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BOR - Papers in Press, published online ahead of print January 22, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.014050
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BIOLOGY OF REPRODUCTION 68, 2207–2214 (2003)
DOI: 10.1095/biolreprod.102.014050
© 2003 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Maintenance of Mouse Male Germ Line Stem Cells In Vitro1

Makoto Nagano3, Buom-Yong Ryu, Clayton J. Brinster, Mary R. Avarbock, and Ralph L. Brinster2

Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The proliferation and differentiation of a stem cell are regulated intrinsically by the stem cell and extrinsically by the stem cell niche. Elucidation of regulatory mechanisms of spermatogonial stem cells (SSCs), the stem cell of the postnatal male germ line, would be facilitated by in vitro studies that provide a defined microenvironment reconstituted ex vivo. We analyzed the effect of in vitro environment on the maintenance of adult and immature SSCs in a 7-day culture system. Allthough the number of adult and immature SSCs decreased in a time-dependent manner, nearly one in four stem cells (24%) could be maintained in vitro for 7 days. Stem cell maintenance was enhanced by coculture with OP9 bone marrow stroma or L fibroblast cell lines, addition of glial cell line-derived neurotrophic factor, or utilization of specific culture medium. In contrast, coculture with TM4 or SF7 Sertoli cell lines and addition of activin A or bone morphogenetic protein 4 (BMP4) reduced stem cell maintenance in vitro. Only 4% of the stem cells remained when cultured with TM4 cells or activin A, and 6% remained when cultured with SF7 cells or BMP4. These results lead to the hypothesis that suppression of germ cell differentiation improves in vitro maintenance of SSCs by interrupting the unidirectional cascade of spermatogenesis and blocking stem cell differentiation.

developmental biology, gamete biology, gametogenesis, spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Stem cells self-renew and differentiate to committed progenitors, providing terminally differentiated cells that perform normal tissue/organ functions throughout life [1]. Because of these unique abilities, stem cells have become an important target in regenerative medicine and gene/cell therapy. To maintain or regenerate normal tissue structure and function, stem cell self-renewal and differentiation must be tightly regulated. Transformation of normal stem cells, which causes distorted potential of stem cells to self-renew and differentiate, may often result in tumorigenesis [2]. However, the mechanism of stem cell fate decision (self-renewal or differentiation) is poorly understood in mammals. Studies using invertebrate models suggest that the stem cell fate decision is regulated by the intrinsic ability of stem cells themselves and close interactions between stem cells and their local microenvironment (stem cell niche). These mechanisms are expected to be similar in the mammalian stem cell system [3–5].

Spermatogenesis is an extremely active and complex process arising from male germ line stem cells (spermatogonial stem cells [SSCs]) that produce spermatozoa [6, 7]. Stem cells in the male germ line confer the long-term reproductive ability of a male after puberty and account for the restoration of spermatogenesis after various types of testicular damage [6]. Sertoli cells, the somatic cells in the seminiferous epithelium, are a major component of the stem cell niche for SSCs [5, 8] and orchestrate the process of spermatogenesis from stem cells to spermatozoa [9, 10]. However, the fate decision mechanism of SSCs has been elusive. The analysis of genetic mutants is an effective approach to investigation of stem cell fate decision as has been widely employed in invertebrate studies. However, despite the generation of numerous mutant mice that exhibit male infertility, no specific model for the study of SSCs has been developed [11]. Another approach to study of the mechanism of stem cell fate decision is to culture SSCs in vitro and then evaluate their responses to a defined culture environment. The analysis of in vitro behavior of SSCs, however, requires a biological assay to detect stem cell activity. Stem cells are functionally defined, and no identification markers are currently available for SSCs.

Development of spermatogonial transplantation has allowed the detection of SSC activity and study of SSC biology [12, 13]. With this technique, male germ cells derived from the testis of a fertile mouse are transferred into the seminiferous tubules of an infertile host mouse. Donor stem cells colonize recipient seminiferous epithelium and establish complete and long-term spermatogenesis. Because spermatogenesis is a unidirectional differentiation event producing a single type of terminally differentiated cell (spermatozoa), nonstem germ cells do not reestablish complete spermatogenesis after transplantation. Therefore, spermatogonial transplantation unequivocally detects SSCs in any donor cell preparation, including germ cells cultured in vitro. Because donor-derived spermatogenesis results in distinctive colonies in recipient seminiferous tubules and each colony is believed to arise from a single stem cell [14, 15], the transplantation technique enables quantitative analyses of SSCs.

