Biol Reprod
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print November 23, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.046441
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
74/3/522    most recent
biolreprod.105.046441v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kanatsu-Shinohara, M.
Right arrow Articles by Shinohara, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kanatsu-Shinohara, M.
Right arrow Articles by Shinohara, T.
Agricola
Right arrow Articles by Kanatsu-Shinohara, M.
Right arrow Articles by Shinohara, T.
BIOLOGY OF REPRODUCTION 74, 522–529 (2006)
DOI: 10.1095/biolreprod.105.046441
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Anchorage-Independent Growth of Mouse Male Germline Stem Cells In Vitro1

Mito Kanatsu-Shinohara 2 34, Kimiko Inoue 6, Jiyoung Lee 4, Hiromi Miki 6, Narumi Ogonuki 6, Shinya Toyokuni 5, Atsuo Ogura 6, and Takashi Shinohara 4

Horizontal Medical Research Organization,3 Departments of Molecular Genetics,4 and Pathology and Biology of Diseases,5 Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan RIKEN,6 Bioresource Center, Ibaraki 305-0074, Japan

ABSTRACT

Spermatogenesis originates from a small number of spermatogonial stem cells that reside on the basement membrane and undergo self-renewal division to support spermatogenesis throughout the life of adult animals. Although the recent development of a technique to culture spermatogonial stem cells allowed reproduction of self-renewal division in vitro, much remains unknown about how spermatogonial stem cells are regulated. In this study, we found that spermatogonial stem cells could be cultured in an anchorage-independent manner, which is characteristic of stem cells from other types of self-renewing tissues. Although the cultured cells grew slowly (doubling time, ~4.7 days), they expressed markers of spermatogonia, and grew exponentially for at least 5 months to achieve 1.5 x 1010-fold expansion. The cultured cells underwent spermatogenesis following transplantation into the seminiferous tubules of infertile animals and fertile offspring were obtained by microinsemination of germ cells that had developed within the testes of recipients of the cultured cells. These results indicate that spermatogonial stem cells can undergo anchorage-independent, self-renewal division, and suggest that stem cells have the common property to survive and proliferate in the absence of exogenous substrata.

developmental biology, gametogenesis, sertoli cells, spermatogenesis, testis

INTRODUCTION

Spermatogenesis depends on a small population of spermatogonial stem cells [1, 2]. These cells are the only cells in the spermatogenic system that can self-renew, and spermatogonial stem cells support male reproduction throughout life. Spermatogonial stem cells reside on the basement membrane and are distributed nonrandomly within seminiferous tubules [3]. Stem cells are thought to grow in a special microenvironment within which factors that regulate proliferation and differentiation are provided [4]. However, despite the importance of spermatogonial stem cells, very little is known about the factors that regulate these cells. Although spermatogonial stem cells can be identified by a functional transplantation assay [5], the small number (20000 to 30000 in mice) of stem cells within a testis has made direct in vivo analysis of spermatogonial stem cells very difficult [2, 6].

To overcome this problem, we recently developed an in vitro system to culture spermatogonial stem cells [7]. In the presence of glial cell line-derived neurotrophic factor (GDNF), which promotes self-renewing division of spermatogonial stem cells [8], germ cells from neonatal testes proliferated to form uniquely shaped colonies on mitomycin-C-treated mouse embryonic fibroblasts (MEFs) [7]. The cultured cells expressed spermatogonia markers and exhibited logarithmic proliferation for several months. When the cells were microinjected into the seminiferous tubules of infertile animals, the transplanted cells colonized the basement membrane and underwent spermatogenesis, and the recipient males sired normal fertile offspring. The cell cultures could be established from testes at various stages of development [7, 9–11], and these cells are an efficient vehicle for the production of transgenic animals [12]. Because of these unique properties, we called these cells germline stem (GS) cells to distinguish them from embryonic stem (ES) and embryonic germ (EG) cells that can be incorporated into the germline following injection into blastocysts [13–16]. Recapitulation of spermatogonial stem cell division in vitro will likely provide valuable information about how the self-renewal division of spermatogonial stem cells is regulated. In addition, the culture technique allows us to obtain large numbers of stem cells for biochemical or molecular analysis. Thus, the establishment of GS cell culture has provided an opportunity to study the factors that control spermatogonial stem cell division.

Generally, stem cells are associated closely with basement membrane [4]. This association probably provides signals via integrins that promote the survival and proliferation of the stem cells [4, 17]. However, several recent reports revealed that stem cells from various types of self-renewing tissue could be cultured in suspension [18–20]. For example, neuronal stem cells grow as neurospheres that do not depend on a particular substrate [18]. Similarly, mammary stem cells or stem cells from skin can also grow in suspension using similar culture conditions [19, 20]. These cells differentiate into various cell lineages after exposure to extracellular matrix molecule (ECM) or serum. These results demonstrate that stem cells can survive and proliferate in anchorage-independent culture conditions. Spermatogonial stem cells preferentially attach to laminin, and they express {alpha}6 and ß1-integrins that in combination comprise the laminin receptor [21]. However, it is not known whether GS cells will remain in an undifferentiated state in suspension or whether suspension will induce differentiation or cell death.

In the present study, we investigated whether self-renewal division of spermatogonial stem cells could occur in the absence of substrate. The functional activity of GS cells cultured in suspension was assessed using a functional transplantation assay.

MATERIALS AND METHODS

Animals and Transplantation

The Institutional Animal Care and Use Committee of Kyoto University approved all animal experimentation protocols. Testis cells were collected from the testes of a transgenic mouse line C57BL6/Tg14(act-EGFP-OsbY01) that was bred on a DBA/2 background (designated Green) (originally provided by Dr. M. Okabe, Osaka University). GS cells were established from newborn animals (0–2 days old), using a two-step enzymatic digestion method described previously [22]. The transgenic mice ubiquitously express the gene for enhanced green fluorescent protein (EGFP) under the control of the ß-actin promoter [23].

