EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS
Birth of Parthenote Mice Directly from Parthenogenetic Embryonic
Stem Cells
ZHISHENG CHEN,a,b ZHONG LIU,a JUNJIU HUANG,a,c TOMOKAZU AMANO,d CHAO LI,a SHANBO CAO,a CHAO WU,a
BODU LIU,a LINGJUN ZHOU,a MARK G. CARTER,d DAVID L. KEEFE,c XIANGZHONG YANG,d LIN LIUc,e
a
School of Life Science, Sun Yet-Sen University, Guangzhou, China; bCollege of Life Science, Foshan University,
Foshan, China; cDepartment of Obstetrics and Gynecology, University of South Florida College of Medicine,
Tampa, Florida; dCenter for Regenerative Biology and Department of Animal Science, University of Connecticut,
Storrs, Connecticut; eCollege of Life Sciences, Key Laboratory of Bioactive Materials of Ministry of Education,
Nankai University, Tianjin, China
Key Words. Parthenogenesis • ESC line • Tetraploid complementation • Mice
ABSTRACT
Mammalian parthenogenetic embryos are not viable and
die because of defects in placental development and
genomic imprinting. Parthenogenetic ESCs (pESCs)
derived from parthenogenetic embryos might advance regenerative medicine by avoiding immuno-rejection. However, previous reports suggest that pESCs may fail to
differentiate and contribute to some organs in chimeras,
including muscle and pancreas, and it remains unclear
whether pESCs themselves can form all tissue types in the
body. We found that derivation of pESCs is more efficient
than of ESCs derived from fertilized embryos, in association with reduced mitogen-activated protein kinase signal-
ing in parthenogenetic embryos and their inner cell mass
outgrowth. Furthermore, in vitro culture modifies the
expression of imprinted genes in pESCs, and these cells,
being functionally indistinguishable from fertilized
embryo-derived ESCs, can contribute to all organs in chimeras. Even more surprisingly, our study shows that live
parthenote pups were produced from pESCs through tetraploid embryo complementation, which contributes to
placenta development. This is the first demonstration that
pESCs are capable of full-term development and can differentiate into all cell types and functional organs in the
body. STEM CELLS 2009;27:2136–2145
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION
Parthenogenetically derived mammalian embryos, with no
paternal genome, are not viable and undergo spontaneous
resorption at midgestation, largely from defective placental
growth [1, 2] attributed to a lack of bi-parental imprinting [1,
3, 4]. Parthenogenetic mouse pups have been produced from
reconstructed parthenogenetic embryos after genetic modification of the imprinted loci Igf2 and H19 [5]. Parthenogenetic
ESCs (pESCs) derived from parthenogenetic embryos are capable of contributing to chimeras [6], although it remains
unclear whether pESCs themselves can form all tissue types in
the body, and the current hypothesis is that parthenogenetic
embryos cannot develop to term without genetic modification
[5]. Previously, pESCs derived from oocytes activated with 67% ethanol, which only triggers a single rise in Ca2þ, showed
limited developmental potential in mice [6–8]. In contrast,
multiple Ca2þ elevations induced by strontium (Sr2þ) treatment fully activate oocytes, similar to fertilization [9, 10], and
significantly enhance parthenogenetic embryo development
[11, 12]. Derivation of pESCs from fully activated oocytes by
Sr2þ may increase the pluripotency of pESCs. Sr2þ-activated
cytoplasts were used for somatic cell nuclear transfer to successfully produce cloned mice [13] and, when using pESCs
for nuclear transfer to regenerate pESCs (NT-enhanced pESC
or NT-pESC), contribution of pESC in the chimeras was considerably improved [14]. We generated new pESC lines, which
are shown indistinguishable from ESCs derived from fertilized
embryos (fESCs), and these pESCs can contribute to all organs
in chimeras. Surprisingly, through tetraploid embryos complementation (TEC) [15], these hybrid pESCs can develop to full
term without gene modification, showing that pESCs are capable of term development and can differentiate into all cell
types and functional organs in the body. Our report provides
scientific support for the possibility of female patient-specific
pESC-based therapeutic applications, because demonstration
of pESC pluripotency is critical to the feasibility of pESC
therapy in regenerative medicine [16, 17]. pESCs are a potential source of histocompatible tissues for transplantation [18],
Author contributions: L.L.: conception and design, financial support, final approval of manuscript; Z.C.: collection and assembly of data,
data analysis and interpretation, manuscript writing; J.H.: collection and assembly of data, data analysis and interpretation; Z.L., C.L.,
B.L., C.W., S.C., T.A.: collection and assembly of data; L.Z.: provision of study material; M.G.C.: collection and assembly of data, data
analysis and interpretation; D.L.K., X.Y., data analysis and interpretation, financial support, final approval of manuscript.
