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BOR - Papers in Press, published online ahead of print December 19, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.065615
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BIOLOGY OF REPRODUCTION 78, 611–617 (2008)
DOI: 10.1095/biolreprod.107.065615
© 2008 by the Society for the Study of Reproduction, Inc.

Reproductive Technology; 

Long-Term Culture of Male Germline Stem Cells From Hamster Testes1

Mito Kanatsu-Shinohara 3, Tomomi Muneto 4, Jiyoung Lee 3, Manami Takenaka 4, Shinichiro Chuma 5, Norio Nakatsuji 5, Toshitaka Horiuchi 4, and Takashi Shinohara 2 3

Department of Molecular Genetics,3 Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan Graduate School of Comprehensive Scientific Research,4 Prefectural University of Hiroshima, Hiroshima 727-0023, Japan Department of Development and Differentiation,5 Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan

ABSTRACT

Spermatogonial stem cells provide the foundation for spermatogenesis in male animals. We recently succeeded in culturing and genetically engineering mouse spermatogonial stem cells, but little is known regarding the culture and growth requirements of spermatogonial stem cells in other animal species. In this study, we report the successful long-term culture of spermatogonial stem cells from hamster testes. Spermatogonial stem cells were purified using an anti-ITGA6 antibody and cultured in the presence of glial cell line-derived neurotrophic factor. The cells continued to proliferate for at least 1 year. During this period, they were genetically modified using a lentivirus and underwent spermatogenesis after transplantation into the testes of immunodeficient nude mice. However, germ cells generated in the surrogate xenogeneic recipients did not differentiate beyond the spermatid stage, and these round spermatids could not produce offspring through in vitro microinsemination. These results suggest that the germ cells may not have acquired characteristics necessary for fertility in the xenogeneic microenvironment. Nevertheless, the successful establishment of culture conditions conducive for hamster spermatogonial stem cell growth and maintenance indicates that this technique can be extended to other animal species in which current genetic modification techniques are impossible or inefficient.

developmental biology, gametogenesis, spermatogenesis, testis

INTRODUCTION

Spermatogonial stem cells divide continuously to support spermatogenesis throughout the life of a male [1, 2]. Because these cells are the only stem cells in the body that can transmit genetic information to the next generation, they have become a prime candidate for genetic modification. However, because of their small population size and the lack of identification methods, spermatogonial stem cells have been extremely difficult to study. In 1994, the germ cell transplantation technique was developed and provided the first functional assay for spermatogonial stem cells [3]. In addition, the development of a germ cell transplantation technique provided the possibility of using spermatogonial stem cells for genetic modification. Unfortunately, the ability to manipulate spermatogonial stem cells was still limited because no long-term culture system existed with which to carry out genetic manipulations.

In 2003, our group succeeded in the long-term culture of spermatogonial stem cells in mice, and named these cells germline stem (GS) cells [4]. The addition of glial cell line-derived neurotrophic factor (GDNF), a self-renewal factor for spermatogonial stem cells [5], enabled the in vitro proliferation of spermatogonial stem cells. These cell lines continued to proliferate for at least 2 years and maintained euploid karyotypes and stable androgenetic imprinting patterns [6]. Additionally, normal, fertile offspring were obtained despite long-term culture. Using these GS cells, we recently produced knockout animals, applying gene trapping and homologous recombination, thus providing a new method for germline modification [7].

The success of mouse GS cells in culture strongly suggests that similar culture techniques may be applicable to other animal species. Indeed, the successful culture of rat spermatogonial stem cells using a similar cocktail of cytokines was reported in 2005 [8, 9]. These cells underwent normal spermatogenesis and produced offspring following transplantation into testes. However, it remains unknown whether spermatogonia from other animal species can be cultured in a similar manner. In this study, we report the successful culture of hamster spermatogonial stem cells. Hamster GS cells were established, and their spermatogenic activity and fertility potential were examined through germ cell transplantation and in vitro microinsemination techniques.