The cultured cells were transplanted into the testes of WBB6F1-W/Wv mice (designated W; purchased from Japan SLC). W mice are congenitally infertile, and lack all stages of differentiating germ cells owing to mutations in the gene that encodes Kit receptor tyrosine kinase [24]. Cultured cells were transplanted into 6- to 10-week-old W mice. As these mice are not histocompatible with the donor cells, the recipient mice were treated with anti-CD4 antibody (clone GK1.5) to induce tolerance to the allogeneic donor cells [25]. For microinjections, approximately 3 µl (1.5 x 104 cells per testis) of the donor cell suspension were introduced into the seminiferous tubules of a W testis by efferent duct injection [22]; this filled 75–85% of the tubules in each recipient testis.

GS Cell Culture and Adhesion Assay

The standard 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, 6 mg/ml D-(+)-glucose, 30 µg/ml pyruvic acid, 1 µl/ml DL-lactic Acid (Sigma), 5 mg/ml BSA (ICN Biomedicals), 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 that were used were 20 ng/ml mouse epidermal growth factor (EGF; BD Biosciences), 10 ng/ml human basic fibroblast growth factor (FGF2; BD Biosciences), 103 U/ml murine leukemia inhibitory factor (LIF; Invitrogen), and 10 ng/ml recombinant rat GDNF (R&D Systems). In some cases (where indicated), cells were cultured in the presence of 300 ng/ml rat GDNF sR{alpha}1/Fc chimera or 10 ng/ml human neurturin (NRTN; both from R&D Systems). The cells were cultured in the presence of 1% or 15% fetal bovine serum (FBS; JRH Biosciences).

To initiate suspension cultures, GS cells that had been maintained on MEFs for 120 days were trypsinized at 37°C for 5 min before being transferred to a 15-ml centrifuge tube. After centrifugation (440 x g for 5 min), the pellet was resuspended in GS cell culture medium, and 3 x 105 cells were plated onto a 35-mm Petri dish (BD Biosciences, cat. no. #351008). To passage the cells, the plated cells were flushed gently by repeated pipetting using a blue tip pipette. The cells were then centrifuged at 440 x g for 5 min. After the removal of the supernatant, 1 ml of 0.25% trypsin/EDTA was added to the pellet, and the cells were incubated for 5 min at 37°C. To stop the trypsin activity, 3 ml of Iscove's modified Dulbecco medium supplemented with 5 mg/ml BSA was added. Dissociated cells were triturated and were then centrifuged at 440 x g for 5 min. Live cells were counted in a hemocytometer, and 3 x 105 cells were resuspended in a Petri dish that contained 1.5 ml of standard GS cell culture medium. Three days after each passage, the cells were fed with 0.5 ml fresh medium (a total of 2 ml), and thereafter were passaged every 4–5 days. Feeder-free GS cell culture was performed as described previously [26]. All cultures were maintained at 37°C in an atmosphere of 5% carbon dioxide in air. Cryopreservation was performed as described previously [27].

For the adhesion assay, GS cells were trypsinized before being plated in GS cell culture medium on MEFs or laminin-coated (20 µg/ml) dishes at a density of 2.5 x 105 cells per 9.5 cm2. After overnight culture, the dishes were washed twice with PBS to collect nonadherent (floating) cells. Adherent cells were collected after 5 min of trypsin digestion. To remove serum in the MEF culture, the plates were washed four times with PBS before the cells were plated. Data were analyzed using the Student t-test.

Flow Cytometry and Immunohistochemistry

Flow cytometric analyses were performed as described previously [28]. Briefly, 106 cells were suspended in 0.1 ml of PBS that contained 1% FBS (PBS/FBS). The suspended cells were then incubated with primary antibodies. The cells were washed twice with PBS/FBS before being stained by exposure to secondary antibodies The stained cells were analyzed using the FACS-Calibur system (BD Biosciences). We collected 10000 events for flow cytometric analysis. For immunohistochemical staining, whole spheres were gently centrifuged onto 3-aminopropyltriethoxy silane-coated slides (Matsunami Glass) at 600 x g for 6 min. After fixation in 3.7% formaldehyde for 10 min, the slides were washed twice, and stained by primary antibodies.

The primary antibodies used were: rat anti-EpCAM (G8.8); mouse anti-SSEA-1 (MC-480) (Developmental Studies Hybridoma Bank, University of Iowa); rat anti-human {alpha}6-integrin (CD49f) (GoH3); biotinylated hamster anti-rat ß1-integrin (CD29) (Ha2/5); biotinylated rat anti-CD9 antigen (KMC8); rabbit anti-human fibronectin (DAKO), rabbit anti-mouse laminin (Sigma); rabbit anti-human POU5F1; rabbit anti-human GFRA1 (Santa Cruz Biotechnology), or allophycocyanin (APC)-conjugated rat anti-mouse KIT (CD117) (2B8) (BD Biosciences). APC-conjugated goat anti-rat IgG (Cedarlane Laboratories); APC-conjugated streptavidin (BD Biosciences); Alexa Fluor 568 goat anti-rabbit IgG; Alexa Fluor 680-allophycocyanin goat anti-rabbit IgG; or Alexa Fluor 633-conjugated goat anti-mouse IgM (Molecular Probes) were used as secondary antibodies.

Analysis of Testes

To quantify the number of donor-derived cell colonies, recipient mice were killed 2 mo after the transplantation of donor cells. Because the donor cells express EGFP, donor-derived colonies were detected by excitation with UV illumination. This method allowed for specific identification of donor cells because the recipient testis does not exhibit endogenous fluorescence. A cluster of germ cells was defined as a colony when it occupied more than 50% of the basal surface of the tubule and was at least 0.1 mm long [29]. The number of colonies was quantified by counting the total number of colonies observed under a stereomicroscope. To prepare histological sections, recipient testes were fixed in 10% neutral-buffered formalin before being embedded in paraffin wax. Sections were stained with hematoxylin-eosin. Data were analyzed using the Student t-test.

