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1 PrimeGen Biotech LLC and 2 New Stem Biosciences, 213 Technology Drive, Irvine, California 92618, USA
Correspondence should be addressed to F Izadyar; Email: fizadyar{at}primegenbiotech.com) www.primegenbiotech.com
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Introduction |
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In the present study, to investigate whether the SSCs or different subpopulations of germ line stem cells generate the mGC lines, we took advantage of a transgenic mouse model expressing green fluorescence protein (GFP) driven by a germ line-specific Pou5f1 (Oct4) promoter (Yeom et al. 1996). POU5F1 (previously known as octamer-binding transcription factor 3/4, OCT4) was originally identified as an ES cell and a germ line-specific marker (Okamota et al. 1990, Scholer et al. 1990). The expression of Pou5f1 is regulated by a proximal promoter, a germ-specific distal enhancer, and a retinoic acid-responsive element (Saiti & Lacham-Kaplan 2007). At gastrulation, Pou5f1 expression is down-regulated and thereafter is maintained only in primordial germ cells (Yeom et al. 1996). PGCs, of both males and females, continue to express Pou5f1 as they proliferate and migrate to the genital ridges. In the males, the expression in germ cells persists throughout fetal development and is maintained postnatally in proliferating gonocytes, prospermatogonia, and undifferentiated spermatogonia, including A single (As), A paired (Apr), and A aligned (Aal) spermatogonia (Pesce et al. 1998, Tadokora et al. 2002). As in the mouse only the As spermatogonia are considered to be spermatogonial stem cells (SSCs), enriched populations of undifferentiated spermatogonia including SSCs can be isolated by sorting the POU5F1-GFP cells from OG2 transgenic mouse model.
Germ line stem cells could further be subdivided based on the expression of c-Kit receptor molecule. c-Kit, a tyrosine kinase receptor, and its ligand stem cell factor (also known as kit ligand or steel factor) are key regulators of PGC growth and survival (De Miguel et al. 2002). c-Kit is expressed in PGCs from their initial segregation to their arrival at the genital ridge. In postnatal mouse testes, it has been shown that c-Kit can be used as a marker for the differentiation of undifferentiated and differentiating type A spermatogonia (Schrans-Stassen et al. 1999). Therefore, the expression levels of POU5F1 and c-Kit were used in this study to isolate distinct populations of germ line stem cells. We then analyzed the molecular and phenotypic characteristics of these cells extensively before and after culture and compared their ability to generate multipotent cell lines under a defined culture condition with a mixture of growth factors. In addition, the functionality of these subpopulations and their descendant mGC lines to repopulate recipient testes was evaluated using the SSC transplantation technique.
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Results |
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Marker and imprinting profiles
The majority of cells in the mGC colonies expressed POU5F1, NANOG, SSEA-1, and VASA (Fig. 4a–d). They also expressed pluripotent genes Sox2, Dppa5, Rex1, eRas, and Crypto along with germ line-specific markers, including Stella, Dazl, Vasa, and cRet (Fig. 4q). In addition, the expression of POU5F1, NANOG, and SOX2 was confirmed by Western blot analysis (Fig. 4p). Among the three mGSC lines tested in this study, only one line showed Nanog expression and this is the line that has been used for Western blot analysis. The mouse cell line at passage 20 showed high telomerase activity (similar to ES cells) and normal karyotype (40, XY) (Fig. 5).
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Testes regeneration
Four weeks after transplantation, testes of the control animals as well as those that received POU5F1-positive c-Kit-positive cells showed no spermatogenesis in the majority of the seminiferous tubules. On the contrary, 80% of those that received freshly isolated POU5F1-positive c-Kit-negative cells showed some degrees of spermatogenesis throughout the testes, indicating the presence of functional SSCs in the cell suspension. Similarly, our short-term transplantation experiment using the cell trace marker carboxyfluorescein diacetate succinimidyl ester (CSFE) showed that only the c-Kit-negative subpopulation of germ line stem cells colonized the recipient testes (Fig. 9). No spermatogenesis was found in the majority of seminiferous tubules of the recipient mice testes transplanted with the mGCs, indicating that these cells do not have SSC properties (Table 1).