MATERIALS AND METHODS

Culture Conditions

Testes were collected from 0-day-old to 14-day-old Syrian golden hamsters (Japan SLC, Shizuoka, Japan). Single cell suspensions were obtained by a two-step enzymatic dissociation procedure using collagenase and trypsin [10]. For culture initiation, ITGA6-expressing cells were enriched by magnetic beads (Dynabeads; Invitrogen, Carlsbad, CA) and were used in culture. The selection procedure was performed as previously described [11]. Briefly, the testis cells were incubated with anti-ITGA6 antibody (GoH3) (BD Biosciences, Franklin Lakes, NJ). After removal of the primary antibody by extensive washing with PBS, anti-rat IgG microbeads (Invitrogen) were used to recover ITGA6-expressing cells.

For testis cell culture initiation, 2–3 x 105 purified cell populations were cultured on mitomycin C-treated mouse embryonic fibroblasts (MEFs) in a six-well culture plate. The basal culture medium 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, 30 µg/ml pyruvic acid, 1 µl/ml DL-lactic acid (Sigma, St. Louis, MO), 2 mM L-glutamine, 5 x 10–5 M 2-mercaptoethanol, minimal essential medium vitamin solution (Invitrogen), minimal essential medium nonessential amino acids solution (Invitrogen), 10–4 M ascorbic acid, and 10 µg/ml d-biotin (Sigma). The growth factors used were 20 ng/ml mouse epidermal growth factor (BD Biosciences), 10 ng/ml human basic fibroblast growth factor (FGF2) (BD Biosciences), 15 ng/ml recombinant rat GDNF (R&D Systems, Minneapolis, MN), and 0.04% fetal calf serum (JRH Biosciences, Lenexa, KS).

After formation of a germ cell colony, the cultured cells were passaged onto dishes that had been coated with laminin (BD Biosciences) at a concentration of 20 µg/ml. The plates were flushed gently by repeated pipetting using a blue pipette, and the floating cells were transferred to a laminin-coated plate at a 1:1 ratio. After three to four rounds of passaging, the culture medium was replaced with TX-WES medium (Thrombogenics, Leuven, Belgium). The same growth factors listed earlier were added at the same concentrations to the medium. The supplemented TX-WES medium was changed every 2–3 days. Under this condition, cells were passaged at either a 1:1 or 1:2 ratio and were maintained at 37°C in an atmosphere of 5% CO2. Mouse GS cells (DBA/2 background) were cultured as previously described [4].

Transduction of Hamster GS Cells by a Lentivirus Vector

Hamster GS cells were transduced by pLenti6/UbC/EGFP, which expresses the enhanced green fluorescent protein (EGFP) gene under a ubiquitin-C promoter (provided by Dr. Shinya Yamanaka, Kyoto University, Japan). The viral supernatant was frozen at –80°C. Germline stem cells were transduced with the virus particles as described previously [12]. In brief, the dissociated GS cells were incubated with virus supernatant in the presence of 10 µg/ml polybrene (Sigma) and centrifuged at 3000 x g for 1 h at 32°C. The cells were then exposed to the lentivirus at a multiplicity of infection of 5.2 overnight, and the medium was changed the next day.

Antibodies and Staining

The primary antibodies used were phycoerythrin-conjugated monoclonal rat anti-human ITGA6 (CD49f) (GoH3), monoclonal biotinylated hamster anti-rat ITGB1 (CD29) (Ha2/5) (BD Biosciences), polyclonal rabbit anti-POU5F1 (H-134), ZBTB16 (H-300), RET (H-300), and GFRA1 (H-70) (Santa Cruz Biotechnology, Santa Cruz, CA) polyclonal rabbit anti-synaptonemal protein 3 (SYCP3) [13]. Allophycocyanin-conjugated streptavidin (BD Biosciences), tetramethyl-rhodamine-conjugated goat anti-rabbit IgG (Biosource, Camarillo, CA), and Alexa Fluor 568-conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR) were used as secondary antibodies. For immunocytochemistry, cells were cultured on a laminin-coated Lab-Tek II Chamber Slide System (Fisher Scientific, Rochester, NY) and fixed in 4% paraformaldehyde at 4°C for 15 min. The cells were then incubated with the primary antibodies overnight at 4°C. Following overnight incubation, cells were incubated with the secondary antibodies at room temperature for 1 h. Slides were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Dojindo, Kumamoto, Japan). The cell-staining technique for flow cytometry was performed as described previously [11]. The cells were analyzed using the FACS-Calibur system (BD Biosciences), and 10 000 events were collected.