Combining in vitro culture and the transplantation technique, we have previously shown that mouse SSCs can be maintained for long periods in culture while retaining their full biological ability to generate complete spermatogenesis upon transplantation [16]. We further demonstrated that a foreign gene can be delivered into the genome of mouse SSCs in vitro using a retroviral vector and that modified stem cells generate spermatogenesis following transplantation [17]. Because retroviral vectors require host cell division for gene delivery, these studies demonstrate that SSCs undergo self-renewal divisions in vitro and suggest the possibility of expanding the stem cell population ex vivo. Recipient mice transplanted with SSCs modified with a retroviral vector subsequently produced transgenic offspring, indicating the feasibility of transgenesis through direct manipulation of the postnatal male germ line [18]

To better understand the SSC fate decision mechanism and the biology of stem cell niche for SSCs, we used spermatogonial transplantation as a functional assay of stem cells to examine the effect on SSCs of extrinsic stimuli exerted by the in vitro microenvironment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Donor Mice and Cell Culture

Donor cells were obtained from the testes of adult and pup ROSA26 transgenic mice, which express lacZ in virtually all cell types including all types of postnatal male germ cells [14, 19]. Adult ROSA26 mice on C57BL/6 (B6) background (B6-Gtrosa26; Jackson Laboratory, Bar Harbor, ME) were designated ROSA26-B6 and were made cryptorchid at 7–8 wk of age and maintained for 2 mo prior to donor cell preparation, which results in 20- to 25-fold enrichment of SSCs [20]. Because the homozygous ROSA26-B6 strain does not breed efficiently, we used hemizygous males as adult donors. Pup donor testis cells were obtained at 5–8 days of age from ROSA26 mice on B6/129 background (B6;129S-Gtrosa26; Jackson Laboratory) and designated ROSA26-B6/129. A donor cell suspension was prepared using a two-step enzymatic digestion as previously described [21].

Donor cells were placed on a feeder layer in a six-well tissue culture plate (9.6 cm2/well) at 2 x 106 cells/well unless indicated otherwise. Although we used various cell lines as feeder layers, a mitomycin C-treated STO (SIM mouse embryo-derived thioguanine and ouabain resistant) fibroblast cell line served as the control to evaluate the effects of other feeder cell lines. The cell lines used in this study and the procedure used for mitomycin C treatment [22] are summarized in Table 1. Mitomycin C-treated feeder cells were placed in the wells coated with 0.1% gelatin 1–3 days before experiments [16]. Donor testis cells were cultured at 32°C for 7 days (unless indicated otherwise) in an atmosphere of 5% carbon dioxide in air. The basic medium for testis cell culture (designated DMEM-B) was prepared with Dulbecco modified Eagle medium (DMEM) (12100; Life Technologies/Invitrogen, Carlsbad, CA) plus 10% fetal bovine serum (FBS), 2.2 g/L sodium bicarbonate, 100 units/ml penicillin (15140; Life Technologies), and 100 µg/ml streptomycin (15140; Life Technologies). DMEM-C medium was prepared by supplementing DMEM-B with 1.25 g/L sodium bicarbonate, 2 mM glutamine (25030-081; Life Technologies), 0.4 mM pyruvic acid (P-5280; Sigma Chemical Co., St. Louis, MO), 6 mM lactic acid (L-4263; Sigma), and 0.1 mM 2-mercaptoethanol (M-7522; Sigma). DMEM-C medium was used throughout this study except in the experiments to evaluate the effect of medium composition on in vitro SSC maintenance. The medium was not changed for 7 days except in the experiments using growth factors. At least two experiments were performed for each condition.


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  		TABLE 1. Preparation of feeder layers using various cell lines.*

To evaluate the effect of soluble factors on in vitro SSC maintenance, growth factors were added to the donor cell culture, which was maintained on the STO feeder layer for 7 days. The growth factor and concentration were as follows: Steel factor, 50 ng/ml (a gift from Amgen, Thousand Oaks, CA) [23, 24]; leukemia inhibitory factor, 10 ng/ml (Life Technologies) [23, 24]; basic fibroblast growth factor, 20 ng/ml (Life Technologies) [23, 24]; bone morphogenetic protein 4 (BMP4), 50 ng/ml (R&D Systems, Minneapolis, MN) [25–27]; activin A, 50 ng/ml (R&D Systems) [28, 29]; Flk-2/Flt-3 ligand, 100 ng/ml (R&D Systems) [30, 31]; and glial cell line-derived neurotrophic factor, 10 and 100 ng/ml (R&D Systems) [32, 33]. These growth factors were added on Day 0, and donor cell culture was replenished with fresh medium and growth factors on Day 3.

