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Testis |
Horizontal Medical Research Organization,3
Department of Pathology and Biology of Diseases,4 Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
The Institute of Physical and Chemical Research (RIKEN),5 Bioresource Center, Ibaraki 305-0074, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, gametogenesis, Sertoli cells, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the experiments described here, we report the in vitro culture of spermatogonial stem cells that proliferate for long periods of time. We used the spermatogonial transplantation technique [11] to evaluate the level of self-renewal activity of stem cells in vitro, and transplantation of the cultured cells restored fertility to congenitally infertile recipient mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Testis cells were collected from a newborn transgenic mouse line C57BL6/Tg14(act-EGFP-OsbY01) that was bred into DBA/2 background (designated Green) (provided by Dr. M. Okabe, Osaka University, Osaka, Japan). The spermatogonia and spermatocytes of these mice express the enhanced green fluorescent protein (EGFP) gene, the level of expression of which decreases gradually after meiosis [12]. Therefore, donor cells can be readily identified following transplantation. Testis cells were collected by two-step enzymatic digestion and used for culture [13]. Briefly, testis cells were digested with 1 mg/ml collagenase (type IV, Sigma, St. Louis, MO) for 15 min, followed by 0.25% trypsin/1 mM EDTA digestion (both from Invitrogen, Carlsbad, CA) for 10 min. Approximately 4 x 105 cells were collected from a neonatal testis by this procedure. The number of dead cells was generally less than 5%, as assessed by trypan blue staining.
Cultured cells were transplanted into BALB/C nude or infertile WBB6F1W/Wv (designated W) pups (5–10 days old, Japan SLC, Shizuoka, Japan). To deplete endogenous spermatogenesis, nude mice were treated with busulfan (44 mg/kg) at 6 wk of age [11] and were subsequently injected with homologous bone marrow cells to reduce mortality [14]. In experiments using W recipients, 50 µg anti-CD4 antibody (GK1.5) was administered intraperitoneally on Days 0, 2, and 4 after transplantation to induce tolerance to the allogeneic donor cells [14]. All animal experimentation protocols were approved by the Institutional Animal Care and Use Committee of Kyoto University.
Culture Conditions
Dissociated testis cells were allocated to 0.2% (w/v) gelatin-coated tissue culture plate (2 x 105 cells/3.8 cm2). The plates were washed twice with PBS before use. Culture medium for the testis cells was StemPro-34 SFM (Invitrogen) supplemented with StemPro supplement (Invitrogen), 25 µg/ml insulin, 100 µg/ml transferrin, 60 µM putrescine, 30 nM sodium selenite, 6 mg/ml D-(+)-glucose, 30 µg/ml pyruvic acid, 1 µl/ml DL-lactic acid (Sigma), 5 mg/ml bovine albumin (ICN Biomedicals, Irvine, CA), 2 mM L-glutamine, 5 x 10-5 M 2-mercaptoethanol, minimal essential medium (MEM) vitamin solution (Invitrogen), MEM nonessential amino acid solution (Invitrogen), 10-4 M ascorbic acid, 10 µg/ml d-biotin, 30 ng/ml ß-estradiol, 60 ng/ml progesterone (Sigma), 20 ng/ml mouse epidermal growth factor (Becton Dickinson, Bedford, MA), 10 ng/ml human basic fibroblast growth factor (Becton Dickinson), 103 U/ml ESGRO (murine leukemia inhibitory factor; Invitrogen), 10 ng/ml recombinant rat glial cell line-derived neurotrophic factor (GDNF) (R&D Systems, Minneapolis, MN) and 1% fetal calf serum (JRH Biosciences, Lenexa, KS). The cells were maintained at 37°C in an atmosphere of 5% carbon dioxide in air.
Antibodies and Staining
Primary antibodies used were: rat anti-EpCAM (G8.8) and mouse anti-SSEA-1 (MC-480) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), rat antihuman 
6-integrin (CD49f) (GoH3), biotinylated hamster antirat ß1-integrin (CD29) (Ha2/5), Allo phyco cyanin-(APC) rat antimouse c-kit (CD117) (2B8) (BD Biosciences, Franklin Lakes, NJ), and rat anti-TDA antigen (EE2) (provided by Dr. Y. Nishimune, Osaka University). APC-conjugated goat antirat-IgG (Cedarlane Laboratories, Ontario, Canada), APC-conjugated streptavidin (BD Biosciences), or Alexa Fluor 633-conjugated goat antimouse IgM (Molecular Probes, Eugene, OR) was used as secondary antibody. The cell staining technique was as previously described [15]. Cells were analyzed with a FACS-Calibur system (BD Biosciences).
