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Characterization of Immortalized Dairy Goat male Germline
Stem Cells (mGSCs)
Haijing Zhu, Jing Ma, Rui Du, Liming Zheng, Jiang Wu, Wencong Song, Zhiwei Niu,
Xin He, Enqi Du, Shanting Zhao, and Jinlian Hua*
College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Key Lab for Animal
Biotechnology of Agriculture Ministry of China, Northwest A&F University, Yangling, Shaanxi 712100, China
ABSTRACT
Male germline stem cells (mGSCs), in charge for the fertility in male testis, are the only kind of adult stem cells that transmit genetic information
to next generation, with promising prospects in germplasm resources preservation and optimization, and production of transgenic animals.
Mouse male germline stem cell lines have been established and are valuable for studying the mechanisms of spermatogenesis. However, there is
a lack of stable mGSC cell lines in livestock, which restricts the progress of transgenic research and related biotechnology. Here, we firstly
established an immortalized dairy goat mGSC cell line to study the biological properties and the signaling pathways associated with mGSCs
self-renewal and differentiation. The ectopic factors SV40 large T antigen and Bmi1 genes were transduced into dairy goat mGSCs, and the
results showed that the proliferation of these cells that were named mGSCs-I-SB was improved significantly. They maintained the typical
characteristics including the expression of mGSC markers, and the potential to differentiate into all three germ layers, sperm-like cells in vitro.
Additionally, mGSCs-I-SB survived and differentiated into three germ layer cell types when they were transplanted into chicken embryos.
Importantly, the cells also survived in mouse spermatogenesis deficiency model testis which seemed to be the golden standard to examine
mGSCs. Conclusively, our results demonstrate that mGSCs-I-SB present the characteristics of mGSCs and may promote the future study on
goat mGSCs. J. Cell. Biochem. 115: 1549–1560, 2014. ©2014 Wiley Periodicals, Inc.
KEY WORDS: MALE GERMLINE STEM CELLS; IMMORTALIZATION; DAIRY GOAT
Male germline stem cells (mGSCs), also named spermatogo-
nial stem cells (SSCs), localized in the basement of
seminiferous tubules, are able of self-renewal to maintain the
number of mGSCs and differentiation into sperm constitutively
which are mainly in charge of supplying the material basis for male
fertility. Male GSCs have been studied for many years, especially in
mouse and human. Importantly, the immortalized mouse mGSCs
have been obtained and the culture system has been developed
[Feng et al., 2002; Kubota et al., 2004a,b; Hofmann et al., 2005].
Pluripotent stem cells have been obtained from mGSCs in mouse and
human, and are a novel resource for pluripotent cells besides
embryonic stem cells (ESCs) and induced pluripotent stem cells
(iPSCs) [Guan et al., 2006; Conrad et al., 2008]. All these progresses
promoted the study of the mechanisms on self-renewal and
differentiation of mGSCs and their application in agriculture and
life science, such as production of transgenic animals [von
Schonfeldt et al., 2004; Kanatsu-Shinohara et al., 2005, 2006; Li
et al., 2005; Ballow et al., 2006; Naughton et al., 2006; Shinohara
and Kanatasu-Shinohara, 2007; He et al., 2008, 2009; Sikarwar
and Reddy, 2008; Izsvák et al., 2010; Kanatsu-Shinohara and
Shinohara, 2010; Shi et al., 2010; Niu et al., 2011; Zhang et al., 2011;
Suzuki et al., 2012].
As we described previously, dairy goat is important for Chinese
people, and studies on dairy goat mGSCs may improve the
preservation and optimization of the germplasm resources [Zhu
et al., 2013] and provide us a great improvement of quality and
quantity of milk and meat. Up to date, there is a lack of stable culture
system and mGSC cell line in livestock [Luo et al., 2006; Bi et al.,
Grant sponsor: National Natural Science Foundation of China; Grant number: 31272518; Grant sponsor: National
Major Fundamental Research Program of China; Grant number: 2013CB947900; Grant sponsor: Doctoral Fund of
Ministry of Education of China (RFDP); Grant number: 20120204110030; Grant sponsor: Fundamental Research
Funds for the Central Universities; Grant number: QN2011012.
