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| Journal of Molecular Cell Biology (2014), 6, 164 –171
doi:10.1093/jmcb/mju004
Article
Conversion of female germline stem cells
from neonatal and prepubertal mice into
pluripotent stem cells
Hu Wang1, Manxi Jiang2, Haiwei Bi1, Xuejin Chen2, Lin He1, Xiaoyong Li1, and Ji Wu1, *
1
Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Bio-X Institutes, Shanghai Jiao Tong University,
Shanghai 200240, China
2
Department of Laboratory Animal Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
* Correspondence to: Ji Wu, E-mail: [email protected]
Pluripotent stem cells derived from neonatal or adult testes are a useful tool to examine the mechanisms of pluripotency and a resource
for cell-based therapies. However, therapies using these cells will only benefit males but not females. Recently, female germline stem
cells (FGSCs) were discovered in ovaries. Whether FGSCs can be converted into pluripotent stem cells, similar to spermatogonial stem
cells, is unknown. Here, we demonstrate that female embryonic stem-like cells (fESLCs) can be generated within 1 month from the
stably proliferating FGSCs cultured in embryonic stem cell (ESC) medium. fESLCs exhibit properties similar to those of ESCs in
terms of marker expression and differentiation potential. Thus, our findings suggest that generation of patient-specific fESLCs is feasible and provides a foundation for personalized regenerative applications.
Keywords: embryonic stem-like cells, female germline stem cells, adult stem cells
Introduction
Pluripotent stem cells such as embryonic stem cells (ESCs) and
embryonic germ cells derived from early embryos have a great potential for use in cell-based therapies (Romeo et al., 2012).
However, their clinical applications are limited because of ethical
issues. In recent years, induced pluripotent stem cells (iPSCs)
have been successfully generated by expression of pluripotency
transcription factors (Takahashi and Yamanaka, 2006; Takahashi
et al., 2007), but their safety in potential therapies is still
unknown. Therefore, new approaches need to be developed for
pluripotent cell generation.
Previous studies have demonstrated that spermatogonial stem
cells (SSCs) from newborn (Kanatsu-Shinohara et al., 2004), adult
(Guan et al., 2006, 2009; Seandel et al., 2007; Conrad et al., 2008;
Kanatsu-Shinohara et al., 2008; Ko et al., 2009, 2010) mouse, and
human testis tissue (Golestaneh et al., 2009; Kossack et al., 2009)
can be converted into ESC-like cells under specific in vitro conditions
without genetic manipulations. This method overcomes the problems associated with the therapeutic use of ESCs and iPSCs by
providing cells that are non-embryonic, patient-specific, and free
from genetic modifications. Although the pluripotent stem cells
converted from SSCs have gained interest in stem cell research,
SSC-based therapies will only benefit males but not females.
Our previous studies have shown that female germline stem cells
Received July 13, 2013. Revised October 12, 2013. Accepted November 5, 2013.
# The Author (2014). Published by Oxford University Press on behalf of Journal of
Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.
(FGSCs) exist in postnatal ovarian tissues. Their isolation from neonatal and adult mice and long-term culture have gained a great
deal of interest in stem cell biology (Zou et al., 2009, 2011; Zhang
et al., 2011). In addition, FGSCs have been found in rat (Zhou
et al., 2014) and lower vertebrate species such as medaka fish
(Nakamura et al., 2010). White et al. (2012) have extended our
work using fluorescence-activated cell sorting (FACS) to isolate
similar FGSCs from human ovarian tissue. Moreover, the shape,
growth pattern, and function of FGSCs during gametogenesis are
similar to those of SSCs. Thus, we hypothesized that FGSCs can
also be converted into pluripotent stem cells using the method for
SSCs conversion.
In the present study, female embryonic stem-like cells (fESLCs)
are generated from FGSCs under ESC culture conditions, and
their pluripotency is confirmed by in vitro differentiation and in
vivo characterizations.
Results
Characterization of FGSCs
FGSCs were isolated from neonatal (nFGSCs) and prepubertal
(pFGSCs) mouse ovaries following the protocols described previously (Wang et al., 2013). After 1 day of culture, most of nFGSCs
were round (Figure 1A). Then, FGSCs showed a typical grape-like
morphology after 3 days of culture. After the initial passage,
FGSCs were subcultured every 5– 7 days at a 1:1 ratio. The
number of FGSCs gradually increased over 4 – 5 passages. After
that, the total number of FGSCs was significantly increased, and
the cells were subcultured every 3–5 days at a 1:2 ratio.
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Figure 1 FGSC characterization. (A) Typical phenotypes of freshly isolated nFGSCs cultured for 1 day, 3 days, 4 weeks, and 8 weeks. nFGSCs showed
a grape-like morphology during the initial 3 days of culture and the number of FGSCs gradually increased during the culture period. Scale bar,
25 mm. (B) Immunostaining of nFGSCs for Mvh and BrdU. IgG was used as negative control. Scale bar, 25 mm. (C) RT –PCR analysis of germlinespecific markers. M, 100-bp DNA marker.
Furthermore, we assessed and confirmed the proliferative potential
and germline-specific protein (Mvh) expression of these cells by immunofluorescence (Figure 1B). More germline markers Oct4,
Blimp-1, Fragilis, Dazl, and Stella were detected by RT–PCR
(Figure 1C). Moreover, FGSCs obtained from ovary of neonatal and
prepubertal mice have a similar growth pattern (Supplementary
Figure S1). These stably proliferating FGSCs were used for a conversion to fESLCs.
Conversion of FGSCs into fESLCs
To obtain fESLCs from FGSCs, ESC-specific culture conditions
were used (Wernig et al., 2007). About 1000–2000 FGSCs were
seeded in a well of a 24-well plate containing a mitotically inactivated STO feeder layer and maintained in ESC culture medium
(DMEM with 15% FBS and 1000 U/ml LIF) (Figure 2A). The
number of cells increased at 325 days after the initial seeding
and small colonies formed after 12– 15 days of culture
(Figure 2B). To passage these colonies, cells were dissociated
mechanically and then plated onto a freshly prepared STO feeder
layer in ESC medium. These cells were then referred as neonatal
female germline-derived ES-like cells (nfESLCs) or prepubertal
female germline-derived ES-like cells (pfESLCs). Our initial
attempt used newly prepared FGSCs for conversion, but failed to
form fESLC colonies. Therefore, in later attempts, FGSCs were cultured for 7– 8 weeks until the cells proliferated stably and actively
before being used for conversion.
The conversion under ESC culture conditions is limited in terms
of speed and efficiency. Several compounds such as vitamin C
(Vc) (Esteban et al., 2010) and valproic acid (VPA) (Zhu et al.,
2010) accelerate the reprogramming process and can be used to
optimize FGSC conversion. After treatment with Vc and/or VPA,
the colonies grew faster (Figure 2C). Moreover, Vc and VPA
showed a synergistic effect on the growth (Figure 2C) and
number (Figure 2D) of fESLC colonies. Also, the initial number of
seeded FGSCs seemed to be a key factor in the conversion
process (Figure 2E). A high seeding density of FGSCs decreased
the conversion efficiency, which is consistent with a previous
study (Ko et al., 2009).
Phenotypic analysis of fESLCs
Alkaline phosphatase (AP) is expressed weakly in FGSCs as shown in
our previous report (Zou et al., 2009), while in fESLCs, there was strong
AP activity (Figure 3A). RT–PCR analysis showed that, under an ESC
culture condition, the cultured fESLCs expressed pluripotency-related
transcription factors similar to mouse ESCs, including Oct4, Sox2,
Nanog, Utf1, Esg1, and Rex1 (Figure 3B). However, only Oct4 was
expressed in FGSC (Supplementary Figure S2A and C). Nanog expression was significantly increased (Supplementary Figure S2B) during
the conversion process, while its expression was undetected in
FGSCs by RT–PCR (Supplementary Figure S2A). Additionally, we
detected the DNA methylation profile of the 5′ -flanking promoter
region of mouse Oct4 and Nanog genes. The analysis showed that
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Wang et al.
Figure 2 nfESLC generation from nFGSC and procedure optimization. (A) Schematic overview of generation procedure for nfESLCs from nFGSCs. (B)
Small nfESLC colonies (white arrow) appeared at 1 week after initiation of ESC culture conditions. nFGSCs grew as clusters (white arrow) in culture,
whereas mouse ESCs grew as compact colonies (white arrow). Scale bar, 25 mm. (C) Vc and VPA accelerated nfESLC colony growth. Scale bar,
25 mm. (D) Vc and VPA synergistically increased the number of nfESLC colonies. Error bars indicate standard deviations from three biological replicates. (E) The number of converted nfESLCs colonies depended on the initial seeding density of nFGSCs. Error bars indicate standard deviations
from three biological replicates.
Figure 3 Characterization of fESLCs. (A) AP staining of nFGSC, nfESLCs, and mouse ESCs. Scale bar, 25 mm. (B) Expression analysis of pluripotency
marker genes. M, 100-bp DNA marker. (C) DNA methylation status of Oct4 and Nanog promoter regions in nFGSC, nfESLCs, and mouse ESCs examined by bisulfite sequencing. (D) Immunofluorescence staining of nfESLCs for pluripotency markers (SSEA-1 and Oct4). Scale bar, 25 mm. (E)
Karyotype analysis of nfESLCs. (F) Distribution of chromosome numbers in nfESLC and pfESLCs.
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Figure 4 Methylation status of DMRs in FGSC, fESLCs, and mouse ESCs. DNA methylation was analyzed by bisulfite genomic sequencing. The DMR
methylation assay examined maternally imprinted regions (Igf2r and Peg10) and paternally imprinted regions (H19 and Meg3 IG). Black and white
circles indicate methylated and unmethylated CpGs, respectively.
all CpG sites in the Oct4 promoter region were unmethylated following
fESLC generation, whereas a change was detected in the methylation
status of the Nanog promoter region (Figure 3C). Immunostaining
showed that cultured fESLCs expressed the cell surface markers,
stage-specific embryonic antigen 1 (SSEA-1) and Oct4 (Figure 3D),
but there was no SSEA-1 expression in nFGSCs (Supplementary
Figure S2C). To examine the chromosome integrity, we conducted a
karyotype assay of fESLCs. The data showed that fESLCs converted
from FGSCs had normal karyotype (40, XX) (Figure 3E) and chromosome number (Figure 3F).
Genomic imprinting analysis of fESLCs
To investigate the genomic imprinting pattern of fESLCs, bisulfite
sequencing analysis was performed to examine the differentially
methylated regions (DMRs) of paternally imprinted regions (H19
and Meg3 IG) and maternally imprinted regions (Igf2r and Peg 10).
In FGSCs, DMRs showed complete methylation of both Peg10 and
Igf2r but no methylation of H19 or Meg3 IG. The DNA methylation
pattern of Peg10 and Igf2r was maintained in fESLCs converted
from FGSCs, although few sites of maternally imprinted regions
(Peg 10 and Igf2r) were demethylated. And paternally imprinted
regions (H19 and Meg3 IG) showed partial methylation in fESLCs.
In contrast, mouse ESCs exhibited a somatic methylation pattern
(Figure 4). Therefore, the DNA methylation status of maternally
imprinted regions was maintained during the FGSC conversion
process under ESC culture conditions.
Differentiation potential of fESLCs
Embryoid bodies (EBs) formed by the hanging drop method were
used to differentiate fESLCs into various cell lineages by standard
ESC differentiation methods (Wernig et al., 2007). The differentiation
procedure used in this study (Figure 5A) showed that fESLCs
formed EBs (Figure 5B) and pluripotency-associated gene expression
decreased during the differentiation period (Figure 5C and D). To test
the capability of EBs to differentiate into derivatives of all three germ
layers, lineage-specific genes and proteins were analyzed during EB
differentiation. Expression of transcription factors specific for mesodermal lineages (cardiac, skeletal muscle, and vascular tissue) was
gradually increased during fESLC differentiation. Neuroectoderm
and endoderm differentiation was also confirmed by the expression
of related markers (Figure 5E). Moreover, we characterized the differentiated cells by immunostaining for Nestin (neuroectoderm lineage
marker), Brachyury (mesodermal lineage marker), and E-cadherin
(endoderm lineage marker) (Figure 5F).
To examine the differentiation ability of fESLCs in vivo, teratomas
formation by subcutaneous injection of fESLCs into nude mice was
monitored. Teratomas were formed in three recipients at 2 months
post-transplantation (Figure 5G), and the three embryonic germ
layers were demonstrated by histological analysis (Figure 5H). No
teratoma was formed by FGSCs transplanted into nude mice
(Figure 5G), which is consistent with previously published data (Zou
et al., 2009). These results indicate that the ESC culture condition is
critical for conversion of unipotent FGSCs to fESLCs.
Chimera assays
A chimera assay was performed to determine the contribution of
fESLCs to all three germ layers and the germline. Enhanced green
fluorescent protein (EGFP)-labeled fESLCs were microinjected (10–
15 cells per blastocyst) into 60 C57BL/6 blastocysts (Figure 6A) that
were then transferred into the uterine horn of 2.5 dpc pseudopregnant
mice (15 blastocysts per mouse). At 2 weeks post-transplantation, two
recipient mice were pregnant. Unfortunately, one litter (named as 1#)
was dead at 18.5 dpc, as the result of mouse dystocia, and the other
litter (named as 2#) was born but dead. Other embryos were not found
after the dissection, which may be absorbed partially or completely.
These dead litters (males; Figure 6B) were used to analyze the chimerism. We detected GFP expression in derivatives of all three germ layers
(including the brain, eye, lung, heart, liver, kidney, spleen, testis,
muscle, skin, and tail) by genotyping (Figure 6C) and immunofluoresence (Figure 6D). We also observed GFP expression in the gonads.
These data indicated that fESLCs converted from FGSCs are fully pluripotent and show germline chimeric.
Discussion
Pluripotent stem cells can be used to regenerate tissues and organs
that are damaged in a broad variety of diseases. ESCs and iPSCs are
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Wang et al.
Figure 5 In vitro and in vivo differentiation of nfESLCs. (A) Experimental scheme of nfESLC differentiation in vitro. (B) Microscopic view of EBs. Scale
bar, 100 mm. (C) RT– PCR analysis of pluripotency-associated transcription factor expression of EBs under differentiation conditions. M, 100-bp
DNA marker. (D) Real-time PCR analysis of Oct4 and Nanog during EBs differentiation (mean + SEM, n ¼ 3). Transcript levels were normalized
to Gapdh expression. (E) RT– PCR analysis of lineage-specific marker genes at various stages during EB differentiation. (F) Immunostaining of
Nestin, Brachyury, and E-cadherin at Day 15 of EB differentiation. IgG was used as negative control. Scale bar, 25 mm. (G) Teratoma formation
assay on nude mice after nfESLCs cells injection (at least n ¼ 3 nude mice per group). Circle indicated a teratoma. (H) Teratoma histological analysis. Teratomas contained the three embryonic germ layers. Scale bar, 50 mm.
the best characterized pluripotent stem cells that not only have selfrenewal capacity, but can also differentiate into various cell types
(Romeo et al., 2012). However, ethical concerns and immunological
rejection have restricted their use in tissue regeneration (Romeo
et al., 2012). Recently, numerous studies have suggested that SSCs
from mouse and human testes can give rise to ESC-like cells under
specific culture conditions in vitro (Kanatsu-Shinohara et al., 2004,
2008; Guan et al., 2006, 2009; Seandel et al., 2007; Conrad et al.,
2008; Golestaneh et al., 2009; Ko et al., 2009, 2010; Kossack et al.,
2009). These ESC-like cells share certain properties with ESCs, including pluripotency gene expression, multilineage differentiation, and
variations in the methylation profiles of imprinted genes (KanatsuShinohara et al., 2004; Guan et al., 2006). Future studies concerning
the conversion process are needed, including optimization of the conditions used for conversion, elucidation of the mechanisms of conversion, and determining whether such conversion is limited to SSCs.
Our study demonstrated that stably proliferating FGSCs from
neonatal or prepubertal mouse ovaries can be converted to
fESLCs within 1 month under ESC culture conditions. The derivation
efficiency of FGSCs into fESLCs was synergistically enhanced by Vc
and VPA. These fESLCs exhibited ESC characteristics such as ESC
morphology and expression of pluripotency markers, and had a
normal karyotype. In addition, fESLCs could differentiate into the
three germ layers in vitro, form teratomas in vivo, and contribute
to chimeras and the germline.
It is important to note that maternal imprinting patterns of Peg10
and Igf2r in fESLCs did not show overall alteration during the conversion process, although the paternal imprinting patterns of H19 and
Meg3 IG became methylated partially. This is consistent with previous finding by Ko et al. (2009) that the methylation status of the
androgenetic pattern is maintained in ESC-like cells. Interestingly,
in ESC-like cells converted from SSCs of newborn or adult testes,
there is a change of the methylation status of DMRs (KanatsuShinohara et al., 2004; Guan et al., 2009). The discrepancy probably
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Figure 6 Analysis of chimera formation. (A) Blastocyst stage of an embryo after nfESLC injection (left) and a hatching blastocyst (right). (B) Chimeric
offspring derived from a chimera at 18.5 dpc. (C) Genomic DNA from various tissues and organs of two male chimeric mice was analyzed by genomic
PCR for the GFP gene. M, 100-bp DNA marker. (D) The liver (bile duct) and testes (seminiferous tubule) of the male chimeric mice were analyzed for
GFP expression by fluorescence microscopy. Scale bar, 50 mm.
came from the use of different derivation protocols. The methylation
status of genomic imprinting genes in ESC-like cells may also depend
on the cells themselves, as freshly prepared FGSCs failed but stably
proliferating FGSCs succeeded to generate fESLCs in our study.
Moreover, fESLCs could not be converted from the stably proliferating FGSCs without ESC culture condition, suggesting that the ESC
culture condition is also a critical factor for FGSCs conversion.
Recent studies indicated that the pluripotent stem cells from SSC
conversion have germline chimeric characterization by blastocyst
injection assay (Kanatsu-Shinohara et al., 2004, 2008; Guan
et al., 2006; Seandel et al., 2007; Conrad et al., 2008; Ko et al.,
2009). Therefore, we did not perform tetraploid complementation
assay in this study, but confirmed previous findings in the germline
chimeric assay by blastocyst injection. Moreover, Ko et al. (2009)
found that the germline-derived pluripotent stem (gPS) cells from
SSC conversion still keep the DNA imprinting methylation patterns,
indicating that gPS cells may have epigenetic memory. It is
unknown whether the epigenetic memory of fESLCs resulted in
the failure of getting live-born chimeric mice in this study.
Thus far, germline-derived pluripotent stem cells are unknown
for the mechanisms of their conversion. One study has demonstrated that somatic cells can be reprogrammed by Oct4 expression
alone under certain conditions (Zhu et al., 2010). There is no clear
evidence whether FGSC and SSC conversion into ESC-like cells is
also related to their maintenance of Oct4 expression. Currently,
male germline-derived ESC-like cells, fESLCs, and iPSCs can be
used as research models for the conversion involved in pluripotency acquisition. This will help us to determine the underlying
mechanisms of pluripotency and why germline cells can be converted to a pluripotent state by culture conditions alone, whereas
somatic cells require exogenous expression of pluripotency-related
factors.
In summary, similar to SSCs, FGSCs from neonatal or prepubertal
mice can be converted into pluripotent stem cells under certain
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| Journal of Molecular Cell Biology
culture conditions. These findings may have important implications
in stem cell reprogramming and regenerative medicine.
Materials and methods
Isolation and culture of FGSCs from mouse ovaries
All animal experiments were approved by the Institutional
Animal Care and Use Committee of Shanghai and performed
according to the National Research Council Guide for Care and
Use of Laboratory Animals.
Ovaries from healthy mice (CD-1 neonatal mice, 6 days of age;
C57BL/6 × CD-1 F1 hybrid prepubertal mice, 16 days of age)
were collected, washed with ice-cold phosphate buffered saline
(PBS), and then cut to small pieces. Two-step enzymatic isolation
and purification of FGSCs was performed as described previously
(Wang et al., 2013). FGSCs medium consists of MEMa (Invitrogen)
supplemented with 10% fetal bovine serum (FBS) (Life
Technologies), 30 mg/ml pyruvate (Amresco), 2 mM L-glutamine
(Amresco), 50 mM b-mercaptoethanol (Biotech), 6 mg/ml penicillin
(Amresco), 1 mM nonessential amino acids (NEAA) (Invitrogen),
20 ng/ml mouse epidermal growth factor (PeproTech), 10 ng/ml
human basic fibroblast growth factor (bFGF) (PeproTech), 10 ng/ml
mouse glial cell line-derived neurotrophic factor (Peprotech), and
1000 U/ml ESGRO (mouse leukemia inhibitory factor [LIF]) (Santa
Cruz Biotechnology).
After 7– 8 weeks of culture, stably proliferating FGSCs were cultured in ESC medium consisting of Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 15% FBS, 1 mM NEAA,
2 mM L-glutamine, 6 mg/ml penicillin, 50 mM b-mercaptoethanol,
and 1000 U/ml ESGRO. R1 mouse ESCs were obtained from the
Institute of Biochemistry and Cell Biology of the Chinese
Academy of Sciences (Shanghai, China). fESLCs were grown on
STO feeder cells in ESC medium at 378C with 5% CO2. Cells were
passaged every 2 days and the medium was changed every day.
RNA isolation, RT– PCR and real-time PCR analysis
Total RNA was extracted using Trizol reagent (Qiagen) according
to the manufacturer’s instructions. Approximately 100 ng RNA was
used to generate cDNA with M-MLV reverse transcriptase. PCR analyses were performed with Taq DNA Polymerase. Primers are listed
in Supplementary Table S1.
The final PCR reaction volume of 20 ml contained 10 ml SYBR
Green PCR Master Mix (Roche), 1 ml cDNA template, 1 ml primer
mixture and 8 ml water. The PCR thermal cycling started with denaturation at 948C for 10 min, followed by 40 two-step cycles:
5 sec at 948C, 10 sec at 608C, and 12 sec at 728C. Primers are
listed in Supplementary Table S1. Amplification data were collected
by the ABI 7300 system (Applied Biosystems) (Huang et al., 2013).
Immunofluorescence and AP staining
For immunofluorescence, cells were fixed with 4% paraformaldehyde (PFA) in PBS (pH 7.4) for 30 min, washed twice with 0.1%
Tween 20 in PBS (PBST), incubated with 1% Triton X-100 in PBS
for 10 min, blocked with 10% goat serum in PBS for 30 min, and
then incubated with primary antibodies diluted in PBS at 48C overnight. Primary antibodies against the following molecules were
used: BrdU (1:200; Chemicon), Mvh (1:200; Abcam), pluripotency
markers Oct4 (1:200; Santa Cruz) and SSEA-1 (1:100; Chemicon),
Wang et al.
and differentiation markers Nestin (1:200; Millipore), Brachyury
(1:200; Abcam), and E-cadherin (1:200; Abcam). After incubation,
the cells were washed twice with PBST for 5 min and then incubated
with secondary FITC- or TRITC-conjugated antibodies (1:200;
Invitrogen) diluted in PBS. After washing with PBST twice,
the cells were incubated with 4′ , 6-diamidino-2-phenlindole dihydrochloride and then viewed under a fluorescence microscope
(Zhang and Wu, 2009).
For AP staining, undifferentiated fESLCs were subjected to an
Alkaline Phosphatase Detection Kit (Millipore) following the manufacturer’s instructions. Briefly, fESLCs growing as colonies were
fixed with 4% PFA in PBS for 2 min, washed three times with PBS,
stained, and then examined under a microscope.
Karyotype analysis
The fESLCs cultured in ESC medium were incubated with colchicine
(100 ng/ml; Sigma) for 3 h. After dissociation to single cell suspension, the cells were incubated with 75 mM KCl for 15 min. Then a solution of methanol:acetic acid (3:1) was added to the cells, followed
by incubation for 30 min. The cells were transferred to slides and airdried for 3–4 days and then stained with Giemsa. Twenty to 30 metaphase spreads were counted for each karyotype analysis.
Bisulfite sequencing analysis
After culturing fESLCs in feeder-free gelatin-coated plates for 2
days, genomic DNA was extracted from the cells. Conversion of
unmethylated cytosines was performed using an EZ DNA
Methylation-Gold KitTM (ZYMO Research). Genomic DNA (15–
20 ng) was used for bisulfite treatment. The converted DNA was
amplified by PCR using specific nested and primary primers
(Supplementary Table S1). PCR products were gel purified,
cloned into a TA vector, sequenced, and then analyzed by methylation analysis software (Xu et al., 2013)
Lineage-specific differentiation of fESLCs
For differentiation of fESLCs into endodermal, mesodermal, and
ectodermal lineages, standard protocols for mouse ESCs were
applied (Wernig et al., 2007). Briefly, feeder cells were removed
by differential sedimentation at 378C for 20 min. Then, the fESLCs
were induced to form EBs. Approximately 400 fESLCs in 20 ml differentiation medium were placed under the lid of a petri dish containing 3 ml PBS and incubated as hanging drops for 3 days. After EB
formation, single EB was plated on a gelatin-coated 24-well plate
or 35-mm culture dish for differentiation. For endodermal differentiation, EBs were transferred onto 0.1% gelatin-coated dishes and
cultured in DMEM supplemented with 0.5% FBS, 5 mg/ml insulin,
10 mg/ml transferrin, and 1 mM glutamine for 3 days. The cells
were then cultured in DMEM/F12 containing 1 mM glutamine and
10 ng/ml bFGF for 5 days, followed by DMEM/F12 containing
50 ng/ml Activin A for another 5 days. For mesodermal differentiation, EBs were cultured in DMEM containing 10% FBS and
50 ng/ml bone morphogenetic protein 4 for 5 days. The EBs were
then plated on gelatin-coated dishes in DMEM containing 20%
FBS and cultured for 10 days. For ectodermal differentiation, EBs
were transferred to dishes and cultured in DMEM/F12 containing
10% FBS, and 1025 M retinoic acid (RA) for 3 days. RA was then
omitted from medium, and the cells were cultured for an additional
Conversion of female germline stem cells into pluripotent stem cells
5 days. After differentiation or at various time points, the cells were
collected and analyzed by RT – PCR and immunofluorescence.
Teratoma formation
fESLC colonies were grown in ESC medium for at least 20 days
and then dispersed into single cells by 0.25% trypsin. Nude mice
were injected subcutaneously with fESLCs (2 × 106) using a 30 G
needle. Tumor formation was monitored daily. After 35 days, palpable tumors were detectable. The mice were sacrificed, and teratomas were removed, fixed in 4% PFA, and embedded in paraffin for
sectioning and hematoxylin/eosin staining.
Chimera formation assay
To further confirm the pluripotency of fESLCs, chimera analysis
was performed to investigate their capability to contribute to all
three germ layers. Before the chimera assay, fESLCs were transduced with lentiviral vectors to express enhanced green fluorescent protein (EGFP) for tracking. EGFP-positive fESLCs were
sorted by FACS. Wild-type blastocysts (3.5 dpc) were collected
from the uteri of C57BL/6 mice, washed, and then cultured in a
5% CO2 incubator. Approximately 10 – 15 EGFP-labeled fESLCs
were microinjected into each blastocyst following standard procedures. After culturing for several hours, the blastocysts were transplanted into the uterine horn of pseudopregnant recipient female
mice. Genomic DNA of various tissues and organs was extracted
from litters and analyzed for EGFP expression by genomic PCR
and fluorescence microscopy.
Supplementary material
Supplementary material is available at Journal of Molecular Cell
Biology online.
Funding
This work was supported by National Basic Research Program of
China (2013CB967401 and 2010CB945001), and the National
Nature Science Foundation of China (81370675 and 81121001).
Conflict of interest: none declared.
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