Analysis of Marker Gene Expression

Total RNA was isolated from whole spheres using Trizol reagent (Invitrogen). For the reverse transcriptase-polymerase chain reaction (RT-PCR), first-strand cDNA was synthesized using Superscript II (RNase H– reverse transcriptase; Invitrogen). The PCR was carried out using appropriate primer sets, as described previously [11, 26, 30]. PCR amplifications of fibronectin1 and laminin {alpha}1 was carried out using specific primers (5'- TGTGACAACTGCCGTAGACC-3' and 5'- TGCTGAAGCTGAGAACATGG-3' for fibronectin1, and 5'- TGTGGACAGGTGCTATGTCG-3' and 5'-AGGTGGCTGTTATCCTTCCG-3' for laminin {alpha}1).

Microinsemination

The seminiferous tubules of recipient testes were dissected under UV illumination when the recipient mice were 7 mo old. EGFP-expressing seminiferous tubules were recovered, and the germ cells were collected mechanically from the tubules by fine forceps. Microinsemination was performed as described previously using elongated spermatids or spermatozoa [31]. Embryos that reached the two-cell stage after 24 h in culture were transferred to the oviducts of Day 1 pseudopregnant Imperial Cancer Research (ICR) female mice. Fetuses that were retrieved on Day 19.5 were raised by ICR foster mothers.

RESULTS

Anchorage-Independent Growth of GS Cells

We first examined whether GS cells adhered to MEFs and/or laminin, both of which can be used for GS cell culture [7, 26]. GS cells were established from a newborn Green mouse using a standard protocol [7]. After dissociation into single-cell suspensions by enzymatic digestion, germ cells from neonatal testis formed uniquely-shaped colonies, and proliferated in the presence of GDNF and 1% FBS. Within 1 mo, germ cells were cultured on mitomycin C-treated MEF. After 120 days in culture, GS cells on MEFs were plated on MEFs or laminin in GS cell culture medium (Fig. 1A, left, top). After overnight culture, although some cells had attached to the substrate, a substantial number of nonadherent cells were observed. The average recoveries of adherent cells from three experiments were 46.2 ± 8.9% and 59.7 ± 5.4% of cells plated on MEFs and laminin, respectively (mean ± SEM, n = 6 for each). By contrast, 48.3 ± 9.5% and 36.8 ± 5.6% of cells failed to adhere to MEFs and laminin, respectively (n = 6 for each) (Fig. 1B). The proportions of cells that adhered to MEFs and laminin were not significantly different (P > 0.3 by t-test). Similar results were obtained under serum-free culture conditions (data not shown).


Figure 1
View larger version (46K):
[in this window]
[in a new window]
  		FIG. 1. Growth of GS cells in suspension. A) GS cells. Left, top: GS cells on MEFs. Note the morula-like structure. Right, top: GS cells in suspension culture, immediately after plating. Left, bottom: GS cells in suspension culture, 4 days after passage. Right, bottom: Higher magnification. Note the chain-type colony (arrow). B) Adhesiveness of GS cells to MEFs and laminin. GS cells were plated overnight on MEFs or laminin (20 µg/ml). Nonadherent (floating) cells were recovered by repeated washing of the culture plates. The values on the vertical axis represent percentages of cells that could be recovered after treatment. Results are from three independent experiments (mean ± SEM, n = 6). C) Expansion of GS cells cultured with various cytokines. The same number of cells (3 x 105 cells) was cultured under the conditions indicated. Cells were passaged three times over 16 days. Abbreviations: E, EGF; F, FGF2; G, GDNF; L, LIF; N, NRTN; GFR, GFR{alpha}1. D) Cumulative growth curves for three separate cultures of nonadherent GS cells. Cells were maintained for 134 or 158 days. The total number of cells was counted at at each passage. Note the steady and consistent exponential increase in total cell number over time. Bar = 100 µm (A)

To examine whether the nonadherent cells would continue to proliferate in vitro, cells were dissociated by trypsin digestion, and 3 x 105 cells were plated as single cells onto uncoated bacteriological Petri dishes that do not bind GS cells (Fig. 1A, right, top). Although GS cells did not grow in serum-free medium, the addition of 1% serum triggered cell growth. Some of the plated cells died, and we did not observe dividing cells 1 day after plating. Within 3 days, the plated cells grew and formed colonies with various morphologies (Fig. 1A, bottom). Although some colonies grew as chains of interconnecting cells, most colonies exhibited a morula-like appearance that resembled GS cells plated on MEFs. These colonies merged to form aggregate when maintained in a small volume of medium. We did not observe sphere-shaped colonies, which is the predominant morphology of cell aggregates in suspension cultures of other types of stem cell [18–20]. As with other types of GS cell cultures, growth of nonadherent cells depended on the presence of GDNF (Fig. 1C). However, cells did not proliferate in the absence of EGF and FGF2, even when a higher concentration of serum (15%) was added to the medium. The action of GDNF was mimicked by NRTN, albeit at a lower degree, which suggested that other members of the GDNF family of ligands can maintain the proliferation of GS cells. The addition of soluble GDNF receptor alpha-1 (GFRA1) did not improve the growth of nonadherent GS cells [9]. Floating colonies could not be derived directly from newborn testes, which suggested that in vitro maturation of gonocytes into spermatogonia is necessary for stating suspension culture.

Although these colonies could be partially dissociated and passaged by trituration into small clumps, it was not possible to obtain a suspension of single cells using trituration alone. We therefore used trypsin digestion to obtain a suspension of single cells to measure the increase in cell number during culture. Cultures were passaged serially every 4–5 days and were replated in fresh medium. The proliferation of viable cells was concentration-dependent, but the cells proliferated efficiently at a constant rate when 2 x 104 to 3 x 105 cells were plated per dish (20–300 cells/mm2). The cells stopped proliferating when 1 x 106 cells were plated per dish. Three independent cultures grew in a logarithmic manner through repeated passages for at least 158 days with a doubling time of ~112 h (Fig. 1D). The GS cells plated on MEFs grew faster and had a doubling time of ~65 h when growing logarithmically after being transferred to MEFs. Nonadherent cells could be frozen successfully at any stage of suspension culture. After thawing, the mean viability index of the freeze-thawed testis cells, as assessed by trypan blue exclusion, was 60.6 ± 3.6% (mean ± SEM; n = 3). The cells could expand 8.8-fold in 24 days after thawing. We did not observe any ES-like cell development during the entire culture period by morphological criteria [11].