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Discussion |
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We have derived multipotent cell lines from postnatal mouse testicular stem cells with some but not all pluripotent characteristics. These cell lines are distinctively different from the mGC lines obtained by the other laboratories (Kanatsu-Shinohara et al. 2004, Seandel et al. 2007), most notably, with regard to the extent of pluripotentiality and teratoma formation. Based on microarray analysis, our mGC lines express pluripotent genes, i.e., Nanog and Crypto respectively 1000 and 5000 times less than ES cells. Similarly, our cell lines express oncogenes, i.e., p53, Eras, Bak, Int2, and c-myc, several folds lower than the ES cells (Supplementary Fig. 3, which can be viewed online at www.reproduction-online.org/supplemental). Comparisons of the present findings with published reports suggest that mGC lines generated by other groups and our cell lines might differ in terms of developmental stages, imprinting profiles, and differentiation potential. Indeed, germ line-derived cells in the present report have properties of ES cells such as marker expression, broad differentiation potential, and limited chimera formation. However, these cells also seem to have retained germ cell-specific imprinting patterns and non-tumorigenic characteristics.
Several lines of evidence support the notion that our cell lines retain their germ cell properties more than they resemble the reported properties of ES cells. First, these cells doubled their cell numbers in about 72 h (determined by both GFP sorting and manual counting). This cell doubling time, similar to that of germ line stem cells, is three times longer than that of the ES cells. Secondly, they seem to have molecular characteristics different from those in ES cells or other mGC lines. Our results on global gene expression analysis show that our cell lines have 65% similarity to ES cells and 87% to germ line stem cells. Among the genes tested, our cell lines showed significantly higher expression level of germ line-specific genes (Vasa, Plzf, Gfra1, Dazl) and lower expression level of pluripotent genes (Pou5f1, Nanog, Dppa5, Sox2, Crypto). Thirdly, our cell lines are more dependent on GDNF for their self-renewal than LIF or FGF2. GDNF has been shown to be the key regulator of the self-renewal of male germ line stem cells (Kubota et al. 2004, Ryu et al. 2005, Oatley et al. 2006), while LIF and FGF2 play crucial role in the self-renewal of the ES cells (Cartwright et al. 2005, Levenstein et al. 2005). Fourthly, the expression level of SSEA-1 in our cell lines was lower that found either in mouse ES cells (Supplementary Fig. 4, which can be viewed online at www.reproduction-online.org/supplemental) or other mGC line as reported by Kanatsu-Shinohara et al. (2004). It has been shown that SSEA-1 may be involved in tumor invasion and metastasis in certain animal model systems (Kajiwara et al. 2005), suggesting that higher expression may reflect higher potential for tumorigenesis. Finally, our multipotent GCs exhibited an androgenic imprinting pattern that is different from mouse ES cells or other mGC lines reported by other laboratories (Kanatsu-Shinohara et al. 2004, Seandel et al. 2007).
Despite all of the similarities to their germ line ancestors, our cell lines did not regenerate testes following transplantation. One possibility is that mGC lines might have changed their phenotypic and/or molecular signature during culture condition. Indeed, a slight difference in gene expression profile between the mGCs and the non-cultured germ line stem cells was observed. As some of the altered genes were cell adhesion molecules, we speculate that at least some of these alterations might be due to the in vitro condition, i.e., attachment to MEF instead of basement membrane of testicular epithelium and the absence of the nursing somatic Sertoli cells. On the contrary, mouse SSCs cultivated for a long period of time are reported to repopulate recipient testes following SSC transplantation, indicating that in vitro condition should not affect SSC properties (Shinohara et al. 2003). On the other hand, some of the growth factors present in our culture medium are reported to reprogram germ line stem cells to embryonic stage. However, the gene profile analysis and imprinting results do not support the reprogramming. Therefore, the most likely explanation would be that our multipotent germ line stem cells might have been generated from a subpopulation of germ line stem cells other than SSCs. Indeed, our transplantation study comparing the POU5F1+/c-Kit+ cells (being the germ line progenitors) versus the POU5F1+/c-Kit– cells (destined to become SSCs) showed that germ line progenitor cells did not repopulate recipient testes. This is also supported by the fact that only the germ line progenitor cells can generate multipotent germ line stem cells.