*Correspondence to: Dr Jinlian Hua, College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering &
Technology, Key Lab for Animal Biotechnology of Agriculture Ministry of China, Northwest A&F University,
Yangling, Shaanxi, China. E-mail: jinlianhua@nwsuaf.edu.cn
Manuscript Received: 19 October 2013; Manuscript Accepted: 27 March 2014
Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 1 April 2014
DOI 10.1002/jcb.24812 ©2014 Wiley Periodicals, Inc. 1549
Journal of Cellular
Biochemistry
ARTICLE
Journal of Cellular Biochemistry 115:1549–1560 (2014)
2007; Aponte et al., 2008; Kaul et al., 2010, 2012; Hua et al., 2011;
Bahadorani et al., 2012; de Barros et al., 2012; Heidari et al., 2012; Li
et al., 2012, 2013; Zhu et al., 2013; McMillan et al., 2013]. The dairy
goat mGSCs cannot survive in vitro under specific culture conditions
for a long period as previously reported by our laboratory [Zhu et al.,
2012]. Thus, it is of great significance to obtain the immortalized
dairy goat mGSC cell line, which will be critical for the long-term
study of dairy goat mGSCs and other aspects of the cells biology,
including dedifferentiation mGSCs into pluripotent state. Feng et al.
[2002] and Hofmann et al. [2005] have shown that mGSCs could be
immortalized by exogenous factors, such as TERT and Simian virus
40 (SV40) T antigen. These cells shared typical SSC characteristics
including the morphology, expression of mGSCs markers such as
DAZL and GFRa1, the ability of differentiating into haploid sperm
cells and rebuilding seminiferous tubules after transplantation into
mouse spermatogenesis deficiency testis.
Immortalization methods in mammals include transduction of
primary cells with vectors carrying viral genes, such as Simian virus 40
(SV40) T antigen, Epstein–Barr virus (EBV), Adenovirus E1A and E1B,
and human Papiloma virus (HPV) E6 and E7. SV40 large T antigen was
regarded as an efficient factor for animal cell immortalization [Lübbe
et al., 1983; Petit et al., 1983; Ozer, 2000], and it has been demonstrated
to be effective for mouse mGSCs immortalization in 2005 [Hofmann
et al., 2005]. Bmi1 is regarded as an marker of mouse mGSCs [Zhang
et al., 2008] and promotes stem cell proliferation [Pietersen et al., 2008].
Being an immortalization factor, Bmi1 has been demonstrated to be
effective in immortalizing somatic cells [Dimri et al., 2002; Saito
et al., 2005]. Conclusively, we believe SV40 large T antigen and Bmi1
are capable of immortalizing dairy goat mGSCs. Here, the lentivirus
particles containing SV40 large T antigen and Bmi1 were transduced
into primary dairy goat mGSCs, and the cells are immortalized and have
been cultured for more than 3 months in vitro. The characteristics of the
immortalized mGSCs were determined through the proliferation test,
marker detection, the differentiation potential assay in vitro and in vivo.
These results suggested that the mGSCs were immortalized by SV40
large T antigen and Bmi1, and these cells will provide unlimited cell
resources for studying male germ cell specification and development.
MATERIALS AND METHODS
ISOLATION AND ENRICHMENT OF MALE GERMLINE STEM CELLS
(mGSCs) FROM GOAT TESTIS
The adult male dairy goats were killed and their testes were collected.
All the procedures were carried on under the supervision of Chinese
Association for Laboratory Animal Science, and approved by
Shaanxi Centre of Stem Cells Engineering & Technology, Northwest
A&F University. Dairy goat mGSCs were isolated and purified as
previously reported [Zhu et al., 2012].
PURIFICATION OF DAIRY GOAT mGSCs
The dairy goat testicular cells were cultured on plates treated with
1mg/ml Fibronectin (Sigma, USA) at 37°C, 5% CO
2
and saturated
humidity for 2 h at the density of 1 10
6
cells/ml [Zhu et al., 2012].
Then the attached cells were dissociated with 0.25% Trypsin
(Invitrogen, USA) for 5 min at 37°C. The cells were collected and
cultured with DMEM/F12 (Invitrogen) supplemented with 10% fetal
bovine serum (FBS, Hyclone, USA), 0.1 mM b-mercaptoethanol
(Sigma) and 2 mM glutamine (Invitrogen).
LENTIVIRUS PREPARATION AND THE IMMORTALIZATION OF DAIRY
GOAT GERMLINE STEM CELLS
Lentivirus production was referred as Anokye-Danso et al. described
[Anokye-Danso et al., 2011]. Briefly, HEK293T cells were seeded on
plates 24 h before transfection, then the plasmids containing SV40
large T antigen and Bmi1 along with the plasmids containing pVSVG
and pPAX2 were incubated respectively. The virus-containing
supernatant was collected at 48 h after transfection, filtered to
remove cell debris, and used for transduction. For cell immortaliza-
tion, dairy goat mGSCs were plated at a density of 1 10
5
cells in a
35 mm dish. Twelve hours later, the cells were transduced with virus-
containing supernatant and 10 mg/ml polybrene (Sigma) and
incubated overnight at 37°C and 5% CO
2
. After 24 h, the medium
was discarded and replaced with fresh DMEM/F12 medium
(Invitrogen) supplemented with 10% FBS (Hyclone, USA), 0.1 mM
b-mercaptoethanol (Sigma) and 2 mM glutamine (Invitrogen), then
cultured for more than 1 month until the cells were immortalized.