To examine the phenotypes of the cultured cells, we performed flow cytometric analysis of surface antigen expression (Fig. 2A). Cultured cells expressed {alpha}6- and ß1-integrins, EpCAM, CD9, and GFRA1 (spermatogonial stem cell markers) [9, 21, 32, 33], but did not express KIT (differentiated spermatogonia marker) or SSEA-1 (ES or primordial germ cell markers) [34, 35], These results indicated that the cultures were relatively enriched with undifferentiated spermatogonia. We also examined other ES and germ cell markers using RT-PCR (Fig. 2B). Floating GS cells expressed the ES cell markers Pou5f1 and Zfp42, but did not express ES cell markers such as Nanog and Tdgf1 [36–40]. In agreement with the flow cytometric analysis, PCR showed that the cells expressed all of the expected germ cell and spermatogonia markers, including Neurog3, Ret, Stra8 (spermatogonia markers), Ddx4, and Dppa3 (germ cell markers) [8, 41–44]. Floating GS cells also expressed Zbtb16 and Taf4b, both of which are essential for spermatogonia self-renewal division [30, 45, 46]. These markers were similarly expressed in other types of GS cell cultures. The cells in the sphere appeared to be relatively uniform by immunohistochemistry (Fig. 2C). On the basis of a recent report that neurospheres expressed ECM [47], we examined whether GS cells expressed ECM. RT-PCR and immunohistological analyses revealed that laminin or fibronectin was expressed under all culture conditions: MEF-based, feeder-free, and nonadherent cultures. These results indicate that nonadherent cells have the spermatogonia phenotype and suggest that these cells also express ECM molecules, which has been reported for neural stem cells [47].


Figure 2
View larger version (45K):
[in this window]
[in a new window]
  		FIG. 2. Phenotypic analysis of nonadherent GS cells. A) Characterization of nonadherent GS cells using flow cytometry. The horizontal axis represents fluorescence (log scale) measured in arbitrary units. Floating GS cells were exposed to trypsin and were incubated with antibodies against {alpha}6-integrin, ß1-integrin, EpCAM, SSEA-1, CD9, KIT, or GFRA1. Unfilled peaks represent staining by control immunoglobulin, whereas filled peaks represent staining by specific antibody against each antigen. B) RT-PCR analysis of nonadherent GS cells. Specific primers were used to amplify cDNA from different types of GS cell cultures. C) Expression of spermatogonia markers and ECM molecules on spheres. EGFP-expressing spheres were stained with specific antibodies. Secondary antibodies with APC or Alexa Fluor 568 were used to detect primary antibodies. Bar = 500 µm (C)

Spermatogonial Stem Cell Activity of the Cultured Cells

To determine whether the GS cells exhibited spermatogonial stem cell activity, we performed germ cell transplantation. In this technique, transplanted spermatogonial stem cells colonize the vacant germline niche in the seminiferous tubules of infertile animals [5]. EGFP-expressing, nonadherent GS cells were collected at four different times after initiation of the suspension culture. The collected cells were trypsinized, and a suspension of single cells was microinjected into the seminiferous tubules of immunosuppressed congenitally infertile W recipient mice. These mice lack endogenous spermatogenesis [24]; therefore, any spermatogenesis in the recipient mice is derived from transplanted donor cells. The recipient mice were killed 2 mo after transplantation, and donor cell colonization was analyzed under UV illumination to detect EGFP expression in donor cells. Donor cell colonization was observed in all recipient testes (Fig. 3A). Assuming that stem cell colonization after transplantation occurred with an efficiency of 10% [29], the concentration of stem cells ranged from 0.2 to 2.0% of the original number of cultured cells (Table 1). Although the total number of cells increased 4 x 106-fold, the number of stem cells increased ~3.2 x 107-fold through 101 days. Histological analysis revealed that normal spermatogenesis occurred within the recipient testis (Fig. 3B). These results indicate that nonadherent GS cells exhibit spermatogonial stem cell activity.


Figure 3
View larger version (99K):
[in this window]
[in a new window]
  		FIG. 3. Production of fertile offspring from suspension-cultured GS cells. A) Macroscopic appearance of a recipient testis after the transplantation of floating GS cells. The green stretches in the seminiferous tubules indicates the presence colonies of spermatogenic donor cells, which express EGFP. B) Histological appearance of a recipient testis. Note the normal appearance of spermatogenesis. Elongated spermatids are observed. C) Spermatogenic cells released from a recipient testis. A spermatozoon (arrow) or a round spermatid (arrowhead) are observed within the cell suspension. D) Expression of the EGFP transgene in F2 offspring (arrow) produced by mating EGFP-expressing F1 males with wild-type females. EGFP fluorescence is not observed in nontransgenic progeny (arrowhead). Bar = 1 mm (A), 50 µm (B). Hematoxylin and eosin staining (B)


View this table:
[in this window]
[in a new window]
  		TABLE 1. Spermatogonial stem cell expansion in suspension conditions.a

Finally, to test whether the germ cells that developed from nonadherent GS cells were functionally normal, we performed in vitro microinsemination, a technique used commonly to derive offspring from animals and humans [31, 48]. At 167 days after transplantation, we killed one of the recipient mice that had received 122-day-old cultured GS cells. The recipient testis was examined under UV illumination, and fragments of the seminiferous tubules that exhibited EGFP fluorescence were dissected before being dissociated mechanically with fine forceps. Spermatozoa and elongated spermatids could be identified in the cell suspension that contained various stages of germ cells (Fig. 3C). These cells were microinjected into oocytes from C57BL/6 x DBA/2 F1 mice. Spermatozoa and elongated spermatids from four different segments were used, and 152 embryos were constructed. Of these, 122 embryos developed to the two-cell stage. These embryos were transferred into the oviducts of pseudopregnant mice after 24 h of culture. A total of 51 offspring were born by cesarean section; 48 of these developed into adults (21 males and 27 females; Table 2). Of these animals, 33 (69%) exhibited EGFP fluorescence that was consistent with the incorporation of the donor cells into the germline. These offspring grew up to be fertile (Fig. 3D).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Development of oocytes fertilized with germ cells derived from floating GS cells

DISCUSSION

Most macromolecular metabolic processes in normal cells require that the cells be adhered to an appropriate substrate [49]. The suspension of fibroblasts in methylcellulose inhibits the synthesis of RNA and proteins. Stem or progenitor cells from various tissues reside on basement membranes [4], and the behavior of these cells is regulated by modulatory signals from the ECM. In particular, signaling via ß1-integrin acts as a negative regulator of stem cell differentiation in various tissues, and cells that express high levels of ß1-integrin have a higher probability of undergoing self-renewal division [17, 47, 50]. Therefore, the close association of stem cells with the basement membrane is considered to be important for the biology of these cells. Nevertheless, stem cells exhibit differential affinities for, and responses to, different ECM molecules. Indeed, the ability to create suspension cultures of stem cells derived from several self-renewing tissues, such as brain, mammary glands, and skin, suggests that close association with the basement membrane may not be a prerequisite for self-renewal division of stem cells.