In summary, the present study demonstrates that a distinct subpopulation of germ line stem cells maintained under defined culture conditions generate multipotent cell lines that has some pluripotent characteristics and do not form teratomas. The findings raise interesting questions about what constitutes pluripotency, as well as an intriguing view into the potentiality of the germ line. Irrespectively, the development of similar cell lines from human germ cells, such as those isolated from an adult testis, could provide a novel and highly valuable autologous cell source for clinical applications, particularly since germ line cells contain some of the best protected DNA in the adult body.
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Materials and Methods |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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30 pups/trial) or adult OG2 mice (2–5 months old, 1 male/trial). After mincing, the testes were digested in DPBS (Dulbecco's 10 mM phosphate-buffered (pH 7.2), 0.14 M saline) containing collagenase (1 mg/ml), DNase-1 (1 µg/ml), and EDTA (5 mM). Testicular cell culture was performed according to the previously published protocols (Izadyar et al. 2003, Shinohara et al. 2003, Kanatsu-Shinohara et al. 2004). In brief, germ cell enrichment was accomplished by differential adhesion. The cells were dispensed into gelatin-coated (0.1%) culture dishes. The following day the floating cells were collected and passed to a secondary culture plate (1x105 cells per 1.2 cm2) in a culture medium used by Kanatsu-Shinohara et al. (2004) with modifications (Supplementary Table, which can be viewed online at www.reproduction-online.org/supplemental). After 2–4 weeks in culture, GFP-positive colonies were mechanically transferred to culture dishes containing mitomycin C-treated murine embryonic fibroblast (MEF) feeder layers (see below). After passage for three to four times, via mechanical transfer, to new MEF cultures, the colonies were established and could be removed from the culture plate enzymatically (collagenase 1 mg/ml, 5–10 min) for further expansion and/or storage in liquid nitrogen. To enhance germ line stem cell derivation, spermatogenesis was arrested in adult OG2 mice (4–6 weeks old, n=4), i.e., testes were surgically secured to the abdominal wall to become cryptorchid as described previously (de Rooij et al. 1999). mGC lines were also generated from neonatal and non-cryptorchid adult OG2 mice by sorting the GFP-positive cells. To further study which subpopulation of germ line stem cells generate these cell lines, the GFP-positive cells were sorted based on their c-Kit expression and were cultured on MEF feeders as described above. Finally, the effect of the growth factor removal on self-renewal or differentiation of mGC lines was investigated.
Preparation of MEF feeders
MEF feeders were made by the standard procedures using 12.5 dpc CD-1 mouse embryos. The embryos were eviscerated before trypsinization and the dissociated cells plated onto 150 mm plates at a plating density of 1.5 embryos per plate. After initial plating, MEFs were split 1:5 and then frozen/thawed (passage 1). Thawed MEFs (P1) were passed only once for expansion purposes prior to mitomycin C treatment. MEF feeders were plated at a density of 50x103 to 60x103 per cm2. New MEF feeders were used for pluripotent germ cell culture every 7–10 days. All animal experiments were conducted in accordance with the National Research Council's Guidelines for the Care and Use of Laboratory Animals.