The immortalized cells were named mGSCs-I-SB.
CELL CYCLE ANALYSIS
For cell cycle analysis, dairy goat mGSCs (5th passage) and
mGSCs-I-SB (10th passage) were cultured for 48 h and then
suspended the cells into single cells and fixed them in 70% ice-
cold ethanol for 30 min. After that, cells were incubated with
propidium iodide (PI, Sigma) solution supplemented RNase H
(Beyotime, China) for 20 min. Cell cycle analysis was determined
by flow cytometry as previously reported [He et al., 2008].
POPULATION DOUBLING TIME (PDT) DETERMINATION
The population doubling time (PDT) of dairy goat mGSCs were
estimated according to the protocol described previously [Zhang
et al., 2011]. Briefly, cells were serially subcultured, the initial seeding
cell number and the total cell number cultured 48h later were all
counted respectively. PDT was calculated according to the formula,
PDT ¼[log
2
/(logNt0 log N0)] t, where Nt is the number of cells
after t h of culturing, N0 is the number of cells seeded.
5-BROMO-2-DEOXYURIDINE (BrdU) INCORPORATION ASSAY
Dairy goat mGSCs proliferative ability was assessed by BrdU
Incorporation. Dairy goat mGSCs (5th passage) and mGSCs-I-SB
(10th passage) were treated with BrdU (30 mg/ml, Sigma) for 3 h
and then subjected to BrdU immunostaining. Cells were fixed with
methanol/acetone (V/V ¼1:1) for 15 min at room temperature and
washed with PBS for three times, incubated with PBS containing
0.1% Triton-100 for 5min. Then the cells were washed three times
with PBS at room temperature. Mouse anti-BrdU (1:100; Santa
Cruz) dissolved in 0.1M PBS (pH 7.4) containing 4% normal goat
serum was added and the cells were incubated overnight at 4°C.
Cells were washed in PBS for three times, and then incubated with
the goat anti-mouse FITC conjugated secondary antibody (1:500,
Chemicon) for 1 h at room temperature. After three washes, cells
were visualized under fluorescent microscope and analyzed for
BrdU staining.
1550 CHARACTERIZATION OFIMMORTALIZED DAIRY GOAT mGSCs JOURNAL OFCELLULAR BIOCHEMISTRY
RT-PCR
Total RNA for RT-PCR analysis was extracted from 6th passage dairy
goat mGSCs and 11th passage mGSCs-I-SB cultured under normal
conditions using RNAiso (TaKaRa, Biotech. Co. Ltd.). The cDNA was
synthesized based on 500 mg RNA with a commercially available kit
(TaKaRa, Biotech. Co. Ltd.). The PCR steps included denature at 94°C
for 5 min, followed by repeated cycles 30 s at 95°C, 55–58°C for 30 s,
72°C for 30–60 s, 35 cycles,and extend at 72°C for 10 min. The primers
were designed basedon the sequences of the open reading frame from
the NCBI GenBank and synthesized by AuGCT Biotechnology
(Beijing). The PCR primers and the length of the amplified products
are shown in Table 1. The PCR products were analyzed by
electrophoresis in 2% agarose (Invitrogen) gel, stained with ethidium
bromide (Invitrogen), and visualized under UV illumination.
IMMUNOFLUORESCENCE STAINING
Dairy goat mGSCs (7th passage) and mGSCs-I-SB (13th passage)
cultured under normal conditions were fixed with 4% PFA for 15 min at
room temperature, followed by three washes in cold PBS for 5 min each.
Washed cultures were treated with blocking solution (PBST þ1% BSA)
for a minimum of 30 min and incubated in primary antibodies including
Oct-4 (1:500, Chemicon), Sox2 (1:500, Chemicon), CD49f (1:500,
Chemicon), TERT (1:200, Santa Cruz), Oct4 (1:500, Abcam), Stra8
(1:500, Abcam), Blimp1 (1:200, Santa Cruz), Nanog (1:200, Chemicon).
The appropriate FITC conjugated secondary antibodies were used
according to the manufacturer’s manual (1:500, Chemicon). The nuclei
of cells were stained by Hoechst 33342. At the same time, the negative
controls were stained with the appropriate fluorescent-conjugated
secondary antibodies [Yu et al., 2014].