In testis, spermatogonial stem cells express ß1- and {alpha}6-integrins and are located at the basal side of seminiferous tubules [1, 2, 21]. Based on the assumption that a cellular component is required for stem cells to proliferate, most previous attempts to culture spermatogonial stem cells included various types of substrates [51–60], such as Sertoli cells or bone marrow stroma cells, and it is possible to culture GS cells on embryonic fibroblasts [7]. However, by taking advantage of the fact that spermatogonial stem cells have an affinity to laminin [21], more recent studies revealed that cellular support is dispensable in GS cell cultures, because feeder cells could be replaced with laminin to support the in vitro expansion of GS cells [26]. One question that was not addressed in the aforementioned studies was whether spermatogonial stem cells can proliferate in suspension, which has been demonstrated for stem cells from other types of self-renewing tissue.

The success of suspension culture in the present study demonstrates that anchorage to the basement membrane is not required for the survival or self-renewal of spermatogonial stem cells. The transplantation experiments showed that GS cells in suspension were functionally normal. These cells formed colonies of spermatogenic cells and could be used to create fertile offspring, thereby satisfying the criteria for classification as spermatogonial stem cells [5]. The proportion of the stem cells that failed to adhere varied among different experiments but was within the range (0.1 to 4.9% of the total cell population) reported for similar studies of adhesion to MEFs and laminin [7, 9–11, 26]. Therefore, it would appear that the type of substrate does not have a significant effect on the germline potential of cultured cells. Interestingly, in the present study, GS cells could be grown on MEFs even after extensive passages under nonadherent culture conditions. These facts indicate that nonadherent GS cells and GS cells on MEFs are convertible to each other and that GS cells retain germline potential regardless of matrix support.

Although functional spermatogonial stem cells were obtained under three different culture conditions (suspended cells and cells grown on MEFs or laminin), several differences in the cells among the different conditions were noted. First, GS cells grew more slowly in suspension than on MEFs (doubling time, 4.7 vs. 2.7 days) [7]. This difference cannot be attributed only to a decrease in cell-matrix interaction, because cell growth on laminin was even slower (doubling time 5.6 days) [26]. One explanation is that MEFs secrete unknown growth factors that are beneficial to GS cells. Second, although the expression of most marker genes was consistent among the three culture conditions, GS cells on laminin aberrantly expressed the embryonic marker, SSEA-1 [26]. SSEA-1 is expressed by ES cells and early germ cells in the fetus, and is not normally expressed in the postnatal testis. GS cells on MEFs and suspension cultures of GS cells did not express SSEA-1. Interestingly, the suspension culture was more similar to the MEF culture than to the laminin culture. In addition, although GS cells cultured on MEFs or laminin express variable levels of Kit [7, 26], Kit expression was suppressed markedly in GS cells from suspension cultures in the present study. This suggests that association with the basement membrane may influence the differentiation of spermatogonia.

The success of suspension culture in the present study was critically dependent on GDNF. GDNF is normally secreted from Sertoli cells in vivo, and a decrease in GDNF expression results in defective spermatogenesis and infertility in mice [8]. Recent studies also showed that GDNF influences spematogonial stem cell activity in species other than rodents [61]. The results of our study confirmed previous reports that GDNF is a crucial regulator of stem cell self-renewal [7–12, 26]. In this study, we also demonstrated that NRTN, another member of the GDNF family of ligands, could also stimulate the growth of GS cells. Although Sertoli cells express NRTN, NRTN is believed to regulate the proliferation and differentiation of spermatogonia, spermatocytes and spermatids [62, 63]. The positive effects of NRTN on GS cells likely reflect cross-talk between NRTN and the GFRA1 receptor [60]. On the other hand, the negative effect of high serum concentration indicates that serum contains unknown factors that may stimulate apoptosis or differentiation of stem cells. However, the requirement of low concentration of serum in suspension culture suggests that it also provides some trace ingredients (e.g., hormones) that are necessary for self-renewal division of spermatogonial stem cells. Although these results concur with those of previous studies, the negative effect of soluble GFRA1 was unexpected. This is in contrast to a previous report that soluble GFRA1 enhanced the growth of GS cells [9]. A possible explanation is that soluble GFRA1 may have interfered with the binding of GDNF to GS cells. Alternatively, this difference could be attributable to the different genetic backgrounds of the mice that were used in the studies (C57 vs. DBA/2) [9]. Further studies are required to confirm the effect of soluble GFRA1 on the self-renewal of spermatogonial stem cells.

Although we succeeded in culturing GS cells in suspension, there are at least three differences between our cultures and other suspension stem cell cultures of stem cells. First, GS cells grew as chains or morula-like clumps in suspension, whereas stem cells from other tissues form spheres in suspension [18–20]. Second, serum generally induces the differentiation of spheres in cultures of other stem cells, but GS cells required serum for propagation [18–20]. Third, GS cells could be passaged by trypsin, whereas trypsin has detrimental effects on sphere-forming stem cells, which are usually passaged using mechanical dissociation [18–20]. These differences may result in part from the fact that stem cells undergo limited proliferation in spheres [19, 64]. Spheres are not composed of a homogeneous population of cells. Indeed, differentiation markers and growth factor receptors are distributed heterogenously within neurospheres, which suggests that stem cells are located nonrandomly in spheres [47]. Generally, the expansion of adult stem cell populations does not occur readily ex vivo, presumably owing to asymmetric cell division kinetics that result in the production of a large number of progenitors and differentiated cells and a small, fixed number of stem cells. By contrast, GDNF induced the symmetric division of spermatogonial stem cells and caused an increase in the total number of these cells [7, 9–12, 26]. The morphology of colonies probably reflects the division patterns of stem cells [65], and this difference in stem cell division kinetics may reflect the unusual colony structure that we observed in the current study.