To evaluate the effect of medium composition on in vitro SSC maintenance, DMEM-based medium and modified Eagle medium (MEM)-{alpha} (12561, Life Technologies)-based medium with or without supplements were used for testis cell culture on STO feeder cells for 7 days. In this series of experiments, donor cells were obtained from cryptorchid testes of ROSA26-B6/129 mice. For DMEM-based media, DMEM-B and DMEM-C were used. Two types of MEM-{alpha}-based medium were also prepared. The basic MEM-{alpha} medium (12561, Life Technologies), designated MEM-B, contained 10% FBS, 2.2 g/L sodium bicarbonate, 100 units/ml penicillin, and 100 µg/ml streptomycin. The second type of MEM-{alpha} medium (MEM-C) was prepared by supplementing MEM-B with 2 mM glutamine, 1.25 g/L sodium bicarbonate, 0.4 mM pyruvic acid, 6 mM lactic acid, and 0.1 mM 2-mercaptoethanol. These modifications were made to examine the effects of basic medium type (DMEM and MEM-{alpha}), metabolic substrates (glutamine, pyruvic acid, and lactic acid), and buffering/oxidative status (sodium bicarbonate and 2-mercaptoethanol).

Recipient Mice and Transplantation Analysis

Recipients used were 129/B6 F1 hybrid mice. At 4–6 wk of age, the recipient mice were injected i.p. with busulfan (50 mg/kg body weight), which destroys endogenous spermatogenesis. Recipients were used for transplantation >=4 wk after injection [14]. Cultured donor cells were harvested using 0.25% trypsin plus 1 mM EDTA, and the cells were suspended in DMEM culture medium supplemented with 0.1 mg/ml DNase I following passage through a 70-µm mesh. Cell concentration for transplantation was 10–30 x 106 cells/ml. Approximately 10 µl of donor cell suspension was transplanted into a recipient testis through the efferent duct [21].

Two months following transplantation, recipient testes were stained with 5-bromo-4-chloro-3-indolyl ß-D-galactoside (X-gal) to visualize the colonies of donor-derived spermatogenesis, and the number of colonies was counted [14, 21]. The number of colonies represents the number of stem cells that successfully colonized recipient testes [14, 15]. The results are expressed as the mean number of colonies ± SEM per 106 donor testis cells that were originally placed in culture, as described previously [17, 18]. Because the number of donor cells recovered at the end of culture varies depending on culture conditions, the colony number obtained after transplantation must be standardized on the basis of initial input of donor cells into the culture for quantitative analyses of cultured SSCs. Data obtained from two or three experiments involving at least 10 recipient testes were pooled for each culture condition, and significance was determined using ANOVA followed by a Tukey multiple range test or Student t-test. Paraffin-embedded sections of the colonies of donor-derived spermatogenesis were prepared following X-gal staining and were counterstained with nuclear fast red. All animal experimentation protocols were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Short-Term Maintenance of Adult SSCs In Vitro

To evaluate the short-term maintenance of adult SSCs in vitro, adult donor cells derived from cryptorchid testes were cultured on STO feeder cells, harvested 0, 2, and 7 days later, and transplanted into adult recipient testes. Two months following transplantation, the number of colonies from donor-derived spermatogenesis was counted. The stem cell number was expressed as the number of colonies per 106 donor cells that were originally placed in culture.

Transplantation of adult donor cells without culture (Day 0) resulted in 357.8 ± 36.7 colonies (mean ± SEM, n = 18) per 106 cells injected into recipient testes. The number of donor-derived colonies significantly decreased during the culture periods and was 164.5 ± 14.2 (n = 17) and 43.9 ± 5.5 (n = 15) colonies per 106 cells originally placed in culture after 2 and 7 days, respectively (P <= 0.002). Thus, 46% of stem cells remained for 2 days in vitro and 12% remained for 7 days.

To evaluate the effect of temperature on stem cell maintenance in vitro, we cultured adult donor cells at 32°C and 37°C for 7 days. Following transplantation, 56.6 ± 9.5 (n = 15) and 54.9 ± 9.9 (n = 15) colonies were produced after in vitro culture at 32°C and 37°C, respectively. There was no significant difference between the two groups, indicating that the culture temperature did not affect maintenance of SSCs for 7 days.

We next examined the effect of donor cell density in culture on SSC maintenance. Donor testis cells were seeded at 1.0, 2.1, and 4.2 x 105 cells/cm2 on the STO feeder layer and cultured at 32°C for 7 days. Transplantation of cultured cells resulted in 41.2 ± 5.2 (n = 14), 32.3 ± 4.3 (n = 13), and 22.4 ± 2.2 (n = 13) colonies per 106 cells at seeding densities of 1.0, 2.1, and 4.2 x 105 cells/cm2, respectively. A significant difference was detected between 1.0 and 4.2 x 105 cells/cm2 (P = 0.007) but not between 1.0 and 2.1 x 105 cells/cm2 or 2.1 and 4.2 x 105 cells/cm2. In subsequent experiments, we used 2.1 x 105 cells/cm2 to provide sufficient stem cells for transplantation and to allow for significant increase and decrease in colony number following various treatments.