Evaluation of telomerase activity and karyotyping
For the determination of telomerase activity, cell extracts were isolated from germ cell lines (passage 10 and higher), freshly isolated POU5F1+/c-Kit+ sorted, and POU5F1+/c-Kit– sorted cells using CHAPS lysis buffer containing 150 U/ml RNase. Cell lysates were centrifuged for 20 min at 12 000 g, 4 °C, and the supernatants were stored at –80 °C. Protein concentration was measured by Bradford assay using BSA as the standard. Telomerase activity was measured by PCR-based assay using TRAPEZE detection kit (Chemicon, Temecula, CA, USA). Two microliters of the cell extract at 750 µg/µl were added to a total volume of 50 µl PCR mix containing the TRAP reaction buffer, dNTPs, substrate oligonucleotide, telomerase primer, internal standard primer, and Taq polymerase. Two microliters of mESC cell extract were added to the reaction mix as positive control, and CHAPS lysis buffer and heat-inactivated telomerase were used as negative control for each experimental sample. Each sample was incubated at 30 °C for 30 min for telomerase extension, followed by PCR amplification. Karyotyping was performed at Coriell Cell Repositories, Cytogenetics Laboratory. For karyotyping, proliferating cells were incubated in culture with 0.1 µg/ml KaryoMAX Colcemid (Invitrogen) for 3–4 h before they were resuspended in hypotonic solution (0.075 M KCl) and incubated at room temperature for 10 min. The cells were then resuspended in a cold fixative (3:1 methanol:acetic acid) and stored at 4 °C for at least 30 min. After washing with the fixative, the cells were applied to clean glass slides and air dried. Metaphase chromosomes were prepared and karyotypes created using an Applied Spectral Imaging Band View digital imaging system.
In vitro differentiation
For generating EBs, mGSC colonies were dissociated with collagenase and plated at a concentration of 1x106/well to 2x106/well on 6-well non-adhesive culture plates in the complete medium containing 15% FBS (Hyclone, Logan ,UT, USA). In some experiments, EBs were formed in hanging drops by aggregating 50x103 to 100x103 cells in 50 µl medium. For differentiation into cells representing the three germ layers, the EBs were cultured for 15 days, and every 3 days 20–30 EBs were collected for RT-PCR analysis and 20–30 EBs were used for histological examinations. For induced differentiation, the EBs were cultured in the complete medium for 4 days before they were cultured in the serum-free N1 medium for lineage selection, i.e., DMEM/F12 (Invitrogen) supplemented with ITS (insulin, 10 mg/l; transferrin, 5.5 mg/l; selenium, 0.67 mg/l) and fibronectin (50 µg/ml). After 5–7 days, N1-treated cell aggregates were transferred to gelatin-coated culture plates (Ying et al. 2003) in the N2 medium for expansion of neural progenitor cells, i.e., N1 medium with ITS, without fibronectin, and supplemented with bFGF (10 ng/ml). For differentiation into cardiomyocytes, the EBs were cultured for 2 weeks in the presence of different cardiogenic compounds including DMSO (0.06 M), 5'-aza-2'-deoxycytidine AZA (5 mM), and Cardiogenol C (25–50 µM; Calbiochem, San Diego, CA, USA) (Paquin et al. 2002, Choi et al. 2004). During the differentiation process, the morphology of cells was analyzed and the samples were taken both for gene expression analysis by RT-PCR and immunohistochemical (IHC) staining (see below). Chondrocyte differentiation (Lee et al. 2004) of mGSCs was induced by adding a chondrogenic induction medium (Chondrogenic SingleQuots, Cambrex, Walkersville, MD, USA) supplemented with transforming growth factor-3β (10 ng/ml) and 20% FBS.
Immunocytochemical and IHC staining
Cultured cells were fixed in 4% paraformaldehyde for 10–30 min at room temperature and stored in PBS at 4 °C. For fluorescent immunocytochemistry, the cells were permeabilized with 1xCytoperm (BD Biosciences, San Jose, CA, USA) or 0.2% Triton X-100 (Sigma) for 15 min and subsequently incubated in 2% (w/v) BSA (Sigma) and 2% (v/v) normal goat serum (GS)/1x Cytoperm–PBS for 30–60 min both at room temperature. Primary antibody was either diluted at the optimal concentration in 2% BSA and 2% GS/1x Cytoperm–PBS and incubated for 3 h at 4 °C or diluted in blocking buffer overnight at 4 °C. After two washes, fluorescent secondary antibody was diluted accordingly (Supplementary Fig. 2) in 2% BSA and 2% goat serum/1x Cytoperm–PBS and incubated for 1 h at 4 °C in the dark. The cells were washed twice with PBS, wrapped in foil, and stored at 4 °C until microscopic analysis. Images were recorded using an Olympus IX71 microscope or Zeiss LSM510 confocal microscope equipped with digital image hardware and software.