Transplantation and Analysis
Approximately 8 µl of the donor cell suspensions were injected into the seminiferous tubules of a nude recipient testis, and 2 µl were introduced into the W pup testis through the efferent duct [13]. The injection filled 75–85% of the tubules in each recipient testis. The ratio between somatic to germ cell types was usually less than 5% at the time of injection, and only GFP-positive cells were counted before transplantation. Adult recipient mice were anesthetized by Avertin injection (640 mg/kg). Pup recipients were placed on ice to cause hypothermia-induced anesthesia [14].
To count colonies, recipient mouse testes were recovered 7–8 wk after donor cell transplantation and analyzed by observing fluorescence under UV light [12, 14]. Donor germ cells were identified specifically because host testis cells had no endogenous fluorescence. A cluster of germ cells was defined as a colony when it occupied the entire circumference of the tubule and was at least 0.1 mm long [14].
Microinsemination
The seminiferous tubules of recipient testes were carefully dissected, and the germ cells were collected mechanically [16]. Microinsemination was performed as previously described [16]. Embryos that reached the four-cell stage after 24 h in culture were transferred to the oviducts of Day 1 pseudopregnant Imperial Cancer Research (ICR) females. Live fetuses retrieved on Day 19.5 were raised by lactating foster ICR mothers.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Neonatal DBA/2 mouse testes were enzymatically dispersed and transferred to gelatin-coated plates in medium containing GDNF, basic fibroblast growth factor, epidermal growth factor, and leukemia inhibitory factor. GDNF was previously shown to stimulate spermatogonial stem cell self-renewal in vivo [17]; other factors are known to affect the proliferation and maintenance of other stem cells, including primordial germ cell (PGC) [18–20]. Many cells attached to the plate after overnight incubation, but a significant number of germ cells, distinguished by their large size and characteristic pseudopod, remained floating. The floating cells were passaged to secondary culture plates after vigorous pipetting. Very few germ cells were left on the original gelatin-coated plate, and cells transferred to secondary plates were relatively germ cell enriched (Fig. 1a). Within 1 wk, the transferred cells proliferated and spread on the bottom of the well, and round proliferating cells formed colonies on top of the flat cell layer (Fig. 1, b and c). Most of these primary colonies consisted of compact clusters of cells with unclear borders (Fig. 1c) but were distinct in appearance from embryonic stem (ES) cell colonies [21]. Cell division and colony formation did not occur without growth factors.
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Cells were dispersed by trypsin treatment and replated at 10–14 days in vitro (DIV) to a fresh culture plate (x 1 dilution). Colonies grew to the original size in about 10 days, and cells were again passaged (x one-half dilution). Although the colonies continued to grow, flat-type somatic cells gradually disappeared after 20 DIV. Therefore, from the second or third passage, the cells were maintained on mitomycin C-inactivated mouse embryonic fibroblasts (MEF) and passaged every 3–5 days to fresh MEF at a one-third to one-fourth dilution. By 3–4 wk, the cultures remained in a relatively steady state and continued to generate colonies of similar morphology (Fig. 1d). Interestingly, chains of proliferating cells, resembling mitotic spermatogonia in vivo, were occasionally observed after passage (Fig. 1e). These results were reproducible, and similar cultures were established from more than 20 different experiments. The derivation of the colonies, however, depended on the genetic background; we could initiate cultures from ICR or C57BL/6 x DBA/2 F1 (BDF1) but not from C57BL/6 or 129/Sv backgrounds. In the latter strains, initial colony formation does not occur. Somatic cells overgrew and germ cells disappeared in a few weeks.
