo, K. C., Hwang,
K., Lamb, D. J., Morris, P. L., Tjian, R. and Richards, J. S.
(2005). Maintenance of spermatogenesis requires TAF4b, a
gonad-specific subunit of TFIID. Genes Dev.
19,794
-803.
Fujiwara, Y., Komiya, T., Kawabata, H., Sato, M., Fujimoto, H.,
Furusawa, M. and Noce, T. (1994). Isolation of a DEAD-family
protein gene that encodes a murine homolog of Drosophila vasa and its specific
expression in germ cell lineage. Proc. Natl. Acad. Sci.
USA 91,12258
-12262.
Gilbert, G. H. (2005). Label-retaining
epithelial cells in mouse mammary gland divide asymmetrically and retain their
template DNA strands. Development
132,681
-687.
Giuili, G., Tomljenovic, A., Labrecque, N., Oulad-Abdelghani,
M., Raaaoulzadegan, M. and Cuzin, F. (2002). Murine
spermatogonial stem cells: targeted transgene expression and purification in
an active state. EMBO Rep.
3, 753-759.
Harle-Bachor, C. and Boukamp, P. (1996).
Telomerase activity in the regenerative basal layer of the epidermis in human
skin and in immortal and carcinoma-derived skin keratinocytes.
Proc. Natl. Acad. Sci. USA
93,6476
-6481.
Humpherys, D., Eggan, K., Akutsu, H., Hochedlinger, K., Rideout
III, W. M., Biniszkiewicz, D., Yanagimachi, R. and Jaenisch, R.
(2001). Epigenetic instability in ES cells and cloned mice.
Science 293,95
-97.
Kanatsu-Shinohara, M., Ogonuki, N., Inoue, K., Ogura, A.,
Toyokuni, S., Honjo, T. and Shinohara, T. (2003a). Allogeneic
offspring produced by male germ line stem cell transplantation into infertile
mouse testis. Biol. Reprod.
68,167
-173.
Kanatsu-Shinohara, M., Ogonuki, N., Inoue, K., Miki, H., Ogura,
A., Toyokuni, S. and Shinohara, T. (2003b). Long-term
proliferation in culture and germline transmission of mouse male germline stem
cells. Biol. Reprod. 69,612
-616.
Kanatsu-Shinohara, M., Ogonuki, N., Inoue, K., Ogura, A.,
Toyokuni, S. and Shinohara, T. (2003c). Restoration of
fertility in infertile mice by transplantation of cryopreserved male germline
stem cells. Hum. Reprod.
18,2660
-2667.
Kanatsu-Shinohara, M., Toyokuni, S. and Shinohara, T.
(2004a). CD9 is a surface marker on mouse and rat male germline
stem cells. Biol. Reprod.
70, 70-75.
Kanatsu-Shinohara, M., Inoue, K., Lee, J., Yoshimoto, M., Ogonuki, N., Miki, H., Baba, S., Kato, T., Kazuki, Y., Toyokuni, S. et al. (2004b). Generation of pluripotent stem cells from neonatal mouse testis. Cell 119,1001 -1012.[CrossRef][Medline]
Kanatsu-Shinohara, M., Toyokuni, S. and Shinohara, T.
(2005a). Genetic selection of mouse male germline stem cells in
vitro: Offspring from single stem cells. Biol. Reprod.
72,236
-240.
Kanatsu-Shinohara, M., Miki, H., Inoue, K., Ogonuki, N.,
Toyokuni, S., Ogura, A. and Shinohara, T. (2005b). Long-term
culture of mouse male germline stem cells under serum- or feeder-free
conditions. Biol. Reprod.
72,985
-991.
Khosla, S., Dean, W., Brown, D., Reik, W. and Feil. R.
(2001). Culture of preimplantation mouse embryos affects fetal
development and the expression of imprinted genes. Biol.
Reprod. 64,918
-926.
Kimura, Y. and Yanagimachi, R. (1995). Mouse
oocytes injected with testicular spermatozoa or round spermatids can develop
into normal offspring. Development
121,2397
-2405.