IN VITRO DIFFERENTIATION OF mGSCs-I-SB
The 15th passage immortalized cells were dissociated into single cell
suspension and resuspended in DMEM (Invitrogen) containing 20%
FBS (Hyclone), 2 mM L-glutamine (Invitrogen), 1% non-essential
amino acids (Millipore), 100 U/ml penicillin and 100 mg/ml
streptomycin at 800–1,000 cells/25 ml to form cell clusters. For
further differentiation, the embryoid bodies (EBs) (d3) cultured in
Petri dishes coated with 0.1% gelatin (Sigma) and DMEM medium
containing 20% FBS (Hyclone), 0.1 mM 2-mercaptoethanol (Sigma),
2 mM glutamine (Invitrogen), 1 mM sodium pyruvate (Invitrogen)
and 0.1 mM non-essential amino acids (Invitrogen) for 3–14 days to
investigate the potentiality of spontaneous differentiation [Zhu
et al., 2012]. And EBs were plated to induce the cells differentiate into
adipocyte, osteoblast and cartilage as previous study [Hua
et al., 2011; Hu et al., 2012; Qiu et al., 2012]. After differentiation,
the cells were determined by histochemistry and immunofluores-
cence staining with the antibodies of all three germ layers: NSE
(Ectoderm), Islet1 (Mesoderm and Endoderm), PDX1 (Endoderm)
[Cao et al., 2011; Hua et al., 2011; Qiu et al., 2012].
DETECTION OF THE DIFFERENTIATION POTENTIAL OF
mGSCs-I-SB IN VIVO
The 17th passage of immortalized cells cultured under normal
conditions were transduced with the lentivirus expressing GFP for
12 h, then the cells were dissociated, collected and 0.5 ml working
solution containing the cells with an concentration of 10
7
cells/ml
injected into dorsal neural tube of 2.5 days chicken embryos (E2.5) to
evaluate whether the cells can differentiate into all three germ layers
in vivo. Then the injection hole was sealed with scotch tape and the
egg was returned to incubator until E6 for analysis.
After 3.5 days development, the chicken embryos were fixed with
4% PFA for 24 h at 4°C, the oscillation sliced slides were produced
and analyzed through immunofluorescence staining. The spinal
cords were coronally section at 80 mm by vibration microtome (VT
1000S, Leica, Germany). The sections were washed three times in
0.1 M PBS for 15 min each and then incubated at 4°C with primary
antibodies for 12 h. All three germ layers markers and the germline
specific markers were detected by immunofluorescence staining
assay [Elena de Bellard and Bronner-Fraser, 2005; Boulland
et al., 2010]. Then sections were incubated with secondary antibodies
for 3 h at room temperature and then washed four times for 15 min
each at room temperature. The sections were then counterstained
TABLE 1. The Primers Were Used in Determining the Immortalized Dairy Goat mGSCs
Gene Sense primer Antisense primer Product size (bps) T
m
(°C) Reference sequence
b-Actin GCGGCATCCACGAAACTAC TGATCTCCTTCTGCATCCTGTC 138 58 NM_001101.3
VASA GCTGGCGTAATAGCGAAGAGG GCACAGATGCGTAAGGAGAAAA 107 58 KC189826.1
Gfra1 ATTTTATTACCTGCTGCCA ATTTCAATCATTCCTTCAT 197 51 NM_010279.2
CD117 TCCCAAACCTCAACACCGACAG GTGTAAGTGCCTCCTTCAGTCCC 153 58 NM_013598
CD90 GATCCAGGACTGAGCTCTCGG TCACGGGTCAGACTGAACTCATAC 195 58 NM_006288
CD49f CGAAGCACGAATCCCGAGAC TGCTCTACACGAACAATCGCTTT 235 58 NM_008397
Oct4A GACACCTGGCTTCCGACTTC GCTGAACACCTTCCCAAAGAG 533 59 NM_001265584.1
Bmi1 CCAGAGGGATGGACTGACGA GGGAACTGTGGGTGAGGAGA 147 59 NM_007552.4
Stra8 AAGGACAGCGGGGTTGAC TCGGGTTTTTTTGAGTTGC 170 56 JQ836663.1
PLZF CACCGCAACAGCCAGCACTAT CAGCGTACAGCAGGTCATCCAG 127 58 JX047313.1
NSE AAGGACAAATACGGCAAGG CAGGTCATCACCCACAATC 371 58 JN887466.1
Cyclin D1 TGAACTACCTGGACCGCT CAGGTTCCACTTGAGYTTGT 212 58 NM_053056.2
Sox2 GGCGGCAACCAGAAGAACAG GCATCTTGGGGTTCTCCTGG 109 58 JQ290347.1
Nanog GGAACTGCTGGGGAAAATTA TACAAATCTTCAGGCTGTATGTTG 118 58 FJ970651.1
CDK2 GCCAGGAGTTACTTCTATGC TGGAAGAAAGGGTGAGCC 180 58 NM_001798.3
PCNA AGTGGAGAACTTGGAAATGGAA GAGACAGTGGAGTGGCTTTTGT 167 58 NM_011045.2
exBmi1 TGTTCCCATAGTAACGCCAATA GGCATCAATGAAGTACCCTCC 644 58
SV40 TGACCTCCATAGAAGACACCG CAAATACCTCAGTTGCATCCC 386 58
JOURNAL OFCELLULAR BIOCHEMISTRY CHARACTERIZATION OFIMMORTALIZED DAIRY GOAT mGSCs 1551
with DAPI (1:1,000, Sigma) for 5 min, washed three times for 5 min
each, and finally mounted with a fluorescent mounting medium
(Dako, Carpinteria, CA) on glass slides.
TRANSPLANTATION OF THE CELLS INTO RECEIPT
SPERMATOGENESIS DEFICIENCY MOUSE TESTIS
Male GSCs-I-SB (20th passage) were dissociated, and the cells
were collected through centrifuging at 1,500 r/min for 5 min. The
cells were counted and diluted with DMEM/F12 (Invitrogen)
supplemented with 10% FBS, and 10
5
cells/testis were then
transplanted into seminiferous tubules of the eight receipt
spermatogenesis deficiency mouse, which were injected with
40 mg/kg weight busulfan (Sigma) and evaluated by previous
study [Choi et al., 2004]. After 2 months, the treated mice were
killed, and the recipient’s testes were collected. The characteristic
of mGSCs-I-SB was analyzed through supervision for GFP
expression of the seminiferous tubules and paraffinslideswhich
were produced from the mouse testis transplanted with the
immortalized cells and untransplanted [Russell et al., 1996]. This
was regarded as the golden standard to determine whether the
putative cells maintain the capabilities of mGSCs which were
established by Brinster and Avarbock [1994] and Brinster and
Zimmermann [1994].
STATISTICAL ANALYSIS
Data are presented as mean SEM. Statistical comparisons were assessed
with analysis of Student’sttest. P<0.05 was considered statistically
difference and P<0.01 was considered significantly difference.
Fig. 1. Dairy goat mGSCs-I-SB maintain greater proliferation ability compared with mGSCs. A: Growth curve. B: PDT analysis. C: BrdU Incorporation assay. D: Cell cycle analysis
for mGSCs-I-SB and wild mGSCs. E: PCR detection of the exogenous gene integration.
1552 CHARACTERIZATION OFIMMORTALIZED DAIRY GOAT mGSCs JOURNAL OFCELLULAR BIOCHEMISTRY
RESULTS
THE CELLS’PROLIFERATION POTENTIAL BEFORE AND AFTER
IMMORTALIZATION
We isolated and enriched dairy goat mGSCs as we described
previously [Zhu et al., 2012]. Then the cells were immortalized by the
lentivirus carrying SV40 large T antigen and Bmi1 genes, and the
mGSCs-I-SB showed an obviously stronger proliferation than wild
mGSCs. Growth curve analysis showed that mGSCs-I-SB had a
higher cell number compared with dairy goat mGSCs (Fig. 1A). PDT
detection showed mGSCs-I-SB hold a lower PDT (28.5 h) to mGSCs
(45 h), which means an improvement of proliferation by immortali-
zation (Fig. 1B). BrdU Incorporation assay showed that over 50% of
mGSCs-I-SB were BrdU positive compared with 20% positive in
Fig. 2. The morphology alteration of mGSCs-I-SB from wild dairy goat mGSCs. The morphology from wild mGSCs (A) (Bar ¼100 mm) to mGSCs-I-SB (B) (Bar ¼50 mm) and
immunofluorescence staining for wild mGSCs with antibodies including CD49f, c-Myc, DAZL, Sox2, and Stra8 (C) (Bar ¼200 mm).
JOURNAL OFCELLULAR BIOCHEMISTRY CHARACTERIZATION OFIMMORTALIZED DAIRY GOAT mGSCs 1553
dairy goat mGSCs (Fig. 1C). Also, cell cycle analysis showed that
dairy goat mGSCs to mGSCs-I-SB alteration accompanied an
increase of S phase cell ratio from 37.7% to 55.0% (Fig. 1D).
Moreover, the cells lifespan also showed a great improvement from
less than 10 passages to be easily subcultured up to 30 passages. In
summary, all these results demonstrated that mGSCs-I-SB maintain
a greater potential in proliferation compared to mGSCs.
To evaluate whether the cells were actually immortalized by SV40
large T antigen and Bmi1, we examined the genomic integration of
the exogenous genes. As indicated in Figure 1E, the exogenous
genes-SV40 large T antigen and Bmi1 were positive in mGSCs-I-SB
and negative in mGSCs. This further demonstrated that the
immortalization of dairy goat mGSCs was mediated by SV40 large
T antigen and Bmi1.
THE MARKERS EXPRESSED IN DAIRY GOAT mGSCs-I-SB
The mGSCs-I-SB exhibited spindle-like cell morphology compared to
the epithelioid-like cell morphology before immortalization (Fig. 2A,
B). Whether mGSCs-I-SB expressed the canonical mGSC markers seem
to be important to supply an intuitive evidence for mGSCs. Wedetected
the mGSC markers through RT-PCR and immunofluorescencestaining
for the cells before and after immortalization. The immunofluores-
cence staining showed dairy goat mGSCs were positive for most of the
male germ cell markers, such as CD49f, DZAL, Stra8, Sox2, andC-Myc
(Fig. 2C) before immortalization, and mGSCs-I-SB were also positive
for CD49f, CD90, Blimp1, C-Myc, Nanog, PLZF, Oct4, Stra8, and TERT
(Fig. 3A). The RT-PCR results showed that the cells were positive for
SSC specific markers such as VASA, GFRa1, CD117, CD90, CD49f,
Stra8, PLZF; they were also positive for pluripotency markers
including Oct4A, Nanog, Sox2; and proliferative markers such as
CDK2, CyclinD1, PCNA, and Bmi1 (Fig. 3B).
THE DIFFERENTIATION POTENTIAL OF mGSCs-I-SB IN VITRO
The cells’pluripotency still need to be elucidated although the
associated markers were expressed. Then we examined the
differentiation potential in vitro by inducing the cells differentiation
into osteoblasts, chondrocytes, neural-like cells and adipocytes. The
EBs formed in suspension were collected and cultured under defined
solutions. When the cells were induced for a defined protocol as we
described previously [Hua et al., 2011], the cells displayed positive
for Oil Red O (adipocyte), Alcian blue (chondrocyte), Alizarin Red
(osteoblast) (Fig. 4A), and immunocytochemical staining showed
Fig. 3. Detection of the markers of mGSCs-I-SB. Immunofluorescence staining for mGSCs-I-SB with antibodies including CD90, CD49f, Blimp1, c-Myc, Nanog, PLZF, Oct4,
Stra8 and TERT (A) (Bar ¼100 mm). RT-PCR detection for markers: Gfra1, CD117, CD90, CD49f, Vasa, Stra8, Sox2, PLZF, Oct4A, Nanog, CDK2, CyclinD1, and PCNA (B).
1554 CHARACTERIZATION OFIMMORTALIZED DAIRY GOAT mGSCs JOURNAL OFCELLULAR BIOCHEMISTRY
that the cells were differentiated into cells positive for NSE (ectoderm
marker), Islet1 (mesoderm and endoderm marker), PDX1 (endoderm
marker), Scp3 and VASA (germ cell marker) (Fig. 4B). All these
results suggested that mGSCs-I-SB maintain the potential to
differentiate into any of the three germ layers in vitro.
THE DIFFERENTIATION POTENTIAL OF mGSCs-I-SB IN VIVO
To determine the differentiation potential of mGSCs-I-SB in vivo,
the GFP labeled cells were transplanted into chicken embryos’dorsal
neural tube under the protocol illustrated in Supplemented Fig. S1.
After 3 days, the GFP positive cells were found in recipient’s neural
Fig. 4. In vitro differentiation potential determination for mGSCs-I-SB. In vitro, mGSCs-I-SB can form EBs (from left to right, Bar ¼400, 200, 100 mm), then differentiate into
oil red O (Bar ¼100 mm), alcian blue (Bar ¼50 mm) and alizarin red (Bar ¼50 mm) positive cells in appropriate conditions (A). The cells exhibited positive for NSE, Islet1, PDX1,
Scp3, and VASA analyzed by immunofluorescence staining (B) (Bar ¼100 mm).
JOURNAL OFCELLULAR BIOCHEMISTRY CHARACTERIZATION OFIMMORTALIZED DAIRY GOAT mGSCs 1555
tube and out everywhere (Fig. 5). The GFP positive cells were also
positive for the markers of all three germ layers including Nestin,
Islet1, and Glut2 (Fig. 5A), and the mGSC markers including CD49f,
GFRa1, and PLZF (Fig. 5B). All these results evidenced that the cells
maintain the differentiation potential to all three germ layers and
male germ cell lineages in vivo.
THE CELLS POTENTIAL TO SURVIVE IN MOUSE IMPAIRED
SEMINIFEROUS TUBULES
The most important characteristic of mGSCs is the potentiality to
reconstitute the impaired seminiferous tubules. The transplantation
assay showed that mGSCs-I-SB maintain the capacity of surviving in
the testis of mouse spermatogenesis deficiency models (3 out of 8), and
no tumor structures was observed for at least two months through
supervision of the recipient’s seminiferous tubules and immunofluo-
rescence staining (Fig. 6A,B). All these results indicated that mGSCs-I-
SB maintain the mGSC unique characteristic of surviving in the receipt
mouse spermatogenesis deficiency model testis.
THE CELLS CAN BE INDUCED TO DIFFERENTIATE INTO
SPERM-LIKE CELLS
Male GSCs maintain the unique characteristic of differentiating into
sperm cells [Olive and Cuzin, 2005]. To further demonstrate the
immortalized cells maintain the capacity of mGSCs, the cells were
Fig. 5. In vivo differentiation potential of mGSCs-I-SB. When mGSCs-I-SB cells were transplanted and developed in chicken embryo for 3 days, the cells expressed all three germ
layers markers including Nestin, Islet1, Glut2 (A) (Bar ¼400 mm) and CD49f, GFRa1, PLZF (B) (Bar ¼63.5 mm).
1556 CHARACTERIZATION OFIMMORTALIZED DAIRY GOAT mGSCs JOURNAL OFCELLULAR BIOCHEMISTRY
cultured in suspension to form EBs (Fig. 7A). Then the EBs were
induced under 2 10
7
mM RA. After 3 days induction, more round
cells emerged in RA stimulation group than control; and this
tendency seemed to be more obvious when the cells were induced for
7 days. Immunocytochemical staining was carried on to determine
whether the induced cells maintain the characters of male germ cells.
The results showed that the cells were positive for Stra8 (pre-meiosis
marker), Scp3 (meiotic marker) and Acr (post-meiosis marker)
(Fig. 7B). These results showed that mGSCs-I-SB maintain the unique
capacity to differentiate into sperm-like cell (Fig. 7).
DISCUSSION
Previous reports on mGSCs were mainly in the mGSCs’establish-
ment, growth, dedifferentiation, and transplantation [Olive and
Cuzin, 2005]. All these progresses were mainly obtained on mouse
and human. For other species, especially livestock, there is a lack of
stable mGSC line. This greatly restricted the further study on the
utilization of mGSCs in preservation and optimization of germplasm
resources. Thus, the success in establishing immortalized dairy goat
mGSC lines may set a model for other species’mGSCs.
Up to date, the immortalized germline cells including C-18-4, GC1,
GC2, etc. were established by exogenous SV40T, hTERT, and/or BmI1
[Bellvé et al., 1977]. SV40 large T antigen, a hexamer protein that is a
proto-oncogene derived from the polyoma virus SV40, is capable of
transforming many cell types [Bellvé et al., 1977]. The transforming
activity of T-tag is due in large part to its perturbation of the
retinoblastoma (pRB) and p53 tumor suppressor proteins [Yang
et al., 2007]. SV40 large T antigen has been shown to be the most
simple and reliable agent for thetransformation of many different cell
types in culture. Most importantly, viral genes induceimmortalization
by inactivating the tumor suppressor genes (p53, Rb, and others) that
can cause a replicative senescent state in cells. Recent studies have
also shown that SV40 large T antigen can induce telomerase activity
in the transduced cells. Additionally, T-tag binds to several other
cellular factors, including the transcriptional co-activators p300 and
CBP, which may contribute to its transformation function [Ali and
DeCaprio, 2001]. BMI1 (BMI1 polycomb ring finger oncogene), a
protein which in human is encoded by the BMI1 gene, has been
reported as an oncogene by regulating p16 and p19 that are cell cycle
inhibitor genes[Alkema et al., 1993]. Studies showed thatinhibition of
p16INK4a and introduction of hTERT can immortalize many types of
human cells with little chromosomal instability [Haga et al., 2007].
Fig. 6. Transplantation assay showed mGSCs-I-SB maintain the unique characteristic of mGSCs. After transplantation mGSCs-I-SB into receipt mouse spermatogenesis
deficiency testis’seminiferous tubules for more than 2 months, the GFP-labeled cells were supervised in the seminiferous tubules (A) (Bar ¼200mm), and the transplanted
mGSCs-I-SB cells existed and also were positive for GFP (B) (Bar¼100 mm).
JOURNAL OFCELLULAR BIOCHEMISTRY CHARACTERIZATION OFIMMORTALIZED DAIRY GOAT mGSCs 1557
Lentiviral vector can be used to integrate both dividing and non-
dividing cells as they can actively pass though nuclei membrane. In
addition, same as retrovirus, lentivirus will integrate into a host cell
genome. Lentivirus is gaining popularity for both in vitro and in vivo
applications of gene transduction. In this study, our results for the
first time showed that overexpression of ectopic factors-SV40 large
T antigen and Bmi1 using lentivirus system may greatly improve the
proliferation of dairy goat mGSCs analyzed by PDT, BrdU
Incorporation assay and cell cycle. Meanwhile, the immortalized
cells maintain the similar biological characteristics as wild mGSCs
and previous reports on mouse mGSCs [Olive and Cuzin, 2005] when
cultured the cells in similar conditions [Feng et al., 2002]. And, the
cells expressed most of the conserved markers used in clarifying of
mGSCs (Supplemented Figs. S2–S4). The immortalized cells also
maintain the capacity to differentiate into all three germ layers as
pluripotent mGSCs did as reported [Olive and Cuzin, 2005].
The golden standard method to examine the mGSCs is to transplant
the cells into the mouse spermatogenesis deficiency model testis to
determine whether the cells can reconstitute the seminiferous tubules
which was established by Brinster and Avarbock [1994]. But this
standard is mainly used in mouse and rat. For other species, whether
the cells can survive for a long time is regularly evaluated [Olive and
Cuzin, 2005]. In our study, the immortalized dairy goat mGSCs
xenotransplantated into rodent model’s seminiferous tubules and
survived in recipient’s testis for more than 2 months, and this greatly
demonstrate the cells maintain the capabilities of mGSCs.
The chicken embryo is a classical animal model for studying
embryonic and fetal development and for xenotransplantation
experiments to study the behavior of cells in a standardized in vivo
environment [Boulland et al., 2010]. Compared with the mammals,
the main advantages of the chicken embryo are low cost, high
accessibility, ease of surgical manipulation and lack of a fully
developed immune system. Xenotransplantation into chicken
embryos can provide valuable information about cell proliferation,
differentiation and behavior, the responses of cells to signals in
defined embryonic tissue niches, and tumorigenic potential
[Boulland et al., 2010]. Transplanting cells into chicken embryos
can also be a step towards transplantation experiments in other
animal models. Recently the chicken embryo has been used to
evaluate the neurogenic potential of human stem and progenitor
cells following implantation into neural anlage [Boulland
et al., 2010]. In this study, we transplanted the GFP-labeled
mGSCs-I-SB into the developing central nervous system of the
chicken embryo. The results evidenced that mGSCs-I-SB cells can
survive, and meanwhile, and they differentiate into three germ
layers in chicken embryos analyzed by immunofluorescence. This
study first demonstrated chicken embryo was an efficient model to
study the behavior and function of mGSCs.
Male GSCs have the ability to differentiate into spermatogenic cells
through in vivo transplantation or through induction in vitro [Hua
et al., 2011]. The immortalized mGSCs-I-SB were differentiated into
sperm-like cells, and the induced cells were positive for Stra8 (a marker for
pre-meiosis germ cells), Scp3 (a marker for meiosis cells) and Acr (a marker
for post-meiosis cells). In combination with the transplantation assay, the
results support that mGSCs-I-SB maintain the characteristic of mGSCs.
Taken together, our results for the first time demonstrate that the
immortalized dairy goat mGSCs were obtained through exogenous
expression of SV40 T antigen and Bmi1, and these cells have the
characteristics of mGSCs compared with the mainly existed studies
on mGSCs.
ACKNOWLEDGEMENTS
The authors appreciate Dr. Steven Gao and Dr. Xiang Chen for their
excellent revision and discussion. This work was supported by the
grants from the Program (31272518) of National Natural Science
Foundation of China, National Major Fundamental Research
Program of China (2013CB947900), Doctoral Fund of Ministry of
Fig. 7. The mGSCs-I-SB cells were differentiated into male germ cells in vitro.
A: EBs were formed (from left to right, Bar ¼50, 20 mm), mGSCs-I-SB were
induced to form large, round cells by RA, also, the sperm-like cells were
observed at 7 day induction by RA (D3, Bar ¼100 mm; D7, Bar ¼50 mm).
B: Identification of the mGSCs-I-SB cells induced by RA through
immunofluorescence staining. The induced mGSCs-I-SB cells were positive
for STRA8, SCP3 (Bar ¼100 mm) and ACR (Bar ¼200 mm).
1558 CHARACTERIZATION OFIMMORTALIZED DAIRY GOAT mGSCs JOURNAL OFCELLULAR BIOCHEMISTRY
Education of China (RFDP, 20120204110030), the Fundamental
Research Funds for the Central Universities (QN2011012).
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