Transplantation and Analysis

For the transplantation of hamster GS cells, we used KSN nu/nu mice (Japan SLC) that were treated with busulfan (44 mg/kg) at age 4 wk. Three days after busulfan treatment, mice were injected with bone marrow cells from syngeneic donors to prevent bone marrow failure. In brief, approximately 106 bone marrow cells from femoral bones of wild-type KSN nu/nu mice were microinjected into the orbital sinuses of busulfan-treated animals, 3 days after busulfan treatment. The recipient was used at least 4 wk after busulfan treatment. Approximately 10 µl of the donor cell suspension was injected into the seminiferous tubules of a busulfan-treated KSN nu/nu recipient testis through the efferent duct [10]. The injection filled 75%–85% of the tubules in each recipient testis. Recipient mouse testes were recovered 5–7 mo after donor cell transplantation and analyzed by observing the amount of fluorescence under ultraviolet light. Donor germ cells were identified specifically, as the host testis cells did not demonstrate any endogenous fluorescence. The Institutional Animal Care and Use Committee of Kyoto University approved all animal experimentation protocols.

Microinsemination

The seminiferous tubules of recipient testes were carefully dissected, and the germ cells were mechanically collected. Microinsemination was performed as described previously [14, 15]. Embryos that developed beyond the morula stage after 72 h in culture were transferred to the uteri of recipient albino females that were naturally mated with a male 3 days prior.

RESULTS

Derivation of Hamster GS Cells

To establish a culture system for spermatogonial stem cells in hamsters, we initially used testis cells collected from 0-day-old to 2-day-old newborn hamsters. The cells were dissociated by enzymatic digestion and were cultured in StemPro medium, as described for mice [4]. Testicular somatic cells were efficiently removed on the day after culture initiation following a differential plating procedure using a gelatin-coated plate. Although germ cells survived for several days, the cells were overwhelmed by rapidly growing testicular somatic cells, and most of the germ cells disappeared from the culture within 7–10 days.

To overcome this problem, we used pup testis cells for initiating cultures. In contrast to neonatal testis cell cultures in which gonocytes must convert into spermatogonia to initiate GS cell colonies [16], pup testis culture allows for the direct cultivation of spermatogonia because it is enriched for spermatogonial stem cells [17]. Hamster testis cells were selected with anti-ITGA6 antibody to enrich putative spermatogonial stem cell populations. In addition, the isolated cells were plated directly on mitomycin C-treated MEFs. We also reduced serum concentrations to 0.04% to inhibit growth of testicular somatic cells. Cell density at the time of plating was 1.2–2 x 105 cells per six-well plate. Within 1 wk, the germ cells started to form colonies (Fig. 1A), which were passaged by gentle pipetting of the culture plate to enrich germ cells. In contrast, the cells did not proliferate in the absence of GDNF, and they gradually disappeared from culture. We did not use trypsin for passaging at this stage because the cultures were easily overwhelmed by the contaminating testicular somatic cells. Moreover, colony formation depended critically on GDNF, a key cytokine involved in the self-renewal division of spermatogonial stem cells.


Figure 01
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  		FIG. 1. In vitro growth of hamster GS cells. Hamster (A) or mouse (B) GS cells cultured on MEFs. Note that the mouse GS cells formed three-dimensional colonies, whereas the hamster GS cells formed two-dimensional colonies on MEFs, and these hamster GS cells somewhat resemble germ cell colonies in bovine or porcine testis cell culture [35, 36]. C) Mouse GS cells cultured on laminin. D–F) Hamster GS cells cultured on laminin. Note the morphological similarities between mouse and hamster GS cellswhen they were cultured on laminin. Hamster GS cells occasionally formed chain-type (E) or branch-type (F) colonies. G, H) Growth requirements of hamster GS cells. Hamster GS cells could make colonies in the presence of GDNF (G), but they disappeared after GDNF removal (H). Hamster GS cells can be maintained in the absence of LIF (I) or GFRA1 (J). These factors did not influence colony formation. K) Long-term proliferation of three independent hamster GS cell cultures. Note the slow growth of GS cells during the initial phase of cultures. Bar = 100 µm (A–F); 200 µm (G–J).