Kolquist, K. A., Ellisen, L. W., Counter, C. M., Meyerson, M., Tan, L. K., Weinberg, R. A., Harber, D. A. and Gerald, W. L. (1998). Expression of TERT in early premalignant lesions and a subset of cells in normal tissues. Nat. Genet 19,182 -186.[CrossRef][Medline]
Kubota, H., Avarbock, M. R. and Brinster, R. L.
(2004). Growth factors essential for self-renewal and expansion
of mouse spermatogonial stem cells. Proc. Natl. Acad. Sci.
USA 101,16489
-16494.
Labosky, P. A., Barlow, D. P. and Hogan, B. L. M.
(1994). Mouse embryonic germ (EG) cell lines: transmission
through the germline and differences in the methylation imprint of
insulin-like growth factor 2 receptor (Igf2r) gene compared with embryonic
stem (ES) cell lines. Development
120,3197
-3204.
Lam, M.-Y. J. and Nadeau, J. H. (2003). Genetic control of susceptibility to spontaneous testicular germ cell tumors in mice. Acta Pathol. Mic. Sc. 111,184 -191.
Lee, H.-W., Blasco, M. A., Gottlieb, G. J., Horner, II, J. W., Greider, C. W. and DePinho, R. A. (1998). Essential role of mouse telomerase in highly proliferative organs. Nature 392,569 -574.[CrossRef][Medline]
Liu, X., Wu, H., Loring, J., Hormuzdi, S., Disteche, C. M., Bornstein, P. and Jaenisch, R. (1997). Trisomy eight in ES cells is a common potential problem in gene targeting and interferes with germ line transmission. Dev. Dyn. 209, 85-91.[CrossRef][Medline]
Longo, L., Bygrave, A., Grosveld, F. G. and Pandolfi, P. P. (1997). The chromosome make-up of mouse embryonic stem cells is predictive of somatic and germ cell chimaerism. Transgenic Res. 6,321 -328.[CrossRef][Medline]
Martin, G. R. (1981). Isolation of a
pluripotent cell line from early mouse embryos cultured in medium conditioned
by teratocarcinoma stem cells. Proc. Natl. Acad. Sci.
USA 78,7634
-7638.
Matsui, Y., Zsebo, K. and Hogan, B. L. M. (1992). Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 70,841 -847.[CrossRef][Medline]
Meistrich, M. L. and van Beek, M. E. A. B. (1993). Spermatogonial stem cells. In Cell and Molecular Biology of the Testis (ed. C. Desjardins and L. L. Ewing), pp. 266-295. New York: Oxford University Press.
Meng, X., Lindahl, M., Hyvönen, M. E., Parvinen, M., de
Rooij, D. G., Hess, M. W., Raatikainen-Ahokas, A., Sainio, K., Rauvala, H.,
Lakso, M. et al. (2000). Regulation of cell fate decision of
undifferentiated spermatogonia by GDNF. Science
287,1489
-1493.
Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K., Maruyama, M., Maeda, M. and Yamanaka, S. (2003). The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113,631 -642.[CrossRef][Medline]
Nagano, M., Avarbock, M. R. and Brinster, R. L.
(1999). Pattern and kinetics of mouse donor spermatogonial stem
cell colonization in recipient testes. Biol. Reprod.
60,1429
-1436.
Niida, H., Matsumoto, T., Satoh, H., Shiwa, M., Tokutake, Y., Furuichi, Y. and Shinkai, Y. (1998). Severe growth defect in mouse cells lacking the telomerase RNA component. Nat. Genet. 19,203 -206.[CrossRef][Medline]
Ogawa, T., Aréchaga, J. M., Avarbock, M. R. and Brinster, R. L. (1997). Transplantation of testis germinal cells into mouse seminiferous tubules. Int. J. Dev. Biol. 41,111 -122.[Medline]
Ogawa, T., Ohmura, M., Tamura, Y., Kita, K., Ohbo, K., Suda, T. and Kubota, Y. (2004). Derivation and morphological characterization of mouse spermatogonial stem cell lines. Arch. Histol. Cytol. 67,297 -306.[CrossRef][Medline]
Ohta, H., Tohda, A. and Nishimune, Y. (2003).