Short-Term Maintenance of Pup SSCs In Vitro

To evaluate the in vitro maintenance of pup SSCs, we cultured testis cells derived from 5- to 8-day-old mice in the same manner used for adult cells. Transplantation of donor pup testis cells without culture resulted in 140.3 ± 18.5 colonies per 106 cells injected into recipient testis (n = 15), which confirmed our previous results that the stem cell concentration in pup testis cells is ~2.5-fold less than that of adult cryptorchid testis cells [8, 20]. Transplantation of pup donor cells cultured for 2 and 7 days resulted in a significant decrease in colony numbers: 90.2 ± 7.7 (n = 14) and 17.3 ± 2.8 (n = 16) colonies per 106 cells originally seeded, respectively (P < 0.001). Thus, 12% of SSCs remained in culture for 7 days regardless of donor ages, although significantly more pup SSCs than adult SSCs remained during the first 2 days (64% vs. 46%, respectively; P = 0.010).

Effects of Feeder Layers on the In Vitro Maintenance of Adult SSCs

Long-term culture of SSCs is feasible only when germ cells are cocultured with feeder cells, suggesting that feeder cells may have a significant impact on in vitro maintenance of SSCs [16]. To evaluate the influence of feeder cells on stem cell maintenance, we cocultured adult donor cells for 7 days with various types of cells as feeder cells. Table 1 summarizes the different cell lines used as feeder cells, which can be divided into three categories based on the origin of tissue: fibroblast, Sertoli cell, and bone marrow stroma cell lines. For each experiment, a coculture using STO feeder cells was prepared as a control, which generated 36.3 ± 1.4 colonies per 106 cells originally placed in culture (n = 174). The results are expressed as the ratio of the number of colonies per 106 cells originally seeded relative to the number of colonies per 106 cells for the control culture with the STO feeder layer (Fig. 1).



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  		FIG. 1. Effect of feeder cells on maintenance of mouse SSCs in vitro. Adult cryptorchid testis cells were cultured on various feeder layers at 32°C for 7 days and transplanted into the recipient testes. The data (mean ± SEM) are expressed as the number of colonies per 106 donor cells originally placed in culture relative to that obtained with the control culture using STO feeder cells (relative colonization activity). A significant effect (asterisk) was observed in coculture with L (2.23- ± 0.33-fold increase, P < 0.001), SF7 (0.50- ± 0.07-fold, P = 0.001), TM4 (0.33- ± 0.06-fold, P < 0.001), and OP9 cells (1.96- ± 0.28-fold, P = 0.011). The average number of colonies obtained using STO feeder cells as the control culture was 36.27 ± 1.44 (n = 174) per 106 donor cells. Two experiments were performed for each condition, and at least 10 recipient testes were analyzed. Sl, Sl/Sl4; m220, Sl/Sl4-m220; m248, Sl/Sl4-m248

Fibroblast cell lines as feeders We first examined the effects of widely used fibroblast cell lines, e.g., L and NIH/3T3 (Fig. 1). Following transplantation, donor cells cultured on L cells produced 2.2-fold more colonies than those cultured on STO feeders (Fig. 2), whereas donor cells cultured on NIH/3T3 cells generated 1.3-fold more colonies than did the STO control culture. Statistical analysis showed that L cells had a significant impact on in vitro SSC maintenance but NIH/3T3 cells did not.



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  		FIG. 2. Coculture of mouse testis cells with L fibroblast or OP9 bone marrow stroma cell lines results in maintenance of a greater number of SSCs and more colonies than control culture with STO feeder cells. A– C) Gross appearance of a recipient testis following transplantation of adult donor cells cultured on STO (A), L (B), and OP9 (C) feeder cells. Colonies of donor-derived spermatogenesis were visualized by X-gal staining (blue). Coculture with L or OP9 cells resulted in a ~2-fold increase in colony number following transplantation, compared with STO control culture. D) Histological section of donor-derived spermatogenesis following coculture with L cells. SSCs cultured in vitro retain the ability to reconstitute complete spermatogenesis and produce spermatozoa (arrows). Counterstained with the nuclear fast red. A–C, bar = 2 mm; D, bar = 20 µm