At present, why spermatogonial stem cells are capable of anchorage-independent growth is not known. Anchorage-independent growth is not a general characteristic of GS cells, because germline cells are associated closely with ECM throughout development and ES, EG, and multipotent GS cells form embryoid bodies and differentiate in suspension [11, 13–16, 66]. However, it is worth noting that very similar clusters of spermatogonia were found in the lumen of seminiferous tubules when GDNF was overexpressed in vivo. This occurred in the testes of GDNF transgenic mice and after in vivo elecctroporation of Sertoli cells with a GDNF transgene [8, 67]. Instead of proliferating as chains or networks on the basement membrane [29], many spermatogonia lost contact with the basement membrane and accumulated within the lumen of seminiferous tubules. In agreement with our findings, these clusters do not express KIT [8], which indicates that the clusters comprise undifferentiated spermatogonia. Furthermore, these cells grew more slowly than did wild-type cells; the peak proliferation index was higher in wild-type mice than in transgenic mice [8]. These results suggest that high concentrations of GDNF can influence cell-substrate adhesion and can create an environment in which stem cells can slowly undergo anchorage-independent growth. Further studies are required to clarify the physiological significance of this phenomenon.

The identification of GDNF as a spermatogonial self-renewal factor was a key to establishing the technique used to culture GS cells [7]. The successful elucidation of the conditions required for the suspension culture of GS cells has provided a simplified method that should facilitate large-scale spermatogonial stem cell culture. However, the GS cell culture technique is not yet perfect, and many improvements are needed to increase the usefulness of this technology. We believe that future refinements in culture technology will lead to the identification of additional factors that are essential for the self-renewal process of spermatogonial stem cells, which will provide greater insight into this unique biological process.

ACKNOWLEDGMENTS

We thank Ms. A. Wada for her technical assistance and Dr. Y. Kaziro for encouragement and critical reading of the manuscript.

FOOTNOTES

2 Correspondence: Mito Kanatsu-Shinohara, Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, 53 Shogoin, Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. FAX: 81 75 751 4169; mshinoha{at}virus.kyoto-u.ac.jp Back

1 Supported by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, and by grants from CREST and the Human Science Foundation (Japanese), and supported in part by Special Coordination Funds for Promoting Science and Technology from MEXT. Back

Received: 11 August 2005.

First decision: 25 September 2005.

Accepted: 22 November 2005.