After at least three passages on MEFs, the cells were plated on a laminin-coated dish. With consecutive passages on laminin, the testicular somatic cells gradually disappeared, and a pure population of hamster germ cells was established. The morphology of hamster GS cells was very similar to that of mouse GS cells on laminin-coated plates (Fig. 1C) [18]; however, unlike mouse GS cells that proliferate actively on MEFs (Fig. 1B), the hamster cells proliferated more actively on laminin-coated dishes. This result suggests that MEFs, like testicular somatic cells, secrete unknown inhibitory molecules against GS cells. Moreover, the cells grew in two-dimensional colonies on both MEFs and laminin (Fig. 1, A and D). We did not observe three-dimensional colonies, which are often found in mouse and rat GS cell culture. Although most of the colonies grew as clusters of germ cells, we occasionally observed chains or branches of germ cells that were found in the initial phase of germ cell colony formation in the seminiferous tubules (Fig. 1, E and F) [19]. The presence of these formations suggests that they were differentiating.

Although StemPro culture medium was used in the initial phase of the culture, we subsequently found that TX-WES medium, which was originally produced for embryonic stem cell culture [20], was more efficient at promoting hamster GS cells. During 8 days, hamster GS cells expanded 2.0-fold in the TX-WES medium. In contrast, the cells expanded 1.1-fold in the StemPro medium (Fig. 2A). Despite the beneficial effects observed in the maintenance of established cells, the use of TX-WES medium was not appropriate for the initiation of hamster GS cell cultures; testicular somatic cells overwhelmed the culture, possibly due to higher serum concentrations. The low growth rates of hamster GS cells observed in StemPro medium did not improve by increasing the serum concentration to 15%. Interestingly, although TX-WES medium could also support the growth of mouse cells (Fig. 2B), it was not possible to maintain these cells for an extended period of time (data not shown). Using this culture condition (TX-WES medium and laminin-coated plates), cultured cells could be maintained for approximately 1 year (364 days) with 27 passages. Unlike mouse GS cells, the hamster GS cells could not be maintained with epidermal growth factor + GDNF supplementation, but required FGF2 + GDNF (Fig. 2C). The growth of hamster GS cells was critically dependent on GDNF, and its removal of GDNF resulted in loss of germ cell colonies (Fig. 1, G and H). Neither leukemia inhibitory factor nor GFRA1 supplementation was essential for hamster GS cells in StemPro or TX-WES medium (Fig. 1, I and J). In the steady phase, the cells were regularly passaged every 8–10 days at a ratio of 1:1 to 1:2 with 0.25% trypsin (Fig. 1K). We did not observe any embryonic stem-like cell development during the entire culture period [21].


Figure 02
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  		FIG. 2. Growth of hamster GS cells in different culture conditions. Comparison of basal culture medium on growth rates of hamster (A) and mouse (B) GS cells. Hamster GS cells proliferated more actively in TX-WES medium, whereas mouse GS cells proliferated more actively in StemPro medium. C) Effects of growth factor stimulation. Proliferation in hamster GS cells was induced by FGF2 + GDNF but not by epidermal growth factor + GDNF. The data are presented as the mean ± SEM for five to eight independent experiments. In these experiments, 2.0 x 105 hamster GS cells or 2.5 x 105 mouse GS cells were plated in a laminin-coated 35 mm plate, and the cells were recovered at 8 or 6 days, respectively. Asterisks indicate results significantly different from the control (P < 0.01 by t-test).

Characterization of Hamster GS Cells

We used flow cytometry to examine the phenotype of the cultured cells. The hamster GS cells were stained with anti-ITGA6 or anti-ITGB1 antibodies, both of which were expressed on spermatogonial stem cells in mice [11]. Flow cytometric analysis showed that more than 95% of the cultured cells expressed both of these antigens (Fig. 3A). We also examined cell phenotypes by immunocytochemistry (Fig. 3B). Because the growth of GS cells was critically dependent on GDNF, we searched for GDNF receptor expression on hamster GS cells. Glial cell line-derived neurotrophic factor signals through the RET-GFRA1 complex, which subsequently activates the phosphoinositide-3 kinase-Akt pathway to promote self-renewal division of spermatogonial stem cells in the mouse [22, 23]. Both RET and GFRA1 were found on mouse and hamster GS cells. The cells also expressed other markers of spermatogonia, including ZBTB16, which is essential for mouse spermatogonial stem cell survival [24, 25], and POU5F1, a known germ cell marker essential for the maintenance of the undifferentiated state in embryonic stem cells [26]. Taken together, these results suggest that the cultured cells possess the spermatogonia phenotype and may depend on the same signaling pathway to undergo self-renewal division as seen in mouse GS cells.