We next examined the effect of fibroblast feeders that express isoforms of Steel factor, which supports differentiating spermatogonia [34–36]. Although c-kit, the receptor for Steel factor, is not expressed on SSCs [37, 38], Steel factor could regulate survival and proliferation of other types of spermatogonia, thereby affecting in vitro maintenance of SSCs. Therefore, prior to transplantation we cocultured SSCs with three embryonic fibroblast cell lines: Sl/Sl4, Sl/Sl4-m220, and Sl/Sl4-m248 [39]. Sl/Sl4 is a fibroblast cell line that expresses no Steel factor. Sl/Sl4-m220 is a variant Sl/Sl4 cell line that expresses a membrane-bound form of Steel factor (220 amino acids), although the secreted form is also produced at a level that is 3- to 5-fold lower than that of the membrane-bound form. Sl/Sl4-m248 is a variant Sl/Sl4 cell line transfected with the full-length Steel factor cDNA (for 248 amino acids) and produces both forms of Steel factor [39]. The transplantation results indicated that cocultures with Sl/Sl4 and Sl/Sl4-m220 feeders produced an approximately 1.4-fold increase in colony numbers, whereas coculture with Sl/Sl4-m248 had no effect (Fig. 1). However, no significant difference was detected among these conditions, indicating that fibroblast feeder layers with or without Steel factor did not significantly affect in vitro maintenance of SSCs.

Sertoli cell lines as feeders To examine the effect of Sertoli cells on in vitro SSC maintenance, we cultured donor cells with four cell lines derived from Sertoli cells: the SF7 cell line, which was generated using the simian virus 40 (SV40) large T antigen [40], the TM4 cell line, which was isolated from a primary culture enriched for Sertoli cells [41, 42], the MSC-1 cell line, which was established from transgenic mice carrying a fusion gene composed of the regulatory sequence of human Müllerian inhibitory substance linked to the SV40 large T antigen [43], and the 15P-1 cell line, which was immortalized with the polyoma virus large T antigen [44]. Following transplantation, coculture with MSC-1 and 15P-1 cells did not produce a significantly different number of colonies from that produced by the STO control culture, whereas coculture with SF7 and TM4 cells resulted in a significant decrease in number of colonies (Fig. 1). The number of colonies established following coculture with SF7 and TM4 cells was 50% and 33% of that derived from the STO control culture, respectively (P <= 0.001). Thus, SF7 and TM4 Sertoli cell lines significantly reduced the stem cell number in vitro, whereas MSC-1 and 15P-1 cells had no effect.

Bone marrow stroma cells as feeders Bone marrow stroma cells have been an important component for the in vitro maintenance of hematopoietic stem/progenitor cells [45]. Because biological characteristics may be conserved among stem cells of various tissues, we examined whether bone marrow stroma cell lines also influence SSC maintenance in vitro. We used three bone marrow stroma cell lines: OP9, which induces hematopoietic differentiation in embryonic stem cells [46, 47], MC3T3-G2/PA6 (designated PA6), which promotes proliferation of hematopoietic progenitors [48], and ST2, which supports myelopoiesis and B lymphopoiesis [49]. Following transplantation, the number of colonies was 2-fold greater for OP9 coculture than for the control culture (Figs. 1 and 2). Although coculture with PA6 cells did not increase the efficiency of SSC maintenance in vitro, coculture with ST2 cells resulted in a 1.7-fold increase in number of colonies (Fig. 1). However, a significant difference was observed only for coculture with OP9 cells (P = 0.011). Therefore, the results indicated that although OP9 bone marrow stroma cell line had a positive impact on in vitro SSC maintenance, ST2 and PA6 did not.

Effects of Soluble Growth Factors on the In Vitro Maintenance of Adult SSCs

To examine the effects of various growth factors on in vitro maintenance of adult SSCs, we cultured donor testis cells on the STO feeder layer with a growth factor for 7 days prior to transplantation. The results were evaluated in the same manner as for experiments using various feeder cells. We first examined the effect of Steel factor, leukemia inhibitory factor (LIF), and basic fibroblast growth factor because these growth factors are known to promote maintenance or proliferation of primordial germ cells in vitro [23, 24]. In addition, LIF has also been reported to increase spermatogonial survival in vitro [50]. However, transplantation results showed that none of these factors individually improved SSC maintenance in vitro, compared with the coculture with STO feeders without any added factors. We also examined the effect of another growth factor, Flk-2/Flt-3 ligand (designated Flk-2L), which is structurally related to Steel factor. Flk-2L stimulates in vitro survival and proliferation of various types of hematopoietic cells, including stem/progenitor cells [51]. Following culture with Flk-2L and transplantation, the colony number was significantly reduced to ~70% that of the control culture (Fig. 3, P = 0.047).