For bright-field immunocytochemistry, the cells were washed once with 1x PBS. Endogenous peroxidase activity was blocked with 3% (v/v) H2O2 for 15 min followed by permeabilization – blocking with 2% BSA and 2% GS/1x Cytoperm–PBS for 30 min. The primary antibody was diluted accordingly (Supplementary Fig. 2) in 2% BSA and 2% GS/1x Cytoperm–PBS and incubated for 3 h at 4 °C. The remainder of the staining was accomplished using ABC staining kits (Vector Labs, Burlingame, CA, USA), according to the manufacturer's instructions. Visualization was done with enhanced diaminobenzidine substrate (Sigma) tablet dissolved in purified water and incubated for 5–10 min. For negative controls, the primary antibody was omitted. The primary antibodies used in this study were obtained from various companies. Each antibody was validated and the concentrations optimized in our laboratory. The source and working dilutions of these antibodies are presented in Table 2.
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Gene expression, imprinting analysis, and GFP amplification
Total RNA was isolated using RNeasy mini kit (Qiagen) and the RNA was used for RT-PCR, quantitative PCR, or microarray analysis. For RT-PCR, cDNA was synthesized with the Sensiscript RT kit (Qiagen), and the PCR was performed with HotStarTaq DNA Polymerase (Qiagen). All PCRs began with an initial incubation at 95 °C for 15 min to activate the enzyme. This was followed by 35 cycles of 95 °C for 15 s, the appropriate annealing temperature for 1 min, and 72 °C for 1 min, which was then followed by 1 cycle of 72 °C for 10 min for final extension. The reactions were carried out using an iCycler Thermal Cycler (Bio-Rad). RT-PCR was carried out using specific primers including Pou5f1, Nanog, Rex1, Dppa5, Dazl, β-actin (Actb), Nkx2.5, nestin, Mab2, and Gfap (primer sequences are presented in Table 3). For internal controls, Gapdh was used as a housekeeping gene for cellular samples and β-actin or interleukin-2 (IL2) was used in mouse embryos.
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Microarray analysis
Total cellular RNA was isolated using RNeasy mini kit (Qiagen Inc.), according to the manufacturer's recommendations. To eliminate DNA contamination, the samples were treated with 2.0 U DNase I (Amplification grade, Invitrogen) at 37 °C for 15 min, and the enzyme is inactivated by the addition of EDTA (2 mM final) at 65 °C for 10 min. The samples were concentrated by ethanol precipitation and resuspended in RNase-free water. Two micrograms of total RNA of each sample were sent to the UCI DNA Microarray Facility, where the samples were prepared and hybridized to Affymetrix Mouse Genome 430 2.0 GeneChips (Affymetrix Inc., Santa Clara, CA, USA) and scanned, according to the manufacturer's protocols.
SSC transplantation
To test the functionality of the subpopulations of germ line stem cells and our mGCs for regeneration of spermatogenesis, we used the SSC transplantation technique. Sixteen 6–8 weeks immune-deficient nude male mice (Harlan, Indianapolis, IN, USA) have been treated with busulfan (40 mg/kg) and used as recipients. One month after busulfan treatment, 2x105 cells were transplanted into the seminiferous tubules via rete testis injection as described previously (Ogawa et al. 2000). Four mice were transplanted with freshly isolated POU5F1-positive c-Kit-positive sorted cells, four mice injected with freshly isolated POU5F1-positive c-Kit-negative sorted cells, and four mice transplanted with mGCs. As the mGCs are cultured on MEFs, they were sorted for GFP to avoid MEF contamination during transplantation. The remaining four mice served as sham control and not injected. One month after transplantation, the animals were killed and the testes harvested and used for histological evaluations. To evaluate the efficiency of transplantation, 100–150 tubule cross sections of each animal was examined and the number of tubules with different stages of spermatogenesis was counted. Statistical analysis was carried out using ANOVA and P<0.05 was considered significant. To better identify the transplanted cells in the recipient testes, a fluorescent cell trace marker, CSFE, (Invitrogen) was used. CSFE is colorless and non-fluorescent until the acetate groups are cleaved by intracellular esterases to yield highly fluorescent product. This fluorescent product is well retained and can be fixed with an aldehyde fixative; however, it diminishes following multiple cell divisions and can be used successfully only in short-term studies. Two mice were transplanted with POU5F1-positive c-Kit-negative cells and two other mice received POU5F1-positive c-Kit-positive cells. Ten days after transplantation, the mice were killed and the number of CSFE-positive colonies was determined. Considering 72 h for each cell doubling of germ line stem cells (Fig. 2), at this time after transplantation, SSCs could have gone through two to three cell doublings. Therefore, colonies of four to eight cells could have been formed.
Tests for teratoma and chimera formation
To test the ability of the mGCs to form teratomas or chimeras, OG2 mice (Jackson Laboratories, Bar Harbor, ME, USA) were bred with Rosa26 mice (Jackson Laboratories) and a new strain (OG2-R26) was generated. These mice have both GFP and LacZ constructs in their germ cells. Culture was performed as described and the new POU5F1-GFP/LacZ germ cell lines were produced for testing teratoma and chimera formation. Mouse POU5F1-GFP/LacZ mGSCs were examined for their ability to form teratomas in vivo by s.c., i.m., or injection into the seminiferous tubules of nude mice (Harlan). As positive controls for teratoma formation, the ES cells were injected into some mice. For s.c., i.m., or testicular injections, 1x106 cells were injected. Mice were killed 6 weeks later and the tissues harvested for morphological and histological analysis.
The ability of mouse POU5F1-GFP/LacZ GSCs to form chimeric cell populations was determined after injection into host blastocysts, or by their aggregation with morula stage embryos or eight-cell stage embryos (Bradley 1987). Blastocyst injections of 15–20 cells were administered using 3.5-day blastocysts collected from CD-1 mice following the procedure as described (Chatot et al. 1990). After injection, the blastocysts were transferred (7–8 blastocysts in each horn of the uterus) to 2.5-day pseudopregnant CD-1 females, previously mated with vasectomized males. Incorporation of LacZ cells was examined in different areas of the chimeric 12.5 dpc embryos by the β-galactosidase staining kit (Sigma). In addition, LacZ and GFP PCRs were performed in DNAs isolated from the brain, heart, liver, and gonadal ridges of the chimeric embryos formed from POU5F1-GFP/LacZ cells.
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Acknowledgements |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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Received 23 October 2007
First decision 5 March 2008
Revised manuscript received 2 January 2008
Accepted 25 March 2008
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References |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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J. Lee, M. Kanatsu-Shinohara, N. Ogonuki, H. Miki, K. Inoue, T. Morimoto, H. Morimoto, A. Ogura, and T. Shinohara Heritable Imprinting Defect Caused by Epigenetic Abnormalities in Mouse Spermatogonial Stem Cells Biol Reprod, March 1, 2009; 80(3): 518 - 527. [Abstract] [Full Text] [PDF]
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 M. Kanatsu-Shinohara, M. Takehashi, and T. Shinohara Brief History, Pitfalls, and Prospects of Mammalian Spermatogonial Stem Cell Research Cold Spring Harb Symp Quant Biol, November 6, 2008; (2008) sqb.2008.73.033v1. [Abstract] [PDF]
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A. Zovoilis, J. Nolte, N. Drusenheimer, U. Zechner, H. Hada, K. Guan, G. Hasenfuss, K. Nayernia, and W. Engel Multipotent adult germline stem cells and embryonic stem cells have similar microRNA profiles Mol. Hum. Reprod., September 1, 2008; 14(9): 521 - 529. [Abstract] [Full Text] [PDF]
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 J. Yu and J. A. Thomson Pluripotent stem cell lines Genes & Dev., August 1, 2008; 22(15): 1987 - 1997. [Abstract] [Full Text] [PDF]
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