Figure 03
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  		FIG. 3. Phenotypic characterization and growth of hamster GS cells. A) Flow cytometric characterization. Hamster GS cells were stained with antibodies against ITGA6 and ITGB1. Both antibodies were reactive with hamster GS cells. Black line, control immunoglobulin; red line, specific antibody. B) Immunocytological analysis. Hamster (top and middle row) and mouse (bottom row) GS cells were cultured on laminin and stained with antibodies against POU5F1, RET, GFRA1, and ZBTB16. The primary antibodies were detected by AlexaFluor 568-labeled anti-rabbit IgG polyclonal antibodies (red). The cells were counterstained with DAPI (blue). Bar = 100 µm.

Transduction of Hamster GS Cells With EGFP-Expressing Lentivirus

The continuous growth and cell staining patterns observed in our study suggest that the cultured testis cells are spermatogonial stem cells. However, in vitro proliferation does not always guarantee the stem cell potential; spermatogonial stem cells are identified only by their activity to recolonize empty seminiferous tubules [3]. It was necessary to examine via a transplantation assay whether the cells were stem cells or differentiated progenitor cells. In a previous study, hamster testis cells were able to seed and proliferate in busulfan-treated immunodeficient nude mouse testes. Additionally, characteristic hamster acrosomes were found in the recipient testes [27]. We took advantage of this system to demonstrate stem cell activity in the cultured cells. In our preliminary experiments, however, the lack of a definitive donor marker made it difficult to confirm the presence of hamster germ cell colonies following transplantation of fresh hamster testis cells into a busulfan-treated nude mouse. Donor cells with characteristic acrosomes were not clearly identified in the mouse seminiferous tubules that were filled with regenerating endogenous germ cells.

To overcome this problem, we introduced a donor cell marker by taking advantage of a lentivirus-mediated gene transfer [28]. The cultured cells (3 x 105 cells) were transduced with a lentivirus vector that expresses the EGFP gene under a ubiquitin-C promoter. The cultured cells were exposed to lentivirus viral particles 10 mo after initiation of culture. The cells were infected overnight, and the viral supernatant was removed the next day. The cells were further cultured in TX-WES medium for 42–47 days to increase cell numbers for transplantation. Approximately 3–4 x 105 cells were microinjected into the seminiferous tubules of a busulfan-treated nude mouse testis. Two separate experiments were performed, and approximately 30% of the cultured cells expressed EGFP-fluorescence at the time of transplantation. The cells were transplanted into a total of three recipients in each experiment.

Three months after transplantation, we killed one of the recipients to examine whether the transplanted cells colonized the recipient testis. Donor cell-derived EGFP fluorescence was found in the recipient testes that received cultured cells maintained for 8 mo in vitro (Fig. 4A). To analyze the degree of donor-derived spermatogenesis, the recipient testes were analyzed histologically. Clear EGFP expression was observed in the seminiferous tubules, and immunocytological staining showed that some of the EGFP-expressing donor germ cells expressed SYCP3, suggesting that they had entered meiosis (Fig. 4B). However, spermatogenesis in the recipient testes was not complete, and we were unable to clearly identify EGFP-expressing haploid germ cells in a histological analysis.


Figure 04
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  		FIG. 4. Spermatogenesis from cultured cells. A) Seminiferous tubules collected from a recipient mouse testis that received hamster GS cells transduced with EGFP-expressing lentivirus. EGFP fluorescence could be found in a segment of seminiferous tubules (arrow). B) Histological appearance of a recipient mouse testis that was stained with anti-SYCP3 antibody (red). The section was counterstained with Hoechst 33258 dye (blue). Only a small number of donor-derived germ cells (green) displayed SYCP3 fluorescence (arrows). C, D) Round spermatids released from a segment of seminiferous tubules used for in vitro microinsemination (arrows). Nomarski (C) and fluorescent (D) images. E, F) Two-cell stage embryos 1 day after in vitro microinsemination. Note the EGFP fluorescence, indicating the donor cell origin of the round spermatids. G) A morula stage embryo that developed 3 days after microinsemination. Bar = 1 mm (A); 100 µm (C, D); 50 µm (B, E–G).