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  		FIG. 3. Effects of growth factors on in vitro mouse SSC maintenance. Adult donor cells obtained from cryptorchid testes were cultured on the STO feeder layer at 32°C for 7 days with or without the growth factor indicated. The data are expressed in the same manner as in Fig. 2. A significant effect (asterisk) was observed with Flk-2L (0.69- ± 0.08-fold increase, P = 0.047), activin A (0.31- ± 0.05-fold, P < 0.001), BMP4 (0.42- ± 0.07-fold, P < 0.001), and 100 ng/ml GDNF (1.57- ± 0.18-fold, P = 0.033). Two experiments were performed, and at least 10 recipient testes were analyzed

We next examined the effect on SSC maintenance in vitro of growth factors that belong to the transforming growth factor ß (TGFß) superfamily, because members of the TGFß superfamily have significant effects on germ cell development and spermatogenesis. BMP4 is an important determinant in germ line specification and germ cell development in the embryo [52]. Activin stimulates proliferation of spermatogonia in vitro [29], and a distantly related member of the TGFß family, glial cell line-derived neurotrophic factor (GDNF), is an important regulator of spermatogonial differentiation in vivo [53]. Thus, we cultured donor testis cells on STO feeders with each of these growth factors and transplanted these cells into recipient testes (Fig. 3). Colony numbers were significantly reduced to 40% and 30% of the colonies produced by control cultures when donor cells were exposed to BMP4 and activin A, respectively (P < 0.001 for both). In contrast, when donor cells were cultured with 100 ng/ml GDNF, the colony number was significantly greater than that for the control culture (~1.6-fold increase, P = 0.033; Fig. 3), although donor cell culture with 10 ng/ml GDNF did not result in a significant difference (~1.2-fold increase, P = 0.276; Fig. 3). Thus, among the factors of the TGFß superfamily examined, BMP4 and activin A had a negative effect whereas GDNF had a positive effect on in vitro maintenance of SSCs for 7 days.

Effects of Medium Composition on the In Vitro Maintenance of Adult SSCs

Because the development of optimal culture medium greatly contributed to the establishment of egg culture [54], we examined the efficiency of in vitro SSC maintenance in DMEM-based medium and MEM-{alpha}-based medium with or without supplement using the STO cell coculture system for 7 days. Following adult testis cell culture using DMEM-based medium, 31.8 ± 3.1 (n = 11) and 32.3 ± 5.7 (n = 11) colonies were observed with and without supplement, respectively. Thus, an average of 32.1 colonies were derived after culture in DMEM-based medium in these experiments. When donor cells were cultured in MEM-{alpha}-based medium, 55.1 ± 5.7 (n = 12) and 51.1 ± 5.3 (n = 10) colonies were obtained with and without supplement, respectively (53.1 colonies on average). Thus, although no effect was observed with medium supplementation (P = 0.656) regardless of the medium type, culture with MEM-{alpha}-based medium resulted in a 1.7-fold (53.1 vs. 32.1) higher number of stem cells maintaining in vitro than culture with DMEM-based medium (P < 0.001).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Because stem cells are defined functionally, a biological assay is needed to detect definitive stem cells of any tissue. Cell transplantation that results in complete regeneration and long-term maintenance of a tissue is an unequivocal functional assay of stem cells. Development of the bone marrow transplantation assay allowed intensive investigation into the biology of hematopoietic stem cells (HSCs) and has contributed to studies of in vitro culture requirements for HSCs [55–57]. The male germ line is the only other cell lineage for which a transplantation assay of stem cell activity has been established. Using this functional assay of SSCs, we determined that the number of stem cells remaining after a 7-day culture period decreased in a time-dependent manner, but manipulation of culture conditions significantly affected the efficiency of in vitro SSC maintenance.

When cocultured with STO feeder cells with no growth factor supplement, adult SSCs rapidly decreased in number. More than half of adult SSCs disappeared after 2 days of culture, and only 12% remained in vitro for 7 days. A similar pattern of decrease in SSC number was also observed with pup donor cells, indicating that regardless of donor age, the majority of SSCs disappear under the current culture conditions. However, significantly more pup SSCs than adult SSCs remained during the first 2 days in culture (64% vs. 46%), probably because of active proliferation of pup stem cells. In mice, spermatogonia are quiescent at birth and initiate active proliferation during the first postnatal week [58]. This active proliferation of pup spermatogonia can also be observed in vitro; these cells rapidly incorporate 3H-thymidine during the initial 48 h in culture [59]. Our previous studies with a retroviral vector have indicated that pup SSCs self-renew more actively in vitro than do adult SSCs [17, 18]. These findings suggest that the active proliferation kinetics of pup SSCs resulted in a better in vitro maintenance of pup than adult stem cells for the first 2 days. However, cell loss occurred more rapidly with pup SSCs thereafter, and a similar efficiency (12%) of SSC maintenance resulted with both donor ages after 7 days in culture.