Correspondence: Lin Liu, Ph.D., College of Life Sciences, Nankai University, Tianjin 300071, China. Telephone: 86-22-23500752;
e-mail: [email protected] Received March 22, 2009; accepted for publication June 9, 2009; first published online in STEM CELLS
C AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1002/stem.158
EXPRESS June 19, 2009. V
STEM CELLS 2009;27:2136–2145 www.StemCells.com
Chen, Liu, Huang et al.
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which will not induce the immuno-rejection that may be generated by bi-parental genes in fESCs.
MATERIALS
AND
METHODS
Production of Parthenogenetic and
Fertilized Embryos
B6C3F1 female mice at 6-10 weeks of age were superovulated
with 5 IU pregnant mare serum gonadotrophin (Calbiochem, San
Diego, http://www.emdbiosciences.com), followed by 5 IU
human chorionic gonadotropin (hCG) 46-48 hours later. Oocytes
enclosed in cumulus masses were collected from oviduct ampullae 14 hours after hCG injection. Cumulus cells were removed by
pipetting, after a brief incubation in 0.03% hyaluronidase prepared in potassium simplex optimized medium (KSOMAA) containing 14 mM HEPES and 4 mM sodium bicarbonate (HKSOM).
Oocytes were reliably activated by a 4-hour treatment with 10
mM SrCl2 and cytochalasin D prepared in Ca2þ-free KSOM [19,
20]. Activated oocytes with two pronuclei were defined as diploid
parthenotes and cultured in 50-ll droplets of pre-equilibrated
KSOM, covered with mineral oil at 37 C in a humidified atmosphere of 6% CO2 in air for 4-5 days. To obtain fertilized F1
embryos, C57Bl/6 females were superovulated and mated individually with C3H/He males of proven fertility. Oviducts of successfully mated females were flushed 3.5 days after mating with
HKSOM using a 30-gauge needle, and blastocysts were obtained
to derive normal fESC lines.
Establishment and In Vitro Culture of ESC Lines
Blastocysts were transferred to mitomycin C-treated murine
embryonic fibroblast feeder (MEF) cell layers and cultured for 45 days in stem cell medium: 80% Knockout Dulbecco’s modified
Eagle’s medium (Gibco, Grand Island, NY, http://www.invitrogen.com) supplemented with 20% fetal bovine serum (FBS),
50 units/ml penicillin, 50 lg/ml streptomycin, 1 mM glutamine,
1% nonessential amino acid stock, 0.1 mM b-mercaptoethanol,
and 1,000 units/ml ESGRO leukemia inhibitory factor (LIF;
Chemicon International, Temecula, CA, http://www.chemicon.
com). Inner cell mass (ICM)-derived outgrowths were mechanically dissociated into clumps with 32-gauge needles and replated
on mitomycin C-treated feeder layers in fresh medium, designated
as P1. Mechanical dissociation was used for passaging until typical ES-like cells appeared on the feeder layer (P1–P3). Afterward,
propagation of colonies was accomplished by exposure to 0.25%
trypsin-EDTA. Fertilized B6C3F1 blastocysts were transferred to
feeder cell layers and cultured in ES-medium supplemented with
50 lM mitogen-activated protein kinase (MAPK) inhibitor
PD98059 (Cell Signaling Technology, Beverly, MA, http://
www.chemicon.com), which was replaced by standard ES medium when typical ES-like cells appeared. Otherwise, the method
used to derive B6C3F1 fESCs was basically the same as for
pESCs.
Production of Tetraploid Embryos for
pESC/fESC Complementation
Tetraploid embryos were produced by electrofusion of blastomeres at the two-cell stage. Two-cell embryos were induced to
fuse by two pulses of 1,200 V/cm DC for 50 ls, after an AC
pulse of 750 V/cm for 5 s, generated from an Eppendorf Multiporator. About 98% efficiency of fusion was routinely achieved 1
hour later. Fused embryos were cultured in KSOMAA to form
blastocysts. pESCs or fESCs were injected into tetraploid blastocysts as done for diploid blastocysts. Injected blastocysts were
transferred into the uteri of 2.5 d.p.c. pseudopregnant Kunming
(KM) mice.