Nevertheless, we killed the remaining recipients 5 mo after transplantation to further examine whether the recipient testes contained any donor-derived haploid germ cells. EGFP-expressing seminiferous tubules were recovered using fine forceps and dissociated mechanically. In the single cell suspension, EGFP-expressing round spermatids were identified by the presence of a small, round nucleus and uniquely shaped chromatin masses (Fig. 4, C and D). We were not able to identify EGFP-expressing, elongated spermatids or spermatozoa. These results suggest that the transplanted hamster cells could not differentiate or may have stopped expressing the EGFP gene. Incomplete spermatogenesis was probably not due to in vitro culture, as we were not able to observe complete spermatogenesis using fresh hamster testis cells following lentivirus transduction (data not shown).

Fertility of Hamster Germ Cells Developed in Mouse Testes

To test whether the round spermatids were fertile, we used in vitro microinsemination, a technique commonly applied to derive offspring from infertile animals and humans [29, 30]. After being frozen in liquid nitrogen for 20–40 days, the freeze-thawed, EGFP-expressing, round spermatids were microinjected into oocytes from golden hamsters (Fig. 4, E–G). In total, 131 eggs were constructed in three experiments. As a control, 134 oocytes were microinjected with round spermatids from wild-type animals. Although more than half of the eggs injected with control round spermatids developed to the eight-cell stage, only 11 eggs that were constructed with the EGFP-expressing, round spermatids developed to the same stage. Although four embryos were transferred to the uterus, we were unable to produce viable offspring (Table 1). In contrast, transfer of the 13 control embryos resulted in production of two normal offspring.


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  		TABLE 1. Embryo development after microinsemination.

DISCUSSION

This report outlines the first successful culture of hamster spermatogonial stem cells. As with mouse and rat GS cell cultures, growth of hamster GS cells critically depends on GDNF; removal of GDNF was detrimental to the growth of hamster GS cells. However, GDNF was not sufficient for active proliferation, and the addition of FGF2 was necessary for adequate growth. Although hamster GS cells generally showed characteristics similar to those of mouse GS cells, culture initiation required slight modifications of the mouse protocol. In our original report on mouse GS cells, we used testis cells from neonatal mice [4]. In the present study, we used pup testis cells because the application of similar methods to hamster testes failed to induce initial germ cell colonies. One of the advantages of using neonatal testes is that they lack differentiated germ cells, and gonocytes are easily recovered without purification. However, conversion from gonocytes to spermatogonia takes several days, and this in vitro process may not be as efficient as the in vivo process [16]. In contrast, germ cells in the pup testes are already dividing as spermatogonia; however, somatic cells proliferate more actively in pup testis cell culture. Because active growth of somatic cells interferes with germ cell proliferation, the initiation of cultures from pup testes was facilitated by the enrichment of germ cell populations. In previous reports on mouse and rat GS cell cultures [4, 8, 9], several enrichment procedures, including FACS sorting and differential plating following gentle pipetting, were used to eliminate testicular somatic cells. In the current experiments, we used the latter procedure because FACS sorting often damages spermatogonial stem cells [31]. By implementation of these methods, hamster GS cells could be successfully established and were able to proliferate for 1 yr. The cultured cells were confirmed to have spermatogonial stem cell activity as shown by the production of germ cell colonies following lentiviral transduction and spermatogonial transplantation. These results indicated that the cells were definitively stem cells and that hamster GS cells can be genetically modified.

Although the general characteristics of hamster GS cells were similar to those of mice and rat GS cells, several differences were noted. For example, hamster GS cells were difficult to maintain on MEFs and were more easily cultured on laminin-coated dishes. This is in contrast to mouse and rat GS cells, which are easily maintained on MEFs [9, 18]. This result was unexpected, as previous studies from our laboratory have shown that GS cells grow more slowly on laminin-coated dishes [18]. Mouse embryonic fibroblasts may negatively affect the growth of hamster GS cells by secreting unidentified cytokines. Moreover, hamster GS cells proliferated more actively in TX-WES medium than in StemPro-based medium. TX-WES medium was not able to maintain mouse GS cells long term. It is currently unclear what constituents of TX-WES medium enhanced GS cell proliferation. Although adverse effects of serum on mouse GS cells are possible, the proliferation of hamster GS cells did not improve after we increased the serum concentration in StemPro-based medium (our unpublished observation), which suggests that other constituents enhanced germ cell growth. These results collectively show that the growth requirements of hamster and mouse GS cells may be similar but are not identical.

Although hamster GS cells initiated spermatogenesis in mouse testes, the degree of spermatogenesis was limited, and we were not able to obtain transgenic offspring. Our failure was not likely due to technical problems with in vitro microinsemination, because the control embryos were able to develop normally. We speculate that the microenvironment of the xenogeneic testes was not sufficient to support hamster stem cells to full competence, which is necessary for fertilization. We employed xenogeneic transplantation in this study because germ cell transplantation protocols for hamsters have not been established [32]. Xenogeneic spermatogenesis is a unique advantage of using spermatogonial stem cells, as spermatogenesis can occur in xenogeneic hosts between species with close genetic distances. For example, rat stem cells are able to complete spermatogenesis in mouse testes [33]. Furthermore, we recently succeeded in the production of xenogeneic offspring using these germ cells [34]. Although hamster spermatogonial stem cells have been reported to colonize mouse testes in a previous study [27], the degree of colonization and differentiation from primary hamster testis cells (uncultured controls) in this study was more limited (data not shown). The difference between this and the previous study may be explained by the different genetic backgrounds of the recipient nude mice (Ncr vs. KSN backgrounds). Although future studies need to clarify this issue, production of hamster offspring by xenogeneic transplantation in mice may not be efficient due to the wide genetic differences between the two species, and mouse environments may not provide the appropriate growth factors or adhesion molecules to allow for complete spermatogenic cell maturation of hamster stem cells. Although hamster GS cells maintained in long-term culture in this study failed to produce complete spermatogenesis in KSN nude mice, they did colonize recipient mouse testes, proliferate, and produce differentiated germ cells. Therefore, the data are consistent with the interpretation that hamster spermatogonial stem cells were maintained in long-term culture. To test the full spermatogenic potential of these hamster GS cells, it probably will be necessary to establish syngeneic transplantation techniques in this species and produce hamster offspring from these GS cells.

Although the development of hamster GS cells strongly suggests that the culture technique can be extended to other animal species, rat and hamster GS cells grew more slowly than mouse GS cells. This is a big impediment for efficient genetic selection, and it must be overcome. Importantly, previous studies have shown that spermatogonial stem cells from rats and hamsters proliferate actively in vivo when transplanted into mouse seminiferous tubules [27, 33, 34]. This suggests that current culture conditions require additional modifications. It appears that GDNF plays a critical role in self-renewal in a wide range of animal species; however, it is likely that these cells may require additional self-renewal factors. The identification of optimal growth conditions will greatly facilitate germline modification and is clearly necessary at this stage of research.

Animal transgenesis is an important method for studying gene functions in vivo. Although embryos/oocytes from females have been used extensively, these conventional techniques have not been applied to several important animal species, including hamsters, due to egg fragility or small numbers. Spermatogonial stem cells in the male germline may provide a solution to these problems. Hamsters have been historically difficult to manipulate genetically, but they may provide a good animal model if current GS cell techniques improve. Future advancements in this technology will allow for germline modification in a diverse array of mammalian species.

ACKNOWLEDGMENTS

We thank Mr. H. Sakashita and Ms. Y. Ogata for their technical assistance. We also thank Dr. Yamanaka for providing the lentivirus vector.

FOOTNOTES

1Supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan, Genome Network Project, Takeda Science Foundation, The Nakajima Foundation, Ichiro Kanehara Foundation, Kowa Life Science, Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation, and Suzuken Memorial Foundation. Back

Correspondence: 2Takashi Shinohara, Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan. FAX: 81 75 751 4169; e-mail: tshinoha{at}virus.kyoto-u.ac.jp

Received: 17 September 2007.

First decision: 11 October 2007.

Accepted: 3 December 2007.

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