REFERENCES

  1. de Rooij DG, Russell LD, All you wanted to know about spermatogonia but were afraid to ask. J Androl 2000 21:776-798[Medline]
  2. Meistrich ML, van Beek MEAB, Spermatogonial stem cells. In: Desjardins C, Ewing LL, (eds.), Cell and Molecular Biology of the Testis. New York: Oxford University Press 1993 266-295
  3. Chiarini-Garcia H, Raymer AM, Russell LD, Non-random distribution of spermatogonia in rats: evidence of niches in the seminiferous tubules. Reproduction 2003 126:669-680[Abstract]
  4. Spradling A, Drummond-Barbosa D, Kai T, Stem cells find their niche. Nature 2001 414:98-104[CrossRef][Medline]
  5. Brinster RL, Zimmermann JW, Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci U S A 1994 91:11298-11302[Abstract/Free Full Text]
  6. Tegelenbosch RAJ, de Rooij DG, A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse. Mutat Res 1993 290:193-200[CrossRef][Medline]
  7. Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, Shinohara T, Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod 2003 69:612-616[Abstract/Free Full Text]
  8. Meng X, Lindahl M, Hyvönen ME, Parvinen M, de Rooij DG, Hess MW, Raatikainen-Ahokas A, Sainio K, Rauvala H, Lakso M, Pichel JG, Westphal H, et al Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 2000 287:1489-1493[Abstract/Free Full Text]
  9. Kubota H, Avarbock MR, Brinster RL, Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc Natl Acad Sci U S A 2004 101:16489-16494[Abstract/Free Full Text]
  10. Ogawa T, Ohmura M, Tamura Y, Kita K, Ohbo K, Suda T, Kubota Y, Derivation and morphological characterization of mouse spermatogonial stem cell lines. Arch Histol Cytol 2004 67:297-306[CrossRef][Medline]
  11. Kanatsu-Shinohara M, Inoue K, Lee J, Yoshimoto M, Ogonuki N, Miki H, Baba S, Kato T, Kazuki Y, Toyokuni S, Toyoshima M, Niwa O, et al Generation of pluripotent stem cells from neonatal mouse testis. Cell 2004 119:1001-1012[CrossRef][Medline]
  12. Kanatsu-Shinohara M, Toyokuni S, Shinohara T, Genetic selection of mouse male germline stem cells in vitro: offspring from single stem cells. Biol Reprod 2005 72:236-240[Abstract/Free Full Text]
  13. Evans MJ, Kaufmann MH, Establishment in culture of pluripotential cells from mouse embryos. Nature 1981 292:154-156[CrossRef][Medline]
  14. Martin GR, Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 1981 78:7634-7638[Abstract/Free Full Text]
  15. Matsui Y, Zsebo K, Hogan BLM, Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 1992 70:841-847[CrossRef][Medline]
  16. Resnick JL, Bixler LS, Cheng L, Donovan PJ, Long-term proliferation of mouse primordial germ cells in culture. Nature 1992 359:550-551[CrossRef][Medline]
  17. Jones PH, Watt FM, Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression. Cell 1993 73:713-724[CrossRef][Medline]
  18. Reynolds BA, Weiss S, Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 1992 255:1707-1710[Abstract/Free Full Text]
  19. Toma JG, Akhavan M, Fernandes KJL, Barnabé-Heider F, Sadikot A, Kaplan DR, Miller FD, Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol 2001 3:778-784[CrossRef][Medline]
  20. Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ, Wicha MS, In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 2003 17:1253-1270[Abstract/Free Full Text]
  21. Shinohara T, Avarbock MR, Brinster RL, ß1- and {alpha}6-integrin are surface markers on mouse spermatogonial stem cells. Proc Natl Acad Sci U S A 1999 96:5504-5509[Abstract/Free Full Text]
  22. Ogawa T, Aréchaga JM, Avarbock MR, Brinster RL, Transplantation of testis germinal cells into mouse seminiferous tubules. Int J Dev Biol 1997 41:111-122[Medline]
  23. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y, ‘Green mice' as a source of ubiquitous green cells. FEBS Lett 1997 407:313-319[CrossRef][Medline]
  24. Ohta H, Tohda A, Nishimune Y, Proliferation and differentiation of spermatogonial stem cells in the W/Wv mutant mouse testis. Biol Reprod 2003 69:1815-1821[Abstract/Free Full Text]
  25. Kanatsu-Shinohara M, Ogonuki N, Inoue K, Ogura A, Toyokuni S, Honjo T, Shinohara T, Allogeneic offspring produced by male germ line stem cell transplantation into infertile mouse testis. Biol Reprod 2003 68:167-173[Abstract/Free Full Text]
  26. Kanatsu-Shinohara M, Miki H, Inoue K, Ogonuki N, Toyokuni S, Ogura A, Shinohara T, Long-term culture of mouse male germline stem cells under serum- or feeder-free conditions. Biol Reprod 2005 72:985-991[Abstract/Free Full Text]
  27. Kanatsu-Shinohara M, Ogonuki N, Inoue K, Ogura A, Toyokuni S, Shinohara T, Restoration of fertility in infertile mice by transplantation of cryopreserved male germline stem cells. Hum Reprod 2003 18:2660-2667[Abstract/Free Full Text]
  28. Shinohara T, Orwig KE, Avarbock MR, Brinster RL, Spermatogonial stem cell enrichment by multiparameter selection of mouse testis cells. Proc Natl Acad Sci U S A 2000 97:8346-8351[Abstract/Free Full Text]
  29. Nagano M, Avarbock M R, Brinster RL, Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient testes. Biol Reprod 1999 60:1429-1436[Abstract/Free Full Text]
  30. Falender AE, Freiman RN, Geles KG, Lo KC, Hwang K, Lamb DJ, Morris PL, Tjian R, Richards JS, Maintenance of spermatogenesis requires TAF4b, a gonad-specific subunit of TFIID. Genes Dev 2005 19:794-803[Abstract/Free Full Text]
  31. Kimura Y, Yanagimachi R, Intracytoplasmic sperm injection in the mouse. Biol Reprod 1995 52:709-720[Abstract]
  32. Ryu B-Y, Orwig KE, Kubota H, Avarbock MR, Brinster RL, Phenotypic and functional characteristics of male germline stem cells in rats. Dev Biol 2004 274:158-170[CrossRef][Medline]
  33. Kanatsu-Shinohara M, Toyokuni S, Shinohara T, CD9 is a surface marker on mouse and rat male germline stem cells. Biol Reprod 2004 70:70-75[Abstract/Free Full Text]
  34. Yoshinaga K, Nishikawa S, Ogawa M, Hayashi S-I, Kunisada T, Fujimoto T, Nishikawa S-I, Role of c-kit in mouse spermatogenesis: identification of spermatogonia as a specific site of c-kit expression and function. Development 1991 113:689-699[Abstract]
  35. Solter D, Knowles BB, Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1). Proc Natl Acad Sci U S A 1978 75:5565-5569[Abstract/Free Full Text]
  36. Pesce M, Schöler HR, Oct-4: gatekeeper in the beginning of mammalian development. Stem Cells 2001 19:271-278[Abstract/Free Full Text]
  37. Hosler BA, LaRosa GJ, Grippo JF, Gudas LJ, Expression of REX-1, a gene containing zinc finger motifs, is rapidly reduced by retinoic acid in F9 teratocarcinoma cells. Mol Cell Biol 1989 9:5623-5629[Abstract/Free Full Text]
  38. Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M, Yamanaka S, The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 2003 113:631-642[CrossRef][Medline]