The 12% efficiency of SSC maintenance was increased ~2-fold when donor cells were cultured with OP9 and L cells (Fig. 1), indicating that ~24% of SSCs, nearly one out of four stem cells, can be maintained for 7 days in our culture system. This efficiency of stem cell maintenance is comparable to that of hematopoietic progenitors (colony forming unit-spleen) in a 7-day culture with bone marrow stroma cells (26%) [45] or in a suspension culture without growth factor supplement (23%) [60].

These cultured SSCs are capable of regenerating complete spermatogenesis following transplantation (Fig. 2) [16], demonstrating that some SSCs always survive in vitro, and when transplanted they function effectively to produce sperm. Our previous research revealed that the sperm arising from cultured SSCs after transplantation is functional and transmits donor genes to offspring [18].

In this study, the efficiency of SSC maintenance in vitro was improved and stem cell activity was retained, but net expansion of SSCs was not achieved under the current culture conditions. In fact, in vitro amplification of stem cells has been hard to achieve. Although in vitro HSC expansion strategies have been intensively studied using various growth factors and cytokines, none have been conclusively shown to activate stem cell-specific proliferation, and the current consensus is that the majority of cells that respond to these factors are committed progenitors [57]. However, if self-renewal is considered an autonomous function of stem cells, then stem cell maintenance and expansion can be achieved by inhibiting cell death and differentiation. Although stem cell death could be the major cause of the loss of SSCs during culture observed in this study, it cannot be evaluated at present because identification of SSCs in culture is not possible because of the absence of stem cell markers. In this study, however, when agents thought or known to block spermatogonial differentiation were added to the cultures, the number of stem cells remaining in vitro increased, whereas addition of agents believed to support proliferation of committed spermatogonia or differentiation processes in spermatogenesis decreased the number of stem cells. These results suggest that differentiation of stem cells may be, at least in part, responsible for the loss of SSCs in vitro, and thus the stem cells were better maintained in culture when differentiation was blocked.

Addition of GDNF in culture resulted in a significant improvement in stem cell maintenance (Fig. 3). Forced expression of GDNF in transgenic mouse testes results in accumulation of undifferentiated spermatogonia in vivo without a change in cell proliferation kinetics, leading to the conclusion that GDNF inhibits spermatogonial differentiation [53]. Likewise, blocking spermatogonial differentiation in culture supplemented with GDNF may have resulted in a better maintenance of SSCs in this study. Because spermatogonial differentiation is unidirectional from stem cells to advanced stages of spermatogonia before entering the meiotic process [9], suppression of any one of the differentiation steps by GDNF could result in accumulation of less advanced spermatogonia. Consequently, the number of SSCs may gradually increase, because of their autonomous self-renewal activity [17, 18], as long as cell death does not exceed proliferation. In the present study, the number of SSCs remaining for 7 days in culture with GDNF was higher than that of the control but lower than the SSC number on Day 0. Therefore, a likely scenario of SSC maintenance in culture with GDNF is that SSCs self-renewed, encouraged by suppression of the differentiation pathway by GDNF, but a significant number of stem cells were depleted, possibly by cell death and differentiation that exceeded the proliferation of SSCs.

The addition of activin significantly reduced the number of SSCs to 30% of those in the control culture (Fig. 3), indicating that only 3.6% (12% x 0.3) of stem cells originally placed in vitro remained for 7 days. Activin binds directly to rat spermatogonia and stimulates mitotic activity, resulting in an increased number of spermatogonia in culture [29]. These results collectively suggest that stimulation of spermatogonial proliferation by activin may be exerted on more advanced spermatogonia rather than on stem cells. The proliferation of committed spermatogonia may reduce in vitro SSC maintenance by recruiting stem cells into the differentiation process to sustain the proliferation of nonstem spermatogonia or by a negative feedback mechanism from the expanding population of advanced spermatogonia to SSCs. Such a negative feedback regulation of stem cells has been proposed for various vertebrate and invertebrate stem cell systems [4, 61].

Donor cell coculture with TM4 and SF7 Sertoli cell lines also resulted in only 4% (12% x 0.33) and 6% (12% x 0.5), respectively, of stem cells remaining after 7 days in culture (Fig. 1). The efficiency of in vitro SSC maintenance was significantly lower in coculture with these Sertoli cell lines than with any other feeder cell type used in this study. These results suggest that TM4 and SF7 cells exhibit the unique function of Sertoli cells to support spermatogonial differentiation, resulting in a significantly lower number of SSCs than that in coculture with any other non-Sertoli cell line. Results of previous studies suggest that the central role of Serotli cells is to support germ cells in differentiation processes [10]. The lack of fully functional Steel factor in Sertoli cells of mutant mice results in disruption of spermatogonial differentiation, even though functional SSCs remain in the testes [34–36]. In organ culture of seminiferous tubules obtained from cryptorchid testes, spermatogonia actively divide but their proliferation coincides with a significant increase in differentiation, and this effect is stimulated by FSH, a Sertoli cell-specific gonadotropin in the testis [62, 63]. In the present study, TM4 and SF7 Sertoli cell lines may have stimulated spermatogonial differentiation or preferentially supported differentiated spermatogonia, resulting in depletion of the stem cell population in vitro.