Karyotype Analysis
Metaphase chromosomes were prepared by exposing cultured
cells to nocodazole (0.4 lg/ml) for 2 hours or colcemid (0.1 lg/
ml) for 1 hour, followed by hypotonic treatment with 75 mM
KCl solution, fixation with methanol:glacial acetic acid (3:1) and
spreading onto slides. The spreads were treated with 0.005% trypsin, stained with Giemsa and 20-30 separate metaphase spreads
were examined for each culture.
DNA Microsatellite Polymorphism Analysis
Polymerase chain reaction (PCR)-based haplotype analysis was
used to determine homo- or heterozygosity at specific loci in
established pESC and fESC lines, as well as in tissues from chimeric mice and pups produced by TEC. Microsatellite markers
D14mit5, D16mit4, and D18mit17 were used to confirm homozygosity in hybrid pESC lines C2 and C3. D1Mit46, D6Mit9,
D14Mit5, and D16Mit4 were used to assess pESC contribution in
pESC chimeras. pESC contribution in tetraploid embryo complemented pup T1 was measured using D8Mit4, D12Mit136,
D16Mit4, and DXMit1. Briefly, genomic DNA was extracted
from tissues collected from chimeras, recipient tissues, and donor
ESCs. By screening >36 microsatellite markers from Mouse Genome Informatics Website (The Jackson Laboratory, Bar Harbor,
ME, http://www.informatics.jax.org), some that are located on
different chromosomes were identified to be polymorphic in our
experiments, and therefore used for genotyping the chimeras.
Microsatellite analysis was performed by PCR amplification with
primers designed from conserved sequences flanking each marker
and subsequent electrophoresis using 15% polyacrylamide gels.
The gels were silver-stained and scanned, and the genotype was
determined. Ratios of pESC to fESC contribution in chimeras
were estimated by quantification of the density of the bands after
subtraction of the background noise using Bio-Rad Quantity One
software (Bio-Rad, Hercules, CA, http://www.bio-rad.com).
Teratoma Formation Assay
Approximately 2 106 ESCs were injected subcutaneously into
4-week-old immunodeficient nude mice to test for teratoma formation. Four weeks after injection, the mice were sacrificed and
the resulting teratomas were excised, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin-eosin for histological examination.
Chimera Production
Alkaline Phosphatase Staining and
Immunofluorescence Microscopy
Recipient blastocysts (3.5 day post coitum [d.p.c.]) were collected
from uteri of superovulated females. The collected blastocysts
were incubated in KSOMAA at 37 C in a humidified atmosphere
of 6% CO2 in air. Approximately 10-20 pESCs or fESCs were
injected into the blastocoels of blastocysts obtained from ICR
mice using a Piezo micromanipulator (PrimeTech, Ibaraki, Japan,
http://www.primetech.org). The blastocysts were transferred into
the uteri of 2.5 d.p.c. pseudo pregnant ICR mice 1-4 hours after
the ESC injection. Chimeras were initially identified by coat
color. The contribution of pESC in chimeras was determined by
standard DNA microsatellite analysis, as described below.
Alkaline phosphatase staining was performed using the Vector
blue kit from Vector Laboratories (DAKO, Glostrup, Denmark,
http://www.dako.com). For immunocytochemistry, cells were cultured in tissue culture grade 4-well plates for 2 days. The media
were removed, the cells washed twice in phosphate-buffered
saline (PBS) and fixed in freshly prepared 4% paraformaldehyde
in PBS for 15 minutes at 4 C. Cells were rinsed with PBS and
blocking buffer (3% goat serum in PBS) and permeabilized in
0.1% Triton X-100 in blocking buffer, washed three times, and
left in blocking solution for 1 hour at room temperature. Cells
were incubated overnight at 4 C with mouse monoclonal
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Pups Produced from Parthenogenetic ES Cells
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antibody OCT3/4, ERK2, pERK (Santa Cruz Biotechnology,
Santa Cruz, CA, http://www.scbt.com) or SSEA-1 (DSHB, The
University of Iowa), diluted 1:100 in blocking solution, washed,
incubated for 1 hour with Alexa Flur 568 (or 488)-conjugated
goat anti-mouse IgG or IgM (Molecular Probes), diluted 1:100 in
blocking solution, washed, and counterstained with 0.2 lg/ml
40 ,6-diamidino-2-phenylindole or Hoechst 33342 in Vectashield
mounting medium. Primary antibodies were omitted from control
samples. Fluorescent images were taken under a Leica microscope using appropriate filters (Leica, Germany, http://www.leicamicrosystems.com).
Western Blots
Cultured cells were washed twice with ice-cold PBS and lysed on
ice for 30 minutes in radioimmunoprecipitation buffer containing
protease inhibitors. The lysates were centrifuged at 20,000g for 30
minutes at 4 C, and the supernatant was collected. The protein
content was determined by Bradford protein assay. The lysates (20
ng protein) were separated by SDS-PAGE (10%) and transferred to
polyvinyldene fluoride membrane (Millipore, Bilerica, MA, http://
www.millipore.com), followed by incubation in PBS containing
0.05% Tween-20 and 2% block powder (Amersham Bioscience,
Piscataway, NJ, http://www.gelifesciences.com) overnight at 4 C.
The blots were incubated with mouse monoclonal antibody OCT3/
4, pERK1, pERK2 (1:200), rabbit polyclonal TERT (1:200; Santa
Cruz Biotechnology), or mouse monoclonal antibody b-actin
(1:200) for 2 hours at room temperature followed by incubation
with horseradish peroxidase-conjugated secondary antibodies
(1:10,000 goat-anti-mouse, goat-anti-rabbit; Pierce, Rockford, IL,
http://www.piercenet.com) for 45 minutes. The bands were
detected by chemiluminescence signals using ECL Advance Western Blotting Detection Kit (Amersham) and scanned to produce
digital images.
Real-Time PCR
Total RNA was isolated with TRIzol (Invitrogen, Shanghai,
CHN, http://www.bioasia.cn/) and treated with DNase I (Invitrogen). After quantitative measurement of the total RNA extracted
from each sample using a spectrophotometer (Eppendorf, Westbury, NY, http://www.eppendorf.com), the samples (1 lg RNA)
were subjected to cDNA synthesis using ReverTra Ace Kit
(Toyobo, Osaka, Japan, http://www.toyobo.co.jp/e/). Total RNA
and PCR products were separated on a 2% agarose gel, stained
with ethidium bromide, visualized, and photographed on a UV
transluminator.
For real-time quantitative PCR, primers were designed using
GeneTool software (supporting information Table S4). Real-time
PCR was performed using F1P9 as controls, and each sample
was analyzed in triplicate with b-actin as the internal control.
The final PCR reaction volume of 20 ll contained 10 ll SYBR
Green PCR Master Mix (Real-time PCR Master Mix; Toyobo),
1 ll cDNA template, 1 ll primer mixture, and 8 ll water. Thermal cycling was carried out with a 5-minute denaturation step at
94 C, followed by 40 three-step cycles: 30 seconds at 94 C,
30 seconds at 60 C, and 30 seconds at 72 C. Amplification data
were collected by the ABI PRISM 7900 and analyzed by the
Sequence Detection System 2.0 software (Applied BioSystems,
Foster City, CA, http://www.appliedbiosystems.com).
RESULTS
pESCs Have Characteristics Similar to fESCs
In our first series of experiments, we generated 17 pESC lines
at various passages from B6C3F1 parthenogenetic blastocysts
(supporting information Fig. 1A; supporting information Table
S1), using methods described in our previous study [19, 20].
We systematically characterized two pESC lines (C2 and C3)
and tested their pluripotency by teratoma formation (supporting information Fig. 1B) and chimera production (supporting
information Fig. 1C) and by generation of pESC pups by
TEC (supporting information Fig. 1D).
The two hybrid F1 pESC lines (C2 and C3) proliferated
in culture for more than 40 passages and maintained morphology typical of ESCs (Fig. 1A–1C). pESCs formed round colonies with well-defined boundaries, a pattern similar to fESCs
(Fig. 1E, 1F). These cells were comparatively small, typically
had large, clear, shiny nuclei containing one or more prominent nucleoli, and were tightly packed within a multilayered,
primary colony (Fig. 1B). These pESC lines maintained normal karyotypes at passages 10 and 50, and G-banding results
showed that pESCs at early passages exhibited the expected
XX karyotype (Fig. 1D). Furthermore, these cell lines were
positive for alkaline phosphatase (AP), SSEA-1, and Oct4 at
early (p9) and late passages (p30), comparable to fESCs (Fig.
1G). Oct4 and TERT protein were present in early and late
passages of pESCs like fESCs, but absent from MEFs served
as negative controls (Fig. 1H). Real-time PCR analysis also
showed that mRNA levels of the stem cell markers Oct4 and
Nanog [21] did not differ between pESCs and fESCs (data
not shown). These data show that expression patterns of stem
cell markers were comparable between pESCs and fESCs.