  39. Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A, Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003 113:643-655[CrossRef][Medline]
  40. Ciccodicola A, Dono R, Obici S, Simeone A, Zollo M, Persico MG, Molecular characterization of a gene of the ‘EGF family' expressed in undifferentiated human NTERA2 teratocarcinoma cells. EMBO J 1989 8:1987-1991[Medline]
  41. Yoshida S, Takakura A, Ohbo K, Abe K, Wakabayashi J, Yamamoto M, Suda T, Nabeshima Y, Neurogenenin3 delineates the earliest stages of spermatogenesis in the mouse testis. Dev. Biol 2004 269:447-458
  42. Fujiwara Y, Komiya T, Kawabata H, Sato M, Fujimoto H, Furusawa M, Noce T, Isolation of a DEAD-family protein gene that encodes a murine homolog of Drosophila vasa and its specific expression in germ cell lineage. Proc Natl Acad Sci U S A 1994 91:12258-12262[Abstract/Free Full Text]
  43. Saitou M, Barton SC, Surani MA, A molecular program for the specification of germ cell fate in mice. Nature 2002 418:293-300[CrossRef][Medline]
  44. Giuili G, Tomljenovic A, Labrecque N, Oulad-Abdelghani M, Raaaoulzadegan M, Cuzin F, Murine spermatogonial stem cells: targeted transgene expression and purification in an active state. EMBO Rep 2002 3:753-759[CrossRef][Medline]
  45. Costoya JA, Hobbs RM, Barna M, Cattoretti G, Manova K, Sukhwani M, Orwig K E, Wolgemuth DJ, Pandolfi PP, Essential role of Plzf in maintenance of spermatogonial stem cells. Nat Genet 2004 36:653-659[CrossRef][Medline]
  46. Buaas FW, Kirsch AL, Sharma M, McLean DJ, Morris JL, Griswold MD, de Rooij DG, Braun RE, Plzf is required in adult male germ cells for stem cell self-renewal. Nat Genet 2004 36:647-652[CrossRef][Medline]
  47. Campos LS, Leone DP, Relvas JB, Brakebusch C, Fässler R, Suter U, ffrench-Constant C, ß1-integrins activate a MAPK signaling pathway in neural stem cells that contributes to their maintenance. Development 2004 131:3433-3444[Abstract/Free Full Text]
  48. Palermo G, Joris H, Devroey P, Van Steirteghem AC, Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992 340:17-18[CrossRef][Medline]
  49. Benecke BJ, Ben-Ze'ev A, Penman S, The control of mRNA production, translation and turnover in suspended and reattached anchorage-dependent fibroblasts. Cell 1978 14:931-939[CrossRef][Medline]
  50. Collins AT, Habib FK, Maitland NJ, Neal DE, Identification and isolation of human prostate epithelial stem cells based on alpha(2)beta(1)-integrin expression. J Cell Sci 2001 114:3865-3872[Abstract/Free Full Text]
  51. Moore TJ, de Boer-Brouwer M, van Dissel-Emiliani FM, Purified gonocytes from the neonatal rat form foci of proliferating germ cells in vitro. Endocrinology 2002 143:3171-3174[Abstract]
  52. Nagano M, Ryu B-Y, Brinster CJ, Avarbock MR, Brinster RL, Maintenance of mouse male germ line stem cells in vitro. Biol Reprod 2003 68:2207-2214[Abstract/Free Full Text]
  53. Kent Hamra F, Schultz N, Chapman KM, Grellhesl DM, Cronkhite JT, Hammer RE, Garbers DL, Defining the spermatogonial stem cell. Dev Biol 2004 269:393-410[CrossRef][Medline]
  54. Jeon D, McLean DJ, Griswold MD, Long-term culture and transplantation of murine testicular germ cells. J Androl 2003 24:661-669[Abstract/Free Full Text]
  55. Izadyar F, den Ouden K, Creemers LB, Posthuma G, Parvinen M, de Rooij DG, Proliferation and differentiation of bovine type A spermatogonia during long-term culture. Biol Reprod 2003 68:272-281[Abstract/Free Full Text]
  56. Feng L-X, Chen Y, Dettin L, Reijo Pera RA, Herr JC, Goldberg E, Dym M, Generation and in vitro differentiation of a spermatogonial cell line. Science 2002 297:392-395[Abstract/Free Full Text]
  57. van Pelt AMM, Roepers-Gajadien HL, Gademan IS, Creemers LB, de Rooij DG, van Dissel-Emiliani FMF, Establishment of cell lines with rat spermatogonial stem cell characteristics. Endocrinology 2002 143:1845-1850[Abstract/Free Full Text]
  58. Hasthorpe S, Barbic S, Farmer PJ, Hutson JM, Neonatal mouse gonocyte proliferation assayed by an in vitro clonogenic method. J Reprod Fertil 1999 116:335-344[Abstract]
  59. Hofmann MC, Braydich-Stolle L, Dym M, Isolation of male germ-line stem cells; influence of GDNF. Dev Biol 2005 279:114-124[CrossRef][Medline]
  60. Hofmann MC, Braydich-Stolle L, Dettin L, Johnson E, Dym M, Immortalization of mouse germ line stem cells. Stem Cells 2005 23:200-210[Abstract/Free Full Text]
  61. Oatley JM, Reeves JJ, McLean DJ, Biological activity of cryopreserved bovine spermatogonial stem cells during in vitro culture. Biol Reprod 2004 71:942-947[Abstract/Free Full Text]
  62. Meng X, Pata I, Pedrono E, Popsueva A, de Rooij DG, Janne M, Rauvala H, Sariola H, Transient disruption of spermatogenesis by deregulated expression of neurturin in testis. Mol Cell Endocrinol 2001 184:33-39[CrossRef][Medline]
  63. Viglietto G, Dolci S, Bruni P, Baldassarre G, Chiariotti L, Melillo RM, Salvatore G, Chippetta G, Sferratore F, Fusco A, Santoro M, Glial cell line-derived neurotrophic factor and neurturin can act as paracrine growth factors stimulating DNA synthesis of Ret-expressing spermatogonia. Int J Oncol 2000 16:689-694[Medline]
  64. Reynolds BA, Rietze RL, Neural stem cells and neurospheres—re-evaluating the relationship. Nat Methods 2005 2:333-336[CrossRef][Medline]
  65. Yamashita YM, Jones DL, Fuller MT, Orientation of asymmetric stem cell division by the APC tumor suppressor and centrosome. Science 2003 301:1547-1550[Abstract/Free Full Text]
  66. Felici MD, Pesce M, Giustiniani Q, Di Carlo A, In vitro adhesiveness of mouse primordial germ cells to cellular and extracellular matrix component substrata. Microsc Res Tech 1998 43:258-264[CrossRef][Medline]
  67. Yomogida K, Yagura Y, Tadokoro Y, Nishimune Y, Dramatic expansion of germinal stem cells by ectopically expressed human glial cell line-derived neurotrophic factor in mouse Sertoli cells. Biol Reprod 2003 69:1303-1307[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
M. Kanatsu-Shinohara, K. Inoue, N. Ogonuki, H. Miki, S. Yoshida, S. Toyokuni, J. Lee, A. Ogura, and T. Shinohara
Leukemia Inhibitory Factor Enhances Formation of Germ Cell Colonies in Neonatal Mouse Testis Culture
Biol Reprod, January 1, 2007; 76(1): 55 - 62.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
D. G. de Rooij
Rapid expansion of the spermatogonial stem cell tool box
PNAS, May 23, 2006; 103(21): 7939 - 7940.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
74/3/522    most recent
biolreprod.105.046441v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kanatsu-Shinohara, M.
Right arrow Articles by Shinohara, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kanatsu-Shinohara, M.
Right arrow Articles by Shinohara, T.
Agricola
Right arrow Articles by Kanatsu-Shinohara, M.
Right arrow Articles by Shinohara, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS