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Horizontal Medical Research Organization3
Department of Pathology and Biology of Diseases,4 Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
Department of Molecular Genetics,5 Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
The Institute of Physical and Chemical Research (RIKEN),6 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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A valuable approach for studying this problem would be to reproduce stem cell self-renewal division in vitro. This would elucidate the minimal external requirement of the self-renewal process and provide important information on the stem cell-niche interaction in vivo. Recently, we described a method of culturing mouse spermatogonial stem cells [11]. In this method, spermatogonial stem cells from neonatal testis were able to proliferate for more than 5 mo on mouse embryonic fibroblasts (MEFs) in the presence of glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), leukemia inhibitory factor (LIF), and fetal calf serum (FCS). After transplantation into infertile recipient testes, the cultured cells retained stem cell activity, underwent spermatogenesis, and produced offspring. In addition to the systems for embryonic stem (ES) and embryonic germ (EG) cells [12–15], this method is the third method for expanding germline cells. Based on these results, we named these cells germline stem (GS) cells to distinguish them from the other two cell types. This GS cell culture system supports the self-renewal of spermatogonial stem cells and opens up a possible way to analyze stem cell-niche interactions in vitro.
However, the use of serum and feeder cells in this culture system is limited by our lack of knowledge of the factors that regulated spermatogonial stem cells. Serum contains complex undefined materials that occasionally affect cell differentiation [16]. For example, neural stem cells will differentiate into progenitor cells when they are cultured in medium containing serum [17]. In addition to providing physical support for stem cell attachment, feeder cells also affect stem cells by producing various undefined factors through their interactions with stem cells. Therefore, the presence of serum or feeder cells complicates the culture conditions, making them uncontrollable.
This study examined the possibility of establishing serum- or feeder-free cultures of germline stem cells. We show that GS cells continued to proliferate in the absence of serum or feeder cells, although they could not proliferate when both of them are absent. The cultured cells were assessed for stem cell activity by using spermatogonial transplantation and by the ability to produce offspring.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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GS cells were established from the transgenic mouse line C57BL6/ Tg14(act-EGFP-OsbY01) bred into the DBA/2 background (designated Green; provided by Dr. M. Okabe, Osaka University) [11]. The spermatogonia and spermatocytes of these mice express the EGFP gene, although the level of EGFP expression decreases gradually after meiosis [18]. Therefore, donor cells can be identified readily following transplantation. All the GS cells used in our studies were derived from 0- to 2-day-old neonatal mice.
GS cells were established according to an existing protocol [11]. In brief, dissociated neonatal testis cells were cultured overnight on a gelatin-coated plate, and floating cells were passaged to secondary plates. These cells were passaged two to three times before they were transferred onto MEFs. For feeder-free culture, fully established GS cells were cultured on dishes that had been coated with laminin (BD Biosciences, Franklin Lakes, NJ) at a concentration of 20 µg/ml. The basal culture medium was StemPro-34 SFM (Invitrogen, Carlsbad, CA) 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, St. Lois, MO), 5 mg/ml BSA (ICN Biomedicals, Irvine, CA), 2 mM L-glutamine, 5 x 10–5 M 2-mercaptoethanol, MEM Vitamin Solution (Invitrogen), MEM nonessential amino acids solution (Invitrogen), 10–4 M ascorbic acid, 10 µg/ml D-biotin, 30 ng/ml ß-estradiol, and 60 ng/ml progesterone (Sigma). The growth factors used were 20 ng/ml mouse EGF (BD Biosciences), 10 ng/ ml human bFGF (BD Biosciences), 103 U/ml ESGRO (a murine leukemia inhibitory factor; Invitrogen), and 10 ng/ml recombinant rat GDNF (R&D Systems, Minneapolis, MN). Growth factors were added as indicated. MEF-conditioned medium was prepared as described [19]. Serum-supplemented medium was prepared by adding fetal calf serum (JRH Biosciences, Lenexa, KS) to the basal culture medium. In the serum-free culture experiments on MEFs, the basal medium was supplemented with B27 (Invitrogen) with the indicated cytokines. The cells were maintained at 37°C in an atmosphere of 5% carbon dioxide in air. Cell number was determined at each passage. The number of cells seeded was 0.2 to 1.0 x 106 cells/9.6 cm2 in a 6-well culture plate. The rest of the cells were discarded.
Antibodies and Staining
The 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), rat anti-human 
6-integrin (CD49f) (GoH3), biotinylated hamster anti-rat ß1-integrin (CD29) (Ha2/5), biotinylated rat anti-CD9 antigen (KMC8), and allophycocyanin (APC)-conjugated rat anti-mouse c-kit (CD117) (2B8) (BD Biosciences). APC-conjugated goat anti-rat-immunoglobulin G (IgG; Cedarlane Laboratories, ON, 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 followed the process described previously [20]. Cells were analyzed using the FACS-Calibur system, and 10 000 events were collected (BD Biosciences).
Analysis of Marker Gene Expression
Total RNA was isolated using Trizol (Invitrogen). For reverse transcription-polymerase chain reaction (RT-PCR), first-strand cDNA was synthesized with Superscript II (Rnase H– Reverse Transcriptase; Invitrogen), and PCR was carried out with rTaq (Takara, Shiga, Japan). RT-PCR for HPRT, Oct-4, Rex-1, neurogenin3 (ngn3), c-ret, Mvh, Fragilis, and Stella were carried out using specific primers, as described [21–25].
Transplantation and Analysis
Approximately 3 µl of the donor cell suspension was injected into the seminiferous tubules of a WBB6F1-W/Wv (designated W; purchased from Japan SLC, Shizuoka, Japan) recipient through the efferent duct [26, 27]. The injection filled 75% to 85% of the tubules in each recipient testis. To induce tolerance to the allogeneic donor cells, the recipient mice received 50 µg if anti-CD4 antibody (GK1.5) i.p. on Days 0, 2, and 4 after transplantation [28].
To count colonies, recipient mouse testes were recovered 2 mo after donor cell transplantation and analyzed by observing the fluorescence under UV light [18]. Donor germ cells were identified specifically because the 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 [29]. Statistical analysis was performed by t-test. The Institutional Animal Care and Use Committee of Kyoto University approved all the animal experimentation protocols.
Microinsemination
The seminiferous tubules of recipient testes were dissected carefully, and the germ cells were collected mechanically. Microinsemination was performed as described previously [30]. Embryos that reached the four-cell stage after 24 h in culture were transferred to the oviducts of Day 1 pseudopregnant 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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To establish a serum-free culture system of spermatogonial stem cells, we initially examined whether serum was required to establish GS cells. Testis cells were prepared from a newborn Green mouse following an established protocol [11], and cultured on a gelatin-coated plate. The medium was supplemented with EGF, bFGF, LIF, and GDNF. In the absence of serum, few cells attached to the plate, and most floated in the medium. Cells occasionally formed aggregates, but there was no apparent growth of germ cells. In the presence of serum, however, some of the cells had attached to the gelatin-coated plate by the next day, and floating cells could be recovered by vigorous pipetting. These cells were relatively enriched for gonocytes and were transferred to a new culture plate for further culture. When the cells were cultured in a low concentration (0.3% to 2%) of serum, fibroblastic somatic cells began to grow on the culture plate by 2–3 days after culture initiation. The majority of germ cells attached to the somatic cells, and began to form germ cell colonies by 5 to 7 days. By contrast, when a high concentration (5% to 15%) of serum was added to the medium, the germ cells still proliferated on somatic cells and formed colonies, but they eventually disappeared owing to the extensive growth of fibroblastic cells. These results indicate that serum is required for establishing GS cells, although the presence of a high concentration of serum abrogates the propagation of germ cell colonies.
Next, we determined whether the established GS cells required serum for in vitro expansion. EGFP-expressing GS cells were established from a newborn Green mouse in the presence of 1% serum. The cells were passaged to a new plate (
x dilution) when the culture became confluent. Within 3 to 4 wk after culture initiation, there were few fibroblastic somatic cells with repeated passages, and the GS cells were transferred onto mitomycin C-treated MEFs for further expansion (Fig. 1A). Sixty-three days after culture initiation, we transferred the EGFP-expressing GS cells to a serum-free culture. In this culture, we removed serum from the medium and supplemented the medium with B27, a proprietary serum-free supplement designed for long-term viability of neuronal cultures [31, 32]. Upon transfer to MEFs, GS cells attached to MEFs, and the cells retained the characteristic morphology of GS cells (Fig. 1B). Similar to GS cells in serum-containing culture, the cells were passaged every 4 to 6 days under serum-free conditions. However, the cell proliferation was more dramatic in the serum-free condition; while the GS cells multiplied approximately 5-fold after 5 days in medium containing 1% serum, they expanded up to 12-fold in the serum-free culture over the same culture period. Flow cytometric analysis showed no significant changes in cell surface marker expression owing to the removal of serum: GS cells strongly expressed EpCAM [33], CD9 [34], and 6- and ß1-integrin [20]; the cells weakly expressed c-kit [35] and did not express SSEA-1 [36] (Fig. 2A). RT-PCR analysis showed that the cultured cells also expressed other primordial germ cell (PGCs) or spermatogonia markers, including Oct-4 [37] (Fig. 2B).
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To examine whether the cultured cells have the ability to colonize seminiferous tubules, we used a spermatogonial transplantation technique [38]. This technique allows competent spermatogonial stem cells to recolonize the empty seminiferous tubules of infertile animals and to differentiate into mature spermatozoa. EGFP-expressing GS that had been cultured for 6 to 52 days in serum-free culture were transplanted into the seminiferous tubules of immune-suppressed W mice [28] (Fig. 3A). W animals lack differentiating germ cells as a result of mutations in the c-kit gene [39] (Fig. 3B); therefore, any spermatogenesis in the recipient testis is derived from cultured donor cells. After different numbers of passages, the cells were transplanted again to measure the increase in stem cell number during this period. Two months after transplantation, the recipient mice were killed, and colonies in the testes were counted under UV light illumination.
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As shown in Table 1, approximately 6.5 x 107-fold expansion of stem cell number was observed during 46 days in culture, during which the total cell number increased 8.5 x 107-fold (Fig. 1G). Assuming 10% colonization efficiency of stem cells [29], the concentrations of stem cells in culture ranged from 0.66% to 1.33%. Histological analysis of the recipient testis confirmed the presence of normal-appearing spermatogenesis (Fig. 3C). Mature spermatogenic cells were observed in the seminiferous tubules. The cells continued to grow in serum-free conditions for at least 3 mo. These results indicate that GS cells can expand in serum-free conditions and that they have the ability to undergo normal spermatogenesis when transferred into the seminiferous tubule environment.
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Feeder-Free Culture of Mouse GS Cells
We then investigated the role of feeder cells in GS cell expansion. We previously showed that freshly prepared spermatogonial stem cells attach preferentially to laminin [20]; the stem cell concentration could be enriched 3- to 8-fold after selection on a laminin-coated plate [40, 41]. Based on this observation, we hypothesized that laminin would replace MEFs in supporting GS cell growth. To test this possibility, EGFP-expressing GS cells that had been cultured on MEFs for 60 days were transferred onto laminin.
In this experiment, we examined various combinations of cytokines and also tested the effect of conditioned medium from MEFs in some experiments, because it is essential for human ES cells to remain undifferentiated under feeder-free culture conditions [19]. As shown in Table 2, GS cells were able to proliferate under all five conditions tested. The cells continued to grow as long as the medium contained GDNF, and either EGF or bFGF was sufficient to promote GS cell growth in medium containing 1% FCS. However, cells could not grow in the absence of GDNF, even when the medium was supplemented with both EGF and bFGF. Only after GDNF supplementation did cells resume proliferation. We did not find statistical differences in the proliferation rate between treatment groups. The presence of serum was essential for the success of feeder-free culture, as GS cells did not attach to laminin when cultured in the serum-free medium.
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Upon transfer to laminin, GS cells gradually changed their morphology. While GS cells generally formed clumps on MEFs (Fig. 1A), they formed various patterns on laminin, including clumps (Fig. 1C), and had a fibroblast-like appearance (Fig. 1D). Occasionally, cells formed chains or networks (Fig. 1E), which resembled the proliferative patterns of spermatogonial stem cells observed in vivo after spermatogonial transplantation [29]. The morphology of cells in feeder-free culture appeared to depend on the cell density, and no particular cytokine combination or MEF-conditioned medium influenced the proliferative patterns. Generally, cells tended to form clumps at low cell density, but they became fibroblastic at high cell density. When seeded at relatively high density, GS cells proliferated and spread on the bottom of the well, and GS cells covered the entire surface of the well (Fig. 1F).
To confirm the phenotype, the cultured cells were analyzed using flow cytometry for the expression of cell surface markers (Fig. 2A). The cells on laminin had a similar phenotype to GS cells on MEFs; however, not only was the expression of c-kit reduced, but the expression of SSEA-1 was initiated. Whereas GS cells on MEFs do not express SSEA-1 [11], a significant proportion of the cells on laminin expressed this molecule. No significant difference was noted in the cell surface marker expression regardless of the type of cytokine (data not shown). RT-PCR analysis showed that the cultured cells not only expressed PGC markers (Fragilis, Stella) but also a spermatogonia marker (ngn3) [24, 42] (Fig. 2B). As SSEA-1 is expressed on PGCs, but not on spermatogonia [43], this suggested that the feeder-free culture changed the cell surface marker expression and induced a partial embryonic phenotype.
To examine whether the cultured cells still retain the ability to colonize seminiferous tubules and produce spermatogenesis, EGFP-expressing GS cells that had been cultured for different periods (ranging from 9 to 39 days) were recovered using trypsin and were microinjected into the seminiferous tubules of immune-suppressed W mice (Fig. 3D). After 4 to 31 passages, the cells were collected again at 35 to 186 days to measure the increase in stem cell numbers during this period. At least two different cultures with the same cytokine combinations were transplanted. The analysis of colony numbers in the recipient testis indicated that stem cell number increased in all five experiments regardless of the cytokine combination. Assuming 10% colonization efficiency of stem cells [29], the concentration of stem cells ranged from 0.04% to 1.26%. The total cell number increased approximately 1.2 x 109-fold during the 6-mo culture period (Fig. 1G). Statistical analyses revealed that colonization of GS cells was most efficient when GS cells were cultured in culture supernatant of MEFs (P < 0.05 by t-test). LIF was also beneficial for colonization (P < 0.05 by t-test), but we did not find significant difference between EGF and bFGF. Histological sections confirmed the presence of spermatogenesis in the recipient testis, and all stages of spermatogenic cells were found (Fig. 3E).
To further confirm that these germ cells are functionally normal, we attempted to derive offspring from the cultured cells using microinsemination, a technique commonly used to derive offspring from infertile animals and humans [30, 44]. EGFP-expressing GS cells were cultured for 4 mo in feeder-free condition, and transplanted into three immune-suppressed W mice. Approximately 1.5 x 104 cells were microinjected into each testis. Four months after transplantation, two of the recipients were killed because they remained infertile after transplantation. Their testes were dissociated mechanically, and live spermatogenic cells were recovered by repeated pipetting of colonized tubule fragments; EGFP expression was identified under UV light. The cell suspension was kept frozen and stored in liquid nitrogen. After storage for 21 days, mature spermatozoa or elongated spermatids were microinjected into oocytes derived from C57BL/6 x DBA/2 F1 mice. A total of 83 eggs were constructed, and 56 eggs that developed to the two-cell stage were transferred to five pseudopregnant female recipients the day after microinsemination. The recipient females sired a total of 11 offspring, and 9 of them grew into adults: 4 males and 5 females. These offspring were fertile and could transmit the EGFP gene to the next generation (Fig. 3F). Therefore, these results indicate that GS cells in feeder-free culture retain the ability to colonize seminiferous tubules and can differentiate normally to produce fertile offspring.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Stem cells usually require stromal cells to maintain their undifferentiated state both in vivo and in vitro [10, 14, 15, 51]. In spermatogonial stem cell culture, it was initially reported that mouse spermatogonial stem cells could survive in vitro for approximately 4 mo on STO feeder, but they disappear within 1 wk when they are cultured directly on a tissue culture dish [52], which suggested that the feeder layer is essential for maintaining spermatogonial stem cells in vitro. However, subsequent studies revealed that the number of spermatogonial stem cells decreased in culture even on STO feeder, and only 10% to 20% of the stem cells could be recovered after 1 wk in vitro [53]. Similar results were recently reported in rats [54, 55]. The type of feeder cells affected stem cell survival; rat spermatogonial stem cells survived longer on Sertoli cells than they did on STO cells [55], but another study showed that Sertoli cells had a negative effect on spermatogonial stem cells in mice [53]. While these culture conditions failed to induce long-term proliferation of spermatogonial stem cells, our results demonstrate that laminin can substitute for the MEFs, indicating that a feeder layer is not essential for inducing the self-renewal division of spermatogonial stem cells [2].
Of interest, several differences were noted between the feeder-free culture condition and MEF-based culture. For example, GS cells on laminin tend to form various types of colonies, ranging from chains to clumps, whereas those on MEFs generally form clumps. The formation of chain-type colonies was reminiscent of spermatogenic colonies after spermatogonial transplantation into a seminiferous tubule environment [29]. Conversely, the formation of clump-type colonies was also observed in vivo, when GDNF was overexpressed in Sertoli cells [56]. GDNF is an essential factor for the self-renewing division of spermatogonial stem cells [57]; the overexpression of GDNF resulted in the loss of differentiation of germ cells and induced clump formation and expansion of spermatogonial stem cells in vivo [56]. These observations suggested that colony morphology is dependent on the amount of GDNF and that spermatogonial stem cells normally proliferate with chain formation, forming clumps when the proliferation of stem cells is strongly stimulated. In our study, because GDNF was used at the same concentration throughout the experiments, the result suggest that MEFs enhance the effect of GDNF, or exert a positive effect by secreting some other molecules that stimulate the proliferation of spermatogonial stem cells. This is supported by our observation that the GS cell growth on laminin was slower than that using MEFs. Thus, the cell-cell interaction between stem cells and MEFs may provide an additional growth stimulus, which was manifested as the changes in the colony morphology.
Another unexpected feature of the feeder-free culture was the changes in surface marker expression. In particular, the expression of SSEA-1 was unexpected. SSEA-1 is expressed on early embryos and PGCs, but its expression disappears after midgestation [43]. Conversely, GS cells on laminin also expressed ngn3, which is a marker of undifferentiated spermatogonia in the postnatal testis [24]. The ratio of cells expressing c-kit was also reduced in the feeder-free culture. Therefore, the pattern of gene expression in GS cells is influenced by the culture environment. Currently, we do not know why GS cells start to express such an embryonic marker in feeder-free culture. Although we cannot exclude the possibility that this reflects the abnormal reaction of spermatogonial stem cells to the unphysiological environment, it is possible that there is a rare population of spermatogonial stem cells that express SSEA-1 in vivo. Because there are very few stem cells (only 2 to 3 cells in 104 testis cells) [2, 58], it is possible that such a rare population escapes detection using conventional immunohistological methods on testis sections. Alternatively, perhaps GS cells have the potential to change into cells with more primitive characteristics.
Despite the different colony morphology and surface marker expression, GS cells on laminin retained the capacity to produce spermatogenic colonies and offspring following transplantation into infertile mice. Given the ability to self-renew as the defining characteristics of stem cells, these results indicate that cultured cells have stem cell activity as spermatogonial stem cells. Although the expression of SSEA-1 suggested a fetal phenotype of GS cells, spermatogenesis occurred efficiently in transplant recipients, which indicates that GS cells on laminin are functionally comparable to spermatogonial stem cells. Although direct comparison was not made, the frequency of stem cells in feeder-free culture was significantly higher than that reported in our previous study using MEFs (Table 3). Because it is possible to collect a large number of stem cells from culture, it provides a new approach for characterizing stem cells. For example, it may be easier to obtain a more pure population of stem cells from culture than currently achieved using primary cells from cryptorchid testis. Combined, our findings demonstrate that direct contact with somatic stromal cells is not necessary for the self-renewal division of spermatogonial stem cells and that cytokines and laminin can replace at least some aspects of niche function.
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While this study provides the first step in reproducing the self-renewal division of spermatogonial stem cells under defined condition in vitro, an improved culture system is clearly necessary to allow more detailed analysis of self-renewal division and its relationship with its niche. Although laminin was able to replace MEFs in the serum-containing culture, it was not possible to culture GS cells on laminin under serum-free condition. It will also be important to establish culture conditions that direct a specific type of self-renewal, such as asymmetric division or self-renewing division [2]. In this sense, our results will be useful because serum-free and feeder-free culture systems will allow more definitive experiments to test the effect of individual factors on stem cells. The identification of critical factors will increase our knowledge of how the self-renewal division of spermatogonial stem cells is regulated and will lead us to develop more efficient techniques for male germline modification.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Takashi Shinohara, Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. FAX: 81 75 751 4169; takashi{at}mfour.med.kyoto-u.ac.jp
Received: 20 September 2004.
First decision: 12 October 2004.
Accepted: 19 November 2004.
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