Phenotype of the Cultured Cells
To evaluate the cultured cell phenotype, we used testis cells from neonatal Green mouse [12]. These mice express GFP gene ubiquitously, including in spermatogenic cells; therefore, cultured GFP-positive cells can be distinguished from feeder cells by observation under a UV light. Cell culture was established from the Green mouse, and the surface phenotype of GFP-positive cells was analyzed by flow cytometry. The cells were positive for 6- and ß1-integrin (spermatogonial stem cell markers) [15], EpCAM (spermatogonia marker) [22], and EE2 (spermatogonia marker) [23]. Although most cells were negative for c-kit (differentiated spermatogonia marker) [15, 24], weak expression was noted, suggesting that some colonies were differentiating (Fig. 2). Nevertheless, adding the c-kit ligand Stem Cell Factor (SCF) to the culture did not change the phenotype or growth characteristics of the colonies. The cells were completely negative for SSEA-1 (PGC marker) [19, 20]. These results indicated that the majority of cells had an undifferentiated spermatogonia phenotype.
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Determination of Stem Cell Activity by Spermatogonial Transplantation
To determine the stem cell activity of cultured cells, we next performed spermatogonial transplantation. Because there are no clear morphological criteria or specific markers for spermatogonial stem cells [2], the only reliable assay is recolonization of the seminiferous tubules of infertile animals to restore spermatogenesis [11]. For these experiments, three separate cultures were established from Green mice. Following culture periods of 29–58 days, cells were harvested and transplanted into the seminiferous tubules of busulfan-treated adult nude mice. After 4–21 passages, cells were collected again at 45–134 DIV for transplantation to measure the increase in stem cell numbers during this period. Colonies in recipient testes were counted under UV light at 7–8 wk after transplantation.
Stem cell numbers increased in all three experiments (Table 1). Because the number of stem cells in neonatal testis is 3.4/105 cells [25], this result also indicates that stem cells expanded approximately 5 x 1012-fold from the initiation of culture to 134 days. At the time of writing, the longest culture with stem cell activity, as confirmed by transplantation assay, has been maintained for 134 days with 27 passages (7 x 1011-fold expansion), and the cells keep growing for over 
160 days (2 x 1014-fold expansion in total cell number) (Fig. 1f), retaining characteristic morphology. These results show that cultured stem cells are actively increasing in number.
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Generation of Offspring from Infertile Males by Transplantation of Cultured Stem Cells
Finally, to confirm whether the germ cells generated in recipient testes were functionally normal, we attempted to restore fertility to W mice by cultured cell transplantation. These mice are congenitally infertile because of a c-kit gene defect [11, 14, 25]. Two experiments were performed, and approximately 105 cells were injected into each testis. Testis cells were cultured for 40 (the first experiment) or 91 days (the second experiment) and transplanted into three immune-suppressed allogeneic W pups (5–10 days old) in both experiments. Forty days after transplantation, one of the recipient W mice in the first experiment was sacrificed and its testes used for histological analysis and in vitro microinsemination, a technique commonly used to derive offspring from infertile animals or humans [16, 26]. The remaining W recipients were mated with wild-type females to determine whether they restored fertility.
Analysis of W recipient testis demonstrated extensive colonization by the cultured cells that filled numerous seminiferous tubules with apparently normal spermatogenesis (Fig. 3, a–c). Spermatogenesis in recipient testes could have come only from cultured donor stem cells because W recipients cannot generate spermatogenesis in their defective stem cells [11, 14, 25]. To generate offspring, 68 live spermatozoa or 139 elongated spermatids were collected from the other W testis and injected into BDF1 oocytes. Of the 207 embryos constructed, 172 (83%) developed to two cells within 24 h in culture. After transfer into the oviducts of 11 pseudopregnant females, a total of 59 live pups were born (17 males and 29 females; not including those that were cannibalized after birth). Offspring were also obtained by natural mating. One of the remaining two recipients in the first experiment sired seven offspring (three males and four females) at 74 days after transplantation, and one of the three recipients in the second experiment sired nine offspring (five males and four females) at 91 days after transplantation. The donor origin of the pups in both experiments was confirmed by fluorescence under UV light (Fig. 3d). The offspring were proved to be fertile. Taken together, these results indicate that germ cells differentiated from cultured cells are capable of producing spermatogenesis and normal offspring.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There are several important differences between ES/EG cells and GS cells. First, although ES/EG cells are available only during the embryonic period, GS cells can be readily harvested from spermatogonia in the postnatal testis. Second, although ES/EG cells are totipotent and differentiate into tissues with diverse phenotypes by forming teratoma [19–21], no such tissues or tumor formation was observed in any GS cell recipient, suggesting that these cells are committed to the germline lineage. In contrast, transplantation of ES cells into seminiferous tubules has resulted in tumor formation and the death of recipients [28]. Third, although ES/EG cells are readily obtained from 129 genetic backgrounds, GS cells were obtained from DBA/2 background. It has been known that genetic differences affect the growth kinetics of hematopoietic stem cells in vivo: Stem cells of DBA/2 mice cycle more rapidly than those of B6 mice [29]. Therefore, it is possible that similar growth difference may exist in the spermatogenic system and influence the initial colony growth in vitro. Thus, GS cells have clearly distinct biological characteristics from ES/EG cells.