The results of the present study therefore lead to the hypothesis that in vitro maintenance of SSCs can be improved by blocking the unidirectional cascade of spermatogonial differentiation. Two in vivo findings support this hypothesis. Forced expression of GDNF in transgenic mouse testes inhibits spermatogonial differentiation and results in accumulation of undifferentiated spermatogonia [53]. Spermatogonial accumulation caused by suppression of differentiation in spermatogenesis has also been observed in transgenic mice that express an anti-apoptotic protein, bcl-2, ectopically in male germ cells [64].

Based on this hypothesis, the results obtained with addition of BMP4 or Flk-2L and from coculture with OP9 or L cells can be interpreted. BMP4 induces proliferation and differentiation of human hematopoietic progenitors [25] and inhibits cell division and promotes differentiation of neuronal restricted precursors [27]. Flk-2L stimulates proliferation of hematopoietic progenitors, and its receptor has been identified as a progenitor-specific marker [51, 65]. Thus, BMP4 and Flk-2L may have also induced SSC differentiation or supported proliferation of progenitors, resulting in stem cell loss in this study. OP9 cells induce the cell fate determination of embryonic stem cells into the hematopoietic cell lineage [46]. The positive effects of OP9 cells on SSC maintenance, together with functions of BMP4 and Flk-2L reported previously and observed in this study, suggest that HSCs and SSCs may require similar mechanisms for in vitro maintenance. The positive and negative effects of growth factors and feeder cells can now be examined using techniques for examining gene expression, such as DNA microarrays.

A notable finding in this study was that MEM-{alpha}-based medium was significantly better (1.7-fold) for in vitro SSC maintenance than DMEM-based medium. Alteration of basal medium components is likely to improve maintenance of stem cells, as demonstrated by the advantage of MEM-{alpha} relative to DMEM. Although more intensive studies are required to elucidate the effective components of MEM-{alpha} and to establish the best medium for SSC culture, the present results encourage the use of MEM-{alpha} medium for efficient SSC culture in future investigations.

Although we observed a significant increase in the efficiency of in vitro SSC maintenance, it was no more than 2.2-fold compared with the control culture, perhaps because donor cells placed in culture were heterogeneous and included testicular somatic cells. These somatic cells may have masked or been involved in the effects observed in this study. Therefore, elimination of testicular somatic cells may be critical for more intensive investigations into the in vitro behavior of SSCs and their proliferation/differentiation mechanisms. Strategies developed recently to enrich SSCs by multiparameter selection will facilitate such investigations [37, 38].

Establishment of an efficient SSC culture system will greatly contribute to our ability to explore the factors involved in the SSC fate decision. As demonstrated in the present study, development of the spermatogonial transplantation technique has now provided the opportunity to evaluate in vitro requirements for SSC maintenance, and this technique together with stem cell enrichment strategies will be important for future investigations. Based on the results obtained in this study, these investigations will allow us to better understand the biology of the stem cell and the stem cell niche, and successful long-term culture will enable efficient expansion, alteration, and selection of SSCs for male fertility restoration and germ line gene modification.


   ACKNOWLEDGMENTS
 
We acknowledge the following sources who generously provided us with the reagents used in this study: Drs. A. Bradley for STO SNL76/7cells, J. Millan for SF7 cells, F. Cuzin for 15P-1 cells, T. Nakano for OP9 cells, D. Williams for Sl4 cells and their variants, RIKEN Institute for PA6 and ST2 cells, and Amgen Co. for Steel factor. We thank Dr. K. Orwig for suggestions, C. Freeman and R. Naroznowski for animal maintenance, and J. Hayden for help with photography. Microscopic sections were produced at the Institute for Human Gene Therapy, Cellular Morphology Core (5-P30-DK-47747-07).


   FOOTNOTES
 
1 Financial support for the research was from the National Institutes of Health (NICHD 36504), The Commonwealth and General Assembly of Pennsylvania, and the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation. Back

2 Correspondence: R.L. Brinster, School of Veterinary Medicine, University of Pennsylvania, 3850 Baltimore Ave., Philadelphia, PA 19104. FAX: 215 898 0667; cpope{at}vet.upenn.edu Back

3 Current address: Department of Obstetrics and Gynecology, Royal Victoria Hospital, McGill University, Montreal, PQ H3A 1A1, Canada Back

Received: 9 December 2002.

First decision: 6 January 2003.

Accepted: 15 January 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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