Our B6C3F1 hybrid pESCs were derived from parthenogenetically activated MII oocytes by Sr2þ, supplemented with
cytochalasin D, which causes retention of the second polar
body. Genotype analysis with markers for D14mit5, D16mit4,
and D18mit17 showed homozygosity in both C2 and C3
pESC lines. Cell line C3 is homozygous at D16mit4 and
D18mit17 for alleles found in C57Bl/6 mice and at D14mit5
for a C3H/He allele (Fig. 1I). Cell line C2 is homozygous at
D16mit4 for an allele in C3H/He mice, homozygous at
D18mit17 for a C57Bl/6 allele, but heterozygous at D14mit5,
which is not surprising as shown in a recent report that human
pESC lines also exhibit heterozygous genotypes by genomewide single nucleotide polymorphism (SNP) analysis [22].
Reduced MAPK Signaling Is Associated with
Efficient Derivation of pESCs from
Parthenogenetic Blastocysts
Maintaining appropriate ICM outgrowth of mouse blastocysts
placed on the MEF feeder in ES medium is the first critical step
for successful derivation of ESC lines. ICM outgrowth of
parthenogenetic blastocysts grew slower than did fertilized
blastocysts. ICM outgrowth formed and appeared large from
fertilized blastocysts than from parthenogenetic blastocysts
(supporting information Fig. 2). However, it seemed that the
growth rate of ICM did not associate with isolation efficiency
of ESCs. Initially, no fESC lines were derived from fertilized
B6C3F1 mouse blastocysts in the ES medium supplemented
with FBS, in contrast to an efficiency of 10% for the derivation
of pESC lines of the same mouse strain (supporting information
Table S1). Notably, fESC lines were generated with efficiency
of up to 29%, when MAPK inhibitor PD98059 was added into
the medium during ICM outgrowth. Therefore, we speculated
that MAPK signaling might be reduced in parthenogenetic blastocysts compared with fertilized blastocysts. Indeed, expression
of pERK protein was significantly lower in parthenogenetic
than fertilized embryos (Fig. 2A). ICM outgrowths of parthenogenetic blastocysts exhibited noticeably reduced fluorescence
intensity of ERK2 and pERK, indicative of decreased MAPK
signaling, compared to those of fertilized blastocysts (Fig. 2B).
Inhibition of MAPK signaling by PD98059 further reduced
expression of ERK2 and pERK and size of ICM outgrowth
from both fertilized and parthenogenetic embryos (Fig. 2B;
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Figure 1. Characterization of
pESC lines. (A–C): pESCs exhibit
morphology typical of undifferentiated embryonic stem cells (E, F).
(A): pES C3 at passage 7. (B): pES
C3 at higher magnification, showing
smooth surface of colony and large
nuclei with shiny nucleoli in individual cells. (C): pES C3 at passage
51. (D): pES C3P7-9 have a normal
40, XX karyotype by G banding
analysis. (E): fES F1P10 derived
from fertilized B6C3F1 blastocysts.
(F): Normal ESC E14.1 as a control
(passage unknown). Bar scale: 100
(A, E), 25 (B), and 50 lm (C, F).
(G): Immunofluorescence of molecular markers specific for identification of mouse ESCs. pESCs at both
early and late passages were
strongly positive for alkaline phosphatase, Oct-4, and SSEA-1, comparable to fES F1P10. (H):
Expression of Oct4 and TERT proteins in pESCs and fESCs shown by
Western blots. (I): Microsatellite
analysis of pESC lines. pES C3P9
cells are homozygous at 164-bp
D14mit5 alleles identical to those in
C3H/He mice and homozygous 132bp D16mit4 and 213-bp D18mit17
alleles identical to those in C57BL/
6 mice. pES C2P8 cells are homozygous 123-bp D16mit4 alleles
identical to those in C3H/He mice
and homozygous 213-bp D18mit17
alleles identical to those in C57BL/
6 mice, but their D16mit4 alleles
are heterozygous. Liver was used to
extract DNA from adult mice. fES,
F1P7. Abbreviations: DAPI, 40 ,6diamidino-2-phenylindole;
fESC,
fertilized embryo-derived ESCs;
MEF, mouse embryonic fibroblasts;
pESC, parthenogenetic ESC.
supporting information Fig. 2). These results show that MAPK
pathway activation was reduced in parthenogenetic blastocysts
and MAPK signaling remained low during ICM outgrowth for
derivation of pESCs. Reduced MAPK signaling in parthenogenetic embryos and ICM outgrowth is associated with increased
efficiency in the derivation of pESC lines. Moreover, inhibition
of MAPK by PD98059 in ICM outgrowth of fertilized blastocysts also increased efficiency in derivation of fESC lines.