The establishment of GS cells provides not only a novel tool to analyze spermatogenesis or stem cell self-renewal but also opens up new areas for practical application. Recently transgenic mice and rats were produced by retroviral infection of spermatogonial stem cells [30–32]; however, exogenous genes were integrated randomly into the genome, and it has not been possible to manipulate the spermatogonial stem cell genome by gene targeting [33]. The development of GS cells provides the opportunity to manipulate the genome and select transfected clones in a manner similar to ES cells. Because germ cells of many species can be successfully transplanted [34–36], it will be important to explore the possibility of deriving GS cells from other species in which genetic engineering methods are inefficient or lacking. Because ES cells with germline potential have been obtained only from mice, GS cells may resolve current challenges with ES cells and greatly contribute to the development of new transgenic technologies [2] or germline mutagenesis. GS cells also have potential clinical value. Patients with malignancies can become infertile following treatment with radiation or chemotherapy [36]; stem cells from a testis biopsy could be used to derive GS cells and increase stem cell numbers before autologous germ cell transplantation, thereby protecting fertility. Ultimately the technique might provide an opportunity for eliminating genetic diseases in humans, although its use should be considered with extreme caution. Thus, GS cells will provide new possibilities in biotechnology and medicine.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Mito Kanatsu-Shinohara, Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan. FAX: 81 75 753 9281; mitos{at}mfour.med.kyoto-u.ac.jp
Received: 9 March 2003.
First decision: 1 April 2003.
Accepted: 11 April 2003.
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 F. K. Hamra, K. M. Chapman, D. Nguyen, and D. L. Garbers Identification of Neuregulin as a Factor Required for Formation of Aligned Spermatogonia J. Biol. Chem., January 5, 2007; 282(1): 721 - 730. [Abstract] [Full Text] [PDF]
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M. Kanatsu-Shinohara, K. Inoue, N. Ogonuki, H. Miki, S. Yoshida, S. Toyokuni, J. Lee, A. Ogura, and T. Shinohara Leukemia Inhibitory Factor Enhances Formation of Germ Cell Colonies in Neonatal Mouse Testis Culture Biol Reprod, January 1, 2007; 76(1): 55 - 62. [Abstract] [Full Text] [PDF]
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J. G. Jung, Y. M. Lee, T. S. Park, S. H. Park, J. M. Lim, and J. Y. Han Identification, Culture, and Characterization of Germline Stem Cell-Like Cells in Chicken Testes Biol Reprod, January 1, 2007; 76(1): 173 - 182. [Abstract] [Full Text] [PDF]
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S. Corallini, S. Fera, L. Grisanti, I. Falciatori, B. Muciaccia, M. Stefanini, and E. Vicini Expression of the adaptor protein m-Numb in mouse male germ cells. Reproduction, December 1, 2006; 132(6): 887 - 897. [Abstract] [Full Text] [PDF]
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 K. Fujita, A. Tsujimura, Y. Miyagawa, H. Kiuchi, Y. Matsuoka, T. Takao, S. Takada, N. Nonomura, and A. Okuyama Isolation of Germ Cells from Leukemia and Lymphoma Cells in a Human In vitro Model: Potential Clinical Application for Restoring Human Fertility after Anticancer Therapy Cancer Res., December 1, 2006; 66(23): 11166 - 11171. [Abstract] [Full Text] [PDF]
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Y. Niki, T. Yamaguchi, and A. P. Mahowald Establishment of stable cell lines of Drosophila germ-line stem cells PNAS, October 31, 2006; 103(44): 16325 - 16330. [Abstract] [Full Text] [PDF]
