CLONING AND STEM CELLS

Volume 9, Number 2, 2007

Mary Ann Liebert, Inc.
DOI: 10.1089/clo.2006.0070

Primate Embryonic Stem Cells Proceed to Early

Gametogenesis In Vitro
TAKESHI TERAMURA,1 TOSHIYUKI TAKEHARA,2 NOBUYUKI KAWATA,2
NAHOKO FUJINAMI,2 TASUKU MITANI,3 MAKOTO TAKENOSHITA,4
KAZUYA MATSUMOTO,2 KAZUHIRO SAEKI,2 AKIRA IRITANI,2
NORIMASA SAGAWA,1 and YOSHIHIKO HOSOI2

ABSTRACT
Embryonic stem cells (ESCs) of nonhuman primates are important for research into human
gametogenesis because of similarities between the embryos and fetuses of nonhuman primates and those of humans. Recently, the formation of germ cells from mouse ESCs in vitro
has been reported. In this study, we established cynomolgus monkey ES cell lines (cyESCs)
and attempted to induce their differentiation into germ cells to obtain further information on
the development of primate germ cells by observing the markers specific to germ cells. Three
cyESCs were newly established and confirmed to be pluripotent. When the cells are induced
to differentiate, the transcripts of Vasa and some meiotic markers were expressed. VASA protein accumulated in differentiated cell clumps and VASA-positive cells gathered in clumps
as the number of differentiation days increased. In the later stages, VASA-positive clumps
coexpressed OCT-4, suggesting that these cells might correspond to early gonocytes at the
postmigration stage. Furthermore, meiosis-specific gene expression was also observed. These
results demonstrate that cyESCs can differentiate to developing germ cells such as primordial germ cells (PGCs) or more developed gonocytes in our differentiation systems, and may
be a suitable model for studying the mechanisms of primate germ cell development.

INTRODUCTION

MBRYONIC STEM CELLS (ESCs) are derived from

the inner cell mass (ICM) of blastocysts prior
to the formation of the epiblast. ESCs can form
all cell lineages when introduced into blastocysts
or give rise to various somatic cell lineages in culture (Evans and Kaufman, 1981; Extavour and
Akam, 2003). In the past decade, ESCs have also
been obtained from nonhuman primate and hu-

man embryos (Thomson and Kalishman, 1998;

Sasaki et al., 2005; Suemori et al., 2001). Human
ES cells (hESCs) are potentially valuable for the
provision of therapeutic cells whose growth and
differentiation can be controlled for the treatment
of disease (Hook et al., 2005; Liew et al., 2005).
However, for ESC-based therapy to become a
clinical reality, translational research involving
nonhuman primates is essential because of the
many regulations and ethical considerations sur-

1Department

of Obstetrics and Gynecology, Graduate School of Medicine, Mie University, Mie, Japan
of Genetic Engineering, Graduate School of Biology Oriented Science and Technology, Kinki University, Wakayama, Japan.
3Institute of Advanced Technology, Kinki University, Wakayama, Japan.
4Wakayama Research Institute, Keari Co., Ltd, Wakayama Japan.
2Department

144

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GAMETOGENESIS FROM PRIMATE ESCs

rounding the use of hESCs. Recently, primate

ESCs have been well characterized, not only in
terms of pluripotency and genetic stability. However, the extent of diversity among these, including humans, is currently unknown.
Primate ESCs differ from mouse ESCs in
colony morphology, growth requirements, and
molecular signatures at various defining developmental stages. For example, leukemia-inhibiting factor (LIF), a component of the STAT-3 signaling pathway indispensable for maintaining an
undifferentiated state, is essential for the culture
of mouse ESCs, but not primate ESCs (Daheron
et al., 2004; Kristensen et al., 2005; Sumi et al.,
2004). To better understand the characteristics of
hESCs, ESCs derived from nonhuman primates
are a useful resource for preclinical research.
Recently, three different groups reported gametes being produced from mouse ESCs in vitro
(Geijsen et al., 2004; Hubner et al., 2003; Toyooka
et al., 2003). These studies have certified that male
and female gametes can be produced in vitro from
ESCs. Hubner et al. (2003) succeeded in producing oocytes from mouse ESCs. These induced
oocytes underwent spontaneous activation and
developed blastocyst-like features. The researchers obtained these structures by sorting primary germ cell fractions spontaneously arising in
cultures, using Oct-4 and c-kit as markers, and
then continuing to culture. Oct-4 is a member of
the POU family of transcription factors, and is expressed in the inner cell mass of blastocysts,
pluripotent ESCs, and embryonic germ cells. After implantation, expression remains only in
PGCs and early gonocytes in vivo (Rajpert-De
Meyts et al. 2004). c-kit is a member of the PDGF
autophosphorylating tyrosine kinase receptor
family, the members of which are expressed on
the surface of germ cells and are essential for PGC
migration or germ cell differentiation (Hutt et al.
2006; Matsui et al. 1990). On the other hand, Toyooka et al. isolated PGC-like cells from differentiated male knock-in ESCs in which GFP/LacZ
was inserted into the mouse Vasa gene. Vasa is a
homeobox gene first isolated in Drosophila, and
expressed in germ cells specifically, and thus in
their experiment the cells expressing GFP or LacZ
also expressed mouse Vasa and differentiated into
germ cells. They succeeded in producing sperm
by isolation of Vasa-positive PGC-like cells from
embryoid bodies (EBs) and subsequent transplantation into testis. Geijisen et al. (2004) confirmed the differentiation of germ cells in vitro

based on the positive expression of markers such

as Oct-4, Piwil2, and Dazl. The differentiated
PGC-like cells underwent limited meiosis, and
were able to fertilize mature oocytes by intracytoplasmic sperm injection) (ICSI). These differentiated germ cells required no specific or significant modification in culture, suggesting that
germ-cell differentiation from ES cells may primarily be a spontaneous event.
In primates, Clark et al. (2004) induced EB formation from hESCs and observed the expression
of Vasa and Scp3, neither of which were expressed
in undifferentiated ESCs or ICM (Clark et al.,
2004). Studying human gametogenesis in culture
is important to understand the biological events
related to germ cell proliferation and differentiation. Even more important is translating the information into improvements in infertility treatment. The use of ESCs to study germ cell
differentiation is indispensable; however, the use
of hESCs precludes observation of development
after fertilization and implantation.
Thus, we constructed nonhuman primate systems for induction of germ cell development and
then examined the mechanisms of this development using cyESCs. We then assessed expression
of the RNA and protein of the germ cell marker
Vasa and other germ cell markers after induction
of differentiation.

MATERIALS AND METHODS

Animals
Mature male and female cynomolgus monkeys
housed individually in cages were used in this
study. All procedures were approved by the Institutional Animal Care and Use Committee at
Kinki University, Japan.

Ovarian stimulation, recovery of oocytes,

injection of sperm, and embryonic culture
Female monkeys were stimulated with 1.8 mg
GnRH agonist (Takeda Pharmaceutical. Co. Ltd.,
Osaka, Japan), and then 2 weeks later the females
were treated with 45 IU per kg PMSG (Serotropin;
Teikoku-Zouki Pharmaceutical. Co. Ltd., Tokyo,
Japan) daily for 9 days, and finally 1000 IU of
hCG(Puberogen; SankyoYell. Co. Ltd., Tokyo,
Japan). Ovarian development was monitored by
ultrasonography (Curnow et al., 2002; Ng et al.,
2002; Sankai, 2000). The oocytes were retrieved

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using laparoscopic control and general anesthesia. Stripped oocytes were placed in CMRL
medium (Invitrogen Corporation, Carlsbad, CA)
modified with 10% fetal calf serum (FCS; JRH Biosciences, Lenexa, KS), 10 mM L-glutamine, 5 mM
pyruvate, 1 mM sodium lactate, 100 IU of penicillin, and 100 mg/mL of streptomycin (mCMRL)
at 37C in 5% CO2, 5%O2, and 90% N2 until experimentation. The preimplantation embryos
were obtained by ICSI using freeze-thawed
sperm and cultured until the blastocyst stage in
mCMRL medium.

Establishment of cyESCs
Inner cell masses were obtained from blastocysts using immunosurgery. After removal of
zonapellucidae by treatment with 0.5% Pronase
(Roche Diagnostics, Basel, Switzerland), embryos
were cultured in a 50-L aliquot of 10% FCS-supplemented DMEM (Invitrogen Corporation) containing 5 L of rabbit anticynomolgus monkey antiserum for 20 min. The embryos were then
transferred to 5 L of guinea pig serum for 20 min.
The isolated ICMs were individually seeded onto
culture dishes containing a layer of mitomycin C
(Invitrogen Corporation)-treated mouse embryonic fibroblast (MEF) feeder cells in ES medium
composed of Knockout-DMEM (Invitrogen Corporation), 1 mM L-glutamine, 0.1 mM -mercaptoethanol (both from Sigma-Aldrich, Inc., St.
Louis, MO), and 0.1 mM MEM nonessential amino
acid solution (Invitrogen Corporation), and supplemented with 4 ng/mL of human recombinant
bFGF (Upstate, Lake Placid, NY) in 20% Knockout serum replacement (Invitrogen Corporation).
The MEF cells were prepared from C57BL/6J
(SLC Co. Ltd., Shizuoka, Japan) fetuses at 13.5
days postcoitum, and used as feeder cells within
three passages. After 10 days, the ICMs developed
morphologically into embryonic stem cell-like
colonies. The putative ES cells were mechanically
transplated onto new dishes with MEF feeder
cells. After the first passage, colonies with an ES
cell-like morphology were selected for propagation, characterization, and storage. All manipulations such as subcloning or routine passage were
also performed by mechanical methods.

Immunocytochemistry of cyESCs
for characterization
The cyESC cultures were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS)

TERAMURA ET AL.

(4% PFA; pH 7.4). For immunocytochemical

staining of OCT-4 and NANOG, fixed ESCs were
then permiabilized in 0.5% Triton-X (SigmaAldrich) diluted in PBS (0.5% PBT) for 5 min,
blocked by incubation in 5% skim milk (SigmaAldrich) diluted in PBS for 1 h, and then incubated with primary antibody overnight at 4C.
The antibodies were anti-OCT-4 rabbit IgG polyclonal antibody (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA) and anti-NANOG rabbit IgG
polyclonal antibody (Chemicon International Inc,
Temecula, CA) diluted 1/100 in 0.1% 0.1% PBT.
For immunostaining of cell-surface molecules,
such as SSEA-1. SSEA-4, TRAI-60, and TRA1-81,
fixed cells were washed with 0.5% BSA (SigmaAldrich) in PBS and blocked by incubation in 5%
skim milk for 1 h. The antibodies were anti-SSEA1 mouse monoclonal IgM antibody (Santa Cruz
Biotechnology), and SSEA-4, TRA1-60, and
TRA1-81 mouse monoclonal IgM antibodies
(Chemicon International) diluted 1/100 in 0.5%
bovine serum albumin (BSA)/PBS. For immunofluorescence microscopy of OCT-4 and NANOG
immunostained samples, sections were incubated
for 1 h at room temperature with rhodamine-conjugated bovine antirabbit IgG antibody (Santa
Cruz Biotechnology) diluted 1/1000 in 0.1% PBT
to detect OCT-4 and NANOG. For observation of
SSEA-1, SSEA-4, and TRA1-60, samples were incubated for 1 h at room temperature with FITCconjugated goat antimouse IgM antibody (Santa
Cruz Biotechnology). For detection of TRA1-81,
we used Cy3-conjugated goat antimouse IgM antibody (Chemicon International) as the secondary
antibody. Samples were then washed three times
for 10 min each in PBS, and incubated in DAPI
(Vector Laboratories, Ltd., Peterborough, England) to counterstain chromosomes. These samples were mounted with Vectorshield mounting
medium (Vector Laboratories, Ltd.) to avoid signal reduction. For alkaline phosphatase (ALP)
staining, we used an ALP kit (Sigma-Aldrich) according to the instructions provided.

Genomic PCR for sex determination

The cyESCs were collected in sampling tubules
and lysed in sodium dodecyl sulfate and EDTA
buffer containing 0.1 mg/mL proteinase K (Roche
Diagnostics) at 55C overnight. PCR amplification of the sequence of the sex-determining region Y (SRY) gene of the cynomolgus monkey
was performed at 95C for 2 min followed by 35

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GAMETOGENESIS FROM PRIMATE ESCs

cycles of 94C, 30 sec; 58C, 30 sec; and 72C, 30

sec using Platinum Taq PCRx DNA polymerase
(Invitrogen Corporation) according to the manufactures instructions The primers were sequences from homologous regions between human and mouse SRY genes. The primer set for
cloning and sequencing was 5-attcctgctctccggagaag-3 and 5-ttgtcgagtggctgtagcgg-3. The
primers for sex determination of cyESCs were
taken from the sequence; Sry, 5-tgtagctctaagtatcagtgtg-3 and 5- agtgcgtggctttggtacag-3. The
putative actin genomic sequences that are homologous between the human genome and the
Macaca fascicularis (cynomolgus monkey) putative actin cDNA (NCBI accession No. AB170391);
Actin, 5-tgacttagttgcgttacacc-3 and 5-agtcagtgtacaggtaagcc-3. We used a cynomolgus testis and
ovary as the positive and negative controls.

Induction of teratoma formation

ESC colonies were injected into the kidney capsule of severe combined immunodeficient (SCID)
mice (Charles River Laboratories Japan, Inc.,
Kanagawa, Japan). At 14 days postinjection, teratomas were recovered and embedded with Tissue-tek O.C.T freezing compound (Sakura
Finetechnical. Co., Ltd, Tokyo, Japan). Cryosections of the teratomas were produced using a
Cryostat CM1800 (Leica, Heidelberg, Germany)
and differentiation capacity evaluated after staining with hematoxylin and eosin (both reagents
from Sigma-Aldrich).

lined below. Primers for the evaluations were Afp

(NCBI accession No. AB158630), 5-atgaaacatatgtccctcctg-3 and 5-tgttcctctgttatttgtggc-3; Desmin
(NCBI accession No. AB168811), 5-tacacctgcgagattgatgc-3 and 5-aggctgtgatgggaggactg-3; and
Neurofilament light polypeptide 68kDa (NF-L, NCBI
accession No. AB170435), 5-tactcggcgccggtgtcttc3 and 5-agcgatttcgtccatcaagc-3.

RNA isolation and reverse transcription

At days 0 (ESCs), 2, 4, 6, 8, 10, 12, 14, and 16,
EBs were collected, centrifuged, and stored in liquid nitrogen for RNA extraction. Total RNA was
extracted using TRIzol (Invitrogen Corporation)
according to the manufactures instructions. Single-strand cDNA was prepared from total RNA
using an oligo-dT primer under standard conditions with the Superscript III reverse transcriptase (Invitrogen Corporation). The cDNA from
each sample was diluted and used for an RTPCR-based assay for Nanog, Oct-4, Vasa, Dmc1,
Scp3, and Gapdh. PCR with total cDNA was performed using Hotmaster-taq (Eppendorf, Hamburg, Germany) and appropriate primers (Table
1). All primers for PCR were designed on two different putative exons so as to span one intron or
designed to span a putative exonexon junction.
The reaction profile was 2 min at 94C followed
by 35 cycles of 94C, 20 sec; 58C, 10 sec; and 70C,
20 sec. Primer designs were based on the sequence of cynomolgus monkey homologs of each
gene. Primers for cloning and sequencing were

Evaluation of in vitro differentiation potency

To induce EB formation, the cyESC colonies
were clipped up using fine needles and cultured
on nonadherent dishes in 10% FCS-supplemented DMEM. The EBs were plated onto
gelatin-coated dishes at day 16 postdifferentiation. After 10 more days of culture, the cells were
fixed in 4%PFA, permiabilized by PBT, blocked
with skim milk, washed, and immunostained as
previously described using antineuronal class III
-tublin (TuJ) monoclonal antibody (Covance Research Products, Richmond, CA) and anticardiac
troponin T monoclonal antibody (Abcam, Cambridge, UK). For immunofluorescent observation,
we used FITC-conjugated goat antimouse IgG antibody (Santa Cruz Biotechnology) and rhodamine-conjugated goat anti mouse IgG antibody
(Santa Cruz Biotechnology). The method of RTPCR for evaluation of differentiated cells is out-

TABLE 1.

PRIMER SEQUENCES CONSTRUCTED

FOR THE EXPERIMENT

Primer

Subject

Forward

Nanog
Nanog
Oct-4
Oct-4
Vasa
Vasa
Vasa
Dmc 1
Dmc 1
Dmc 1
Sycp3
Gapdh
-actin

Sequence
RT-PCR
Sequence
RT-PCR
Sequence
RT-PCR
Real time PCR
Sequence
RT-PCR
Real time PCR
Sequence/RT-PCR
RT-PCR
Real-time PCR

CCTGATTCTTCTAC
ACTCAAATACCTAC
GCCAAGCTCCTGAA
TGAAGCAGAAGA
GTGCTCAAACAG
CTACTCCTGGAA
AAGTTAATTTCTTG
TCTTTGTTTCAAG
GTGTGACAGCTCA
ATTCAATAATGGCA
TCTAGAATTGTTCA
GTGAAGGTCGGA
CCAGAGCAAGAGA

aThe

sequences are arranged by 5-sides to 3 -sides.

primers were constructed in the regions prepared
using sequence primers.
bThe

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designed on conserved sequences in mice, humans, and rats. After PCR, amplicons were
cloned using the pGEM-T Easy Vector System
(Promega Corporation, Madison, WI) and then
sequenced using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster
City, CA).

TERAMURA ET AL.

RESULTS
ICSI and embryonic culture
We collected 105 oocytes from superovulated
females. By sperm injection, 57 (54%) embryos
successfully developed, and 31 (30%) of these
reached the blastocyst stage.

Quantitative PCR

Establishment and characteristics of cyESCs

Quantitative PCR with cyESCs and EBs

cDNA was performed using Perfect Real-Time
SYBR green (Takara Bio, Inc., Shiga, Japan). The
PCR amplifications were performed with the
7300 Real-Time PCR System (Applied Biosystems) at 95.C for 5 min followed by 35 cycles of
95C, 30 sec; 60C, 30 sec; and 72C, 30 sec. Reactions were replicated three times for two different samples independently differentiated
and prepared. The results were analyzed using
7300 system SDS software (Applied Biosystems)
and Microsoft Excel (Microsoft Corporation,
Redmond, WA). All experiments included negative controls consisting of no cDNA for each
primer pair. Primers were designed to span exons to distinguish cDNA from genomic DNA
products.

In this experiment, we used 11 of the 31 blastocysts to establish cyESCs and the remaining 20
embryos were cryopreserved.
To establish the cyESCs, we conducted immunosurgical isolation and mechanical maintenance, which maintains rhesus monkey ESCs
undifferentiated and karyotypically normal
(Hanson and Caisander, 2005; Mitalipova et al.,
2005; Pau and Wolf, 2004). As a result of immunosurgery, six ICMs were successfully obtained from all the embryos used (55%), and all
developed on feeder layers. The primary outgrowths consisted of various genealogical cells, a
few of which had an undifferentiated shape. Finally, three cyESC-lines were established by repeated transfer of undifferentiated fractions and
selective culturing (27%).
The rhesus monkey ESC expresses pluripotent
markers, including SSEA-3 and SSEA-4, glycoproteins TRA1-60, and TRA1-81 (Thomson et al.,
1998), ALP activity, and transcription factors Oct4 and Rex-1 (Ginis et al., 2004; Rogers et al., 1991)
in an undifferentiated state. In contrast, the expression of markers such as SSEA-1 and Sox-2
(Ambrosetti et al., 2000; Yuan et al., 1995) indicates the differentiated state of ESCs in primates.
Moreover, SSEA-1 and SSEA-3 are negative in
some cyESCs (Suemori et al., 2001). Thus, we examined ALP activity, Oct-4 and Nanog expression, and SSEA-4, TRA1-60, and TRA1-81 expression to evaluate the undifferentiated state,
and used SSEA-1 as a negative marker for characterization of our established cyESCs. Our
strains showed the same characteristics as previous pluripotent cyESCs, and expressed Oct-4,
Nanog, SSEA-4, TRA1-60, and TRA1-81 (Figs. 1A
and C). SSEA-1 was not observed (data not
shown). PCR-based sex determination revealed
all three cell lines to be male (Fig. 1C). Similar to
the case in humans, cyESCs may be vulnerable to
enzymatic treatment, and difficult to culture
when dispersed into single cells (Hanson and
Caisander, 2005; Mitalipova et al., 2005). How-

Immunohistochemistry for cryosections

The gonadal tissues of adult cynomolgus
monkeys and differentiated EBs were fixed in
4% PFA, and embedded in tissue-tek compound. The tissues were cut into 8-m pieces
and the EBs were cut into 5-m serial sections.
Slides were washed in PBS for 15 min and
blocked by incubation in skim milk for 1 h. The
slides were then incubated with primary antibody overnight at 4C. The primary antibodies
were rabbit antihuman VASA polyclonal antibody (R&D systems, Inc., Minneapolis, MN)
used at 1/100 and anti-OCT-4 rabbit IgG polyclonal antibody diluted 1/100 with 0.1% PBT.
For immunofluorescence, sections were incubated for 1 h at room temperature with rhodamine-conjugated anti-goat IgG antibody
(Santa Cruz Biotechnology) to detect VASA, or
with FITC-conjugated antirabbit IgG antibody
following incubation with the primary antibodies. Sections were then washed twice for 10
min in 0.1% PBT, and incubated in DAPI. Sections were mounted under glass coverslips using Vectorshield mounting medium.

FIG. 1. Characteristics of cynomolgus ESDs (cyESCs). (A) Chemical and immunocytochemical characterization of established ESCs. CYK1, BS1, and MEC1 expressed all ESC markers. Scale bars: 100 m. (B) Evaluation of pluripotency in cyESCs
by in vivo and in vitro differentiation. All cell lines formed teratomas in the kidney capsules of sCID mice. Teratomas derived from CYK1 (B-a), BS1 (B-b), and MEC1 (B-c). Scale bars: 200 m. When EBs formed by transferring floated cultures
of CYK1 to cell culture dishes, various kinds of cells were derived. (B-d) shows nerve cells (ectoderm) immunostained with
antineuronal class III -tubulin antibody. (B-e) Putative cardiomyocytes (mesoderm) immunostained with anticardiac troponin T antibody. Scale bars: 50 m. (B-f) Evaluation of the differentiation potency to three embryonic layers by RT-PCR.
Positive control (PC) of Afp is a CYK1 derived teratoma, PC of desmin is a muscle and PC of Nf-L is a brain. Negative controls (NC) of all lanes are cynomolgus monkey fibroblast cells. (C) PCR-based characterization of cyESCs. The upper panel
shows RT-PCR analysis for confirmation of the pluripotency of cyESCs. Nanog and Oct-4 were expressed in all ES cell lines,
and also in the testis, as reported in other animals such as humans. In the ovary, Oct-4 was weakly expressed, but Nanog
expression was not detected. The lower panel shows sex determination by genomic-PCR of the Sry gene. All cells had putative sry sequences, and the cells lines are all male.

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ever, all three cell lines underwent over 70 passages, and 50% of the cells maintained the normal chromosome number of 42 in over 50 passages when maintained mechanically. These
cyESCs formed teratomas when injected into the
kidney capsule of SCID mice (Fig. 1B-a to -c). Furthermore, one of these lines, CYK1, was able to
differentiate into neural cells and putative-cardiomyocytes (Fig. 1B-d to -f), and expressed Afp
(expressed in early endoderm), Desmin (expressed in mesodermal cells), and Nf-L (expressed in early neural cells) after differentiation.
Thus, our cyESC lines possess differentiation potency to three embryonic layers, and also good
potential for differentiation into germ cells.

ESCs germinal differentiation

We observed Vasa, Dmc1, and Scp3 as germ
cell-specific markers, and OCT-4 protein as a
supporting marker. VASA protein was detected
in male gonocytes in the seminiferous tubules
and oocytes in preantral follicles (Fig. 2A). Dmc1
is detected only in spermatocytes during meiosis
in mouse (Yoshida et al., 1998) and Scp3 is one of
the axial/lateral elements of the synaptonemal
complex and functions to attach chromatin loops
(Yuan et al., 2002). Thus, they are superior markers for the detection of more advanced stages in
germ cell development. Oct-4 is expressed in both
undifferentiated ESCs and early germ cells, and
we premised the protein as supported markers
for germ cell foundation.
Upon in vitro differentiation, we selected the
cell lines CYK1 and BS1, as these two lines show
good reproducibility in the timing of EB development and EB morphology. Vasa mRNA expression was detected at differentiation day 4, in-

TERAMURA ET AL.

creased until day 8, and continued to at least day

16 (Fig. 2B-a). VASA protein was first observed
between day 4 to day 8 in a scattered pattern over
a broad area, in about 30% of EBs. At the later
stages, the number of VASA-positive cells had reduced to 10%, and the cells were localized with
promotion of differentiation until day 16 (Fig. 2Bb). To confirm germ cell foundation, we performed double staining with OCT-4 in day 16
EBs. Interestingly, OCT-4 expression was also observed in the area where the cells expressing
VASA were concentrated (Fig. 3A). Higher magnification showed that the cells expressing both
VASA and OCT-4 were located on the surfaces of
the EBs, and were compactly colonized (Fig. 3B).
Furthermore, to determine whether more developed cells were present in the EBs, we examined
the expression of the meiotic markers Scp3 and
Dmc1. Both markers were expressed on differentiation day 16 (Fig. 4a). Quantitative analysis for
Vasa and Dmc1 mRNA revealed that the genes
were expressed at significantly higher levels compared with the undifferentiated state in day 16
EBs, to the same high levels as in the ovary. These
results suggest that some cells in EBs might be in
meiosis at day 16.

DISCUSSION
In the present study we found the same expression patterns of Vasa as seen in other animals
in cryosections of the gonads. In the cynomolgus
monkey, VASA proteins are observed in the seminiferous tubules and oocytes in preantral follicles, as in the mouse and humans. Vasa is a member of the DEAD-box family of proteins of

FIG. 2. Vasa expression in gonadal tissue and EBs. (A) Gonadal tissue-specific expression of VASA. VASA expression in a cynomolgus monkey testis and ovary. VASA was detected as red fluorescent images. The proteins were detected in germ cells in the testis (A-a to A-d). (A-a) shows the DIC image of a seminiferous tubule. (A-b) DAPI stained
nuclei, (A-c) depicts the fluorescent images by anti-VASA antibody and rhodamine-conjugated secondary antibody.
(A-d) A merging of the images. In the ovary, the proteins were recognized in ooplasm in developing follicles (A-e to
A-h). (A-e) DIC, (A-f) DAPI stained nuclei. (A-g) fluorescent images of VAS containing oocytes, and (A-h) merged
image. In both cases, VASA was expressed in the cytoplasm. Scale bars: 50 m. (B) Vasa expressions in EBs. (B-a)
Transition of Vasa mRNA quantity accompanied with progression of EB development by real-time PCR analysis using total cDNA from EBs. Black lines show the results for BS1, and gray lines show the results for CYK1. The Y-axis
represents relative values for the quantity of -actin (Actinb) mRNA as an internal control. The error bars show SE.
Each point depicts the standard scores calculated from three independent experiments and more than two replications for each sample. (B-b) Immunocytochemical observation of VASA-positive cells in cryosections of EBs formed
from CYK1 on each differentiation day. VASA signals are shown as red fluorescent images. At differentiation day 4
(data not shown) to day 8, VASA was weakly expressed in many cells contained in the EBs. When differentiation
reached day 12, mainly surface cells strongly expressed VASA and localization became clear, as in some stages of
gonocytes in the testis. Scale bars: 50 m.

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GAMETOGENESIS FROM PRIMATE ESCs

FIG. 2.

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ATP-dependent RNA helicase, and is conserved

in invertebrate and vertebrate species alike, including Caenorhabditis elegans, Xenopus laevis, Mus
musculus, and Homo sapiens (Noce et al., 2001). In
all species, Vasa homologous genes are specifically expressed in the cytoplasm of germ cells undergoing the gametogenic process until the postmeiotic stage. Our results suggest that the
specificity and localization of VASA protein is
also conserved in the cynomolgus monkey, and
that VASA is a specific marker of germ cell foundation in our system.
After differentiation induction, we detected
Vasa mRNA expression at differentiation day 4,
and expression increased until differentiation day
8. The quantity of Vasa transcript was then maintained until differentiation day 16. On differentiation days 12 to 16, we observed no remarked
changes in the number of VASA protein-expressing cells. Interestingly though, there was a
localization change in the VASA-expressing cells.
The VASA-positive cells were scattered at the beginning, but then became focused as the number
of differentiation days increased. To observe transitions of putative germ cells in vitro, we performed double staining with VASA and OCT-4.
In vivo, VASA is first detected in PGCs during
colonization of the gonadal ridge in the mouse
and humans (Castrillon et al., 2000; Noce et al.,
2001). OCT-4 is a transcription factor that is expressed in pluripotent ESCs and only in undifferentiated gonocytes. In the present study, OCT4 signals were also detected in the region where
VASA-positive cells had concentrated. In humans, OCT-4 is mainly immunolocalized at 79
weeks postgestation (Gaskell et al., 2004). Thus,
cells expressing both VASA and OCT-4 may be
consistent with postmigrated PGCs or early
gonocytes. We speculate that germ cell determination might be complete at around 8 days after
differentiation in vitro, and that only cells with
continued VASA and OCT-4 expression are destined to germ lineage and possibly colonization
as primary germ cells. In the present study, we
did not detect other cell types such as VASA-positive/OCT-4-negative or VASA-negative/OCT-4positive cells. In addition, VASA-negative/OCT-

TERAMURA ET AL.