Proliferation and differentiation of spermatogonial stem cells in the
W/Wv mutant mouse testis. Biol. Reprod.
69,1815
-1821.
Pesce, M. and Schöler, H. R. (2001).
Oct-4: gatekeeper in the beginning of mammalian development. Stem
Cells 19,271
-278.
Potten, C. S. (1992). Cell lineages. In Oxford Textbook of Pathology (ed. J. O. McGee, P. G. Isaacson and N. A. Wright), pp. 43-52. Oxford: Oxford University Press.
Potten, C. S., Owen, G. and Booth, D. (2002).
Intestinal stem cells protect their genome by selective segregation of
template DNA strands. J. Cell. Sci.
115,2381
-2388.
Resnick, J. L., Bixler, L. S., Cheng, L. and Donovan, P. J. (1992). Long-term proliferation of mouse primordial germ cells in culture. Nature 359,550 -551.[CrossRef][Medline]
Rubin, H. (2002). The disparity between human cell senescence in vitro and lifelong replication in vivo. Nat. Biotechnol. 20,675 -681.[CrossRef][Medline]
Ryu, B.-Y., Orwig, K. E., Kubota, H., Avarbock, M. R. and Brinster, R. L. (2004). Phenotypic and functional characteristics of male germline stem cells in rats. Dev. Biol. 274,158 -170.[CrossRef][Medline]
Saitou, M., Barton, S. C. and Surani, M. A. (2002). A molecular program for the specification of germ cell fate in mice. Nature 418,293 -300.[CrossRef][Medline]
Sasaki, H., Ferguson-Smith, A. C., Shum, A. S. W., Barton, S. C.
and Surani, M. A. (1995). Temporal and spatial regulation of
H19 imprinting in normal and uniparental mouse embryos.
Development 121,4195
-4202.
Shinohara, T., Avarbock, M. R. and Brinster, R. L.
(1999). ß1- and 6-integrin are surface markers on
mouse spermatogonial stem cells. Proc. Natl. Acad. Sci.
USA 96,5504
-5509.
Shinohara, T., Orwig, K. E., Avarbock, M. R. and Brinster, R.
L. (2000). Spermatogonial stem cell enrichment by
multiparameter selection of mouse testis cells. Proc. Natl. Acad.
Sci. USA 97,8346
-8351.
Shinohara, T., Orwig, K. E., Avarbock, M. R. and Brinster, R.
L. (2001). Remodeling of the postnatal mouse testis is
accompanied by dramatic changes in stem cell number and niche accessibility.
Proc. Natl. Acad. Sci. USA
98,6186
-6191.
Solter, D. and Knowles, B. B. (1978).
Monoclonal antibody defining a stage-specific mouse embryonic antigen
(SSEA-1). Proc. Natl. Acad. Sci. USA
75,5565
-5569.
Takahashi, K., Mitsui, K. and Yamanaka, S. (2003). Role of Eras in promoting tumor-like properties in mouse embryonic stem cells. Nature 414,122 -128.[CrossRef]
Tatematsu, T., Nakayama, J., Danbara, M., Shionoya, S., Sato, H., Omine, M. and Ishikawa, F. (1996). A novel quantitative `stretch PCR assay', that detects a dramatic increase in telomerase activity during the progression of myeloid leukemias. Oncogene 13,2265 -2274.[Medline]
Xiong, Z. and Laird, P. W. (1997). COBRA: a
sensitive and quantitative DNA methylation assay. Nucleic Acids
Res. 25,2532
-2534.
Yoshida, S., Takakura, A., Ohbo, K., Abe, K., Wakabayashi, J., Yamamoto, M., Suda, T. and Nabeshima, Y. (2004). Neurogenenin3 delineates the earliest stages of spermatogenesis in the mouse testis. Dev. Biol. 269,447 -458.[CrossRef][Medline]
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