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T. Shinohara, M. Kato, M. Takehashi, J. Lee, S. Chuma, N. Nakatsuji, M. Kanatsu-Shinohara, and M. Hirabayashi Rats produced by interspecies spermatogonial transplantation in mice and in vitro microinsemination PNAS, September 12, 2006; 103(37): 13624 - 13628. [Abstract] [Full Text] [PDF]
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C. Itman, S. Mendis, B. Barakat, and K. L. Loveland All in the family: TGF-{beta} family action in testis development. Reproduction, August 1, 2006; 132(2): 233 - 246. [Abstract] [Full Text] [PDF]
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 S. Fouchecourt, M. Godet, O. Sabido, and P. Durand Glial cell-line-derived neurotropic factor and its receptors are expressed by germinal and somatic cells of the rat testis. J. Endocrinol., July 1, 2006; 190(1): 59 - 71. [Abstract] [Full Text] [PDF]
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M. Kanatsu-Shinohara, K. Inoue, H. Miki, N. Ogonuki, M. Takehashi, T. Morimoto, A. Ogura, and T. Shinohara Clonal Origin of Germ Cell Colonies after Spermatogonial Transplantation in Mice Biol Reprod, July 1, 2006; 75(1): 68 - 74. [Abstract] [Full Text] [PDF]
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B.-Y. Ryu, K. E. Orwig, J. M. Oatley, M. R. Avarbock, and R. L. Brinster Effects of Aging and Niche Microenvironment on Spermatogonial Stem Cell Self-Renewal Stem Cells, June 1, 2006; 24(6): 1505 - 1511. [Abstract] [Full Text] [PDF]
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M. Kanatsu-Shinohara, M. Ikawa, M. Takehashi, N. Ogonuki, H. Miki, K. Inoue, Y. Kazuki, J. Lee, S. Toyokuni, M. Oshimura, et al. From the Cover: Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells PNAS, May 23, 2006; 103(21): 8018 - 8023. [Abstract] [Full Text] [PDF]
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D. G. de Rooij Rapid expansion of the spermatogonial stem cell tool box PNAS, May 23, 2006; 103(21): 7939 - 7940. [Full Text] [PDF]
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M. Kanatsu-Shinohara, K. Inoue, J. Lee, H. Miki, N. Ogonuki, S. Toyokuni, A. Ogura, and T. Shinohara Anchorage-Independent Growth of Mouse Male Germline Stem Cells In Vitro Biol Reprod, March 1, 2006; 74(3): 522 - 529. [Abstract] [Full Text] [PDF]
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D. R. Lee, K.-S. Kim, Y. H. Yang, H. S. Oh, S. H. Lee, T. G. Chung, J. H. Cho, H. J. Kim, T. K. Yoon, and K. Y. Cha Isolation of male germ stem cell-like cells from testicular tissue of non-obstructive azoospermic patients and differentiation into haploid male germ cells in vitro Hum. Reprod., February 1, 2006; 21(2): 471 - 476. [Abstract] [Full Text] [PDF]
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F. K. Hamra, K. M. Chapman, D. M. Nguyen, A. A. Williams-Stephens, R. E. Hammer, and D. L. Garbers Self renewal, expansion, and transfection of rat spermatogonial stem cells in culture PNAS, November 29, 2005; 102(48): 17430 - 17435. [Abstract] [Full Text] [PDF]
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A. Buageaw, M. Sukhwani, A. Ben-Yehudah, J. Ehmcke, V. Y. Rawe, C. Pholpramool, K. E. Orwig, and S. Schlatt GDNF Family Receptor alpha1 Phenotype of Spermatogonial Stem Cells in Immature Mouse Testes Biol Reprod, November 1, 2005; 73(5): 1011 - 1016. [Abstract] [Full Text] [PDF]
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B.-Y. Ryu, H. Kubota, M. R. Avarbock, and R. L. Brinster Conservation of spermatogonial stem cell self-renewal signaling between mouse and rat PNAS, October 4, 2005; 102(40): 14302 - 14307. [Abstract] [Full Text] [PDF]
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M. Kanatsu-Shinohara, N. Ogonuki, T. Iwano, J. Lee, Y. Kazuki, K. Inoue, H. Miki, M. Takehashi, S. Toyokuni, Y. Shinkai, et al. Genetic and epigenetic properties of mouse male germline stem cells during long-term culture Development, September 15, 2005; 132(18): 4155 - 4163. [Abstract] [Full Text] [PDF]
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M. Kanatsu-Shinohara, H. Miki, K. Inoue, N. Ogonuki, S. Toyokuni, A. Ogura, and T. Shinohara Germline niche transplantation restores fertility in infertile mice Hum. Reprod., September 1, 2005; 20(9): 2376 - 2382. [Abstract] [Full Text] [PDF]
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N. Sofikitis, E. Pappas, A. Kawatani, D. Baltogiannis, D. Loutradis, N. Kanakas, D. Giannakis, F. Dimitriadis, K. Tsoukanelis, I. Georgiou, et al. Efforts to create an artificial testis: culture systems of male germ cells under biochemical conditions resembling the seminiferous tubular biochemical environment Hum. Reprod. Update, May 1, 2005; 11(3): 229 - 259. [Abstract] [Full Text] [PDF]
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M. Kanatsu-Shinohara, H. Miki, K. Inoue, N. Ogonuki, S. Toyokuni, A. Ogura, and T. Shinohara Long-Term Culture of Mouse Male Germline Stem Cells Under Serum-or Feeder-Free Conditions Biol Reprod, April 1, 2005; 72(4): 985 - 991. [Abstract] [Full Text] [PDF]
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M.-C. Hofmann, L. Braydich-Stolle, L. Dettin, E. Johnson, and M. Dym Immortalization of Mouse Germ Line Stem Cells Stem Cells, February 1, 2005; 23(2): 200 - 210. [Abstract] [Full Text] [PDF]
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M. Kanatsu-Shinohara, S. Toyokuni, and T. Shinohara Genetic Selection of Mouse Male Germline Stem Cells In Vitro: Offspring from Single Stem Cells Biol Reprod, January 1, 2005; 72(1): 236 - 240. [Abstract] [Full Text] [PDF]
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M. M. Matzuk Germ-line immortality PNAS, November 23, 2004; 101(47): 16395 - 16396. [Full Text] [PDF]
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H. Kubota, M. R. Avarbock, and R. L. Brinster Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells PNAS, November 23, 2004; 101(47): 16489 - 16494. [Abstract] [Full Text] [PDF]
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M. Kanatsu-Shinohara, S. Toyokuni, and T. Shinohara Transgenic Mice Produced by Retroviral Transduction of Male Germ Line Stem Cells In Vivo Biol Reprod, October 1, 2004; 71(4): 1202 - 1207. [Abstract] [Full Text] [PDF]
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H. Kubota, M. R. Avarbock, and R. L. Brinster Culture Conditions and Single Growth Factors Affect Fate Determination of Mouse Spermatogonial Stem Cells Biol Reprod, September 1, 2004; 71(3): 722 - 731. [Abstract] [Full Text] [PDF]
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O. Lacham-Kaplan In vivo and in vitro differentiation of male germ cells in the mouse Reproduction, August 1, 2004; 128(2): 147 - 152. [Abstract] [Full Text] [PDF]
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M. Kanatsu-Shinohara, T. Morimoto, S. Toyokuni, and T. Shinohara Regulation of Mouse Spermatogonial Stem Cell Self-Renewing Divisionby the Pituitary Gland Biol Reprod, June 1, 2004; 70(6): 1731 - 1737. [Abstract] [Full Text] [PDF]
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S. Kishigami, S. Wakayama, N. Van Thuan, and T. Wakayama Similar Time Restriction for Intracytoplasmic Sperm Injection and Round Spermatid Injection into Activated Oocytes for Efficient Offspring Production Biol Reprod, June 1, 2004; 70(6): 1863 - 1869. [Abstract] [Full Text] [PDF]
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Y. Hong, T. Liu, H. Zhao, H. Xu, W. Wang, R. Liu, T. Chen, J. Deng, and J. Gui Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro PNAS, May 25, 2004; 101(21): 8011 - 8016. [Abstract] [Full Text] [PDF]
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