4-negative cells, which are reported, attach to

VASA-positive colonies as observed in a previous experiment in mice (Hubner et al., 2003). It is
possible that germ cell determinations are complete in day 16 EBs, but a large number of these
cells did not proceed to the next stage. Another
possibility is that longer culture is required to
produce more differentiated cells.
To investigate whether more developed germ
cells exist in the EBs, we analyzed the expression
of two meiotic markers. The transcription of Scp3
and Dmc1 was observed at differentiation day 16.
Vasa, Dmc1, and Scp3 mRNA were all expressed,
suggesting the formation of various types of germ
cells in meiosis in our differentiation system, although this was not observed immunocytochemically.
Importantly, VASA-expressing cells were detected from differentiation day 4 in early experiments. In humans, the interval spanning the arrival of PGCs corresponds to 67 weeks after
implantation, and VASA-positive PGCs are first
detected in the gonadal ridge at 7 weeks after implantation (Castrillon et al., 2000). Thus, the timing of appearance of primary germ cells in this
system is abnormally early. Also, in humans, it
has been reported that Vasa expression is initiated
considerably earlier in vitro than in vivo (Clark et
al., 2004).
Interestingly, PGCs appear at the first day after
differentiation when mouse ESCs are cocultured
with BMP4-expressing cells (Toyooka et al., 2003).
Some cytokines, such as BMP4 and BMP8b, which
are associated with germ cell induction from mouse
epiblasts in vivo (Okamura et al., 2005; Ying et al.,
2001), are expressed in trophectodermal cells. It is
known that hESCs can spontaneously differentiate
to a trophectodermal layer, unlike mouse ESCs
(Gerami-Naini et al., 2004; Tesar., 2005; Xu et al.,
2002). The cyESC are phylogenetically closely related to humans, and there is similarity with physiological characteristics such as flat colony morphology, loose cell aggregates, expression patterns
of some cell surface markers such as some stagespecific embryonic antigens and glycoproteins, and
instability when dispersed to single cells. More importantly, it is possible that the cyESCs have simi-

FIG. 3. Double staining with VASA and OCT-4 in cryosections of EBs. (A) The area colonized by VASA-expressing
cells was strongly immunostained by OCT-4 antibody. These pictures were obtained from day 16 EBs form CYK1.
Scale bars: 50 m. (B) High magnification of images of double straining. VASA expressing cells coexpressed OCT-4.
These cells were detected in cryosections of day 16 EBs of CYK1. Scale bars: 50 m.

FIG. 3.

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TERAMURA ET AL.

FIG. 4. Confirmation for more developed germ cell differentiation by RT-PCR for two meiotic markers. (a) Scp3 and
Dmc1 were expressed in day 16 EBs of both lines. Dmc1 was weakly expressed in the undifferentiated state. It is possible that some differentiated fractions of cyESCs or unusual expression events caused these results. (b) Comparison
of the quantity of Vasa and Dmc1 mRNA expression between tissue, cyESCs, and EBs. The Y-axis depicts relative values for the quantity of Actinb mRNA as the internal control. The error bars show SE. The rectangles depict the standard scores of three independent experiments and more than two replications for each sample.

lar differentiation potency to hESCs, and may develop trophectodermal layers as in humans. In fact,
cdx2, which is a marker of primary trophectodermal differentiation, was observed by spontaneous

differentiation in cynomolgus monkey ESCs (Yasuda et al., 2006).

A different interpretation is that the culture
conditions we used, involving numerous supple-

GAMETOGENESIS FROM PRIMATE ESCs

ments, produced an abnormal internal environment that may have influenced gene regulation,
thereby causing divergence from the in vivo developmental schedule. Another possibility is that
in EBs, which clearly differ in structure from a fetus, cell-to-cell interactions that may be necessary
for normal development may not work as they
should. To develop the system to better represent
in vivo conditions, we may need to coculture with
cells that secrete factors that promote the formation of germ cells at various developmental
stages. Further maturation, such as complete
meiosis or feature differentiation, might enable
these primitive cells to proceed to the next steps,
such as fertilization or development.
In conclusion, we have established pluripotent
ESCs from cynomolgus monkeys and constructed
a model system to induce putative germ cells that
show expression of the germ cell makers Vasa,
Dmc1, and Scp3. Furthermore, a change in localization, and coexpression of OCT-4 protein, was
also observed, as is seen in the mouse. cyESCs
and hESCs share some characteristics, and thus
these germ cell induction systems using cyESC
are important models to elucidate the mechanism
of early gametogenesis in humans. Effective germ
cell induction or completion of meiosis should
improve assisted reproductive medicine for infertile individuals.

ACKNOWLEDGMENTS
This study was supported by a Grant-in-Aid
for the 21st Century COE Program of the Japan
Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.
We thank Dr. Hirofumi Suemori and Dr. Norio
Nakatsuji, Department of Development and Differentiation, Institute for Frontier Medical Science, Kyoto University, for technical advice in
culture and handling of cynomolgus ESCs. We
also thank Dr. Toshiaki Noce, Mitsubishi Kagaku
Institute of Life Sciences, for advice on Vasa.

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Address reprint requests to:

Yoshihiko Hosoi
Department of Genetic Engineering
Graduate School of Biology Oriented Science
and Technology
Kinki University
Nishimitani 930
Kinokawa, Wakayama 649-6493, Japan
E-mail: hosoi@waka.kindai.ac.jp