Thus, reduced MAPK pathway found in parthenogenetic
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embryos also plays critical role in the efficient derivation of
pESC lines, as in the derivation of fESCs.
Differentiation Capacities In Vivo Are
Indistinguishable Between pESCs and fESCs
pESCs have been shown to retain an extensive differentiation
capability in vitro [16, 23]. To assess the in vivo differentiation potential of pESC lines, we injected C3 pESC cells at
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Pups Produced from Parthenogenetic ES Cells
Figure 2. Reduced mitogen-activated protein kinase signaling in parthenogenetic
embryos and the inner cell mass (ICM)
outgrowth during derivation of parthenogenetic ESC (pESCs). (A): Detection of pERK2 (42 kDa) and p-ERK1 (44 kDa) in
FEs and PAs at 120 hours in culture as
shown by Western blot analysis. (B): Representative immuonofluorescence images
showing expression of ERK2 and pERK in
ICM outgrowth from B6C3F1 PA and FE
blastocysts cultured for 7 days on feeder
cells with or without PD98059. ICM outgrowth from FE blastocysts showed higher
expression of ERK2 and p-ERK proteins
compared with outgrowth from PA blastocysts cultured in the same condition. In
addition, the outgrowths from FE blastocysts proliferated more rapidly and formed
larger colonies than those from PA blastocysts. Expression of ERK2 and p-ERK in
the ICM outgrowth was reduced in both
types of embryos in the presence of PD
and even undetectable in the outgrowth of
PA blastocysts. Abbreviations: FE, fertilized blastocysts; PA, parthenogenetic blastocysts; PD, ES media plus PD98059.
early, middle, and late passages into the hypodermic cavity of
immunodeficient nude mice. After injection of C3 cells at
early passages [5–10] under the skin of nude mice (n ¼ 4),
all nude mice produced teratomas, weighing 0.8-1.9 g, comparable to teratomas from fESCs (Fig. 3A, 3B). By contrast,
injection of MEF into nude mice (n ¼ 5) did not produce teratomas (Fig. 3C). Four weeks after injection, teratomas were
sectioned and analyzed by histology, showing mature tissues
and low mitotic frequencies, indicating their benign nature.
Furthermore, the teratomas contained derivatives of all three
germ layers, including cartilage, neurons, and skin (ectoderm),
intestinal epithelia (endoderm), and muscle (mesoderm; Fig.
3D–3I). Injection of pESCs at passages 22 and 30 also led to
teratoma formation in nude mice (n ¼ 2).
To further assess the pluripotency of pESC in vivo, we
injected C3 pESC cells carrying the agouti coat color marker
into the blastocoels of albino ICR diploid blastocysts and
transferred them into the uteri of pseudopregnant surrogate
mice. We generated 18 chimeras from these pESCs, including
10 chimeras from early passages (passages 6-9) and 8 from a
late passage (passage 30; Table 1). These chimeras developed
well postnatally, with no growth retardation. The contributions of pESCs in these chimeras varied from as little as 5%
to more than 70%, as judged by coat color (Fig. 3J, 3K). We
showed participation of pESCs in various tissues of chimeras
at ages of 3 weeks to 9 months, estimated by density measurement of bands resolved by microsatellite gel electrophoresis (Table 2; Fig. 3L–3N). pESC generally did not exhibit
tissue-specific distribution, except for that pESCs contributed
to pancreas at a higher ratio than to other tissues. Distribution
of pESCs in the chimeric tissues correlated with the extent of
coat color. Remarkably, in our study, the contribution of
pESCs to skeletal muscle (up to 31% vs. 34% for fESCs) and
gonads (up to 39% vs. 40% for fESCs) did not differ from
other tissues (Table 2; Fig. 3L, 3M). This is in contrast to a
previous report showing limited pESC contributions to pancreas, muscle, and gonads in pESC chimeras [6]. Repressed
expression of Igf2 was associated with defects in contributions
by pESCs to muscle [6]. Moreover, the distribution and extent
of pESC contribution to chimeras produced in our study were
Chen, Liu, Huang et al.
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Figure 3. Teratoma and chimeras generated from parthenogenetic ESC (pESC) lines. (A):
Teratoma 1-2 cm in size (arrowhead) formed in a nude mouse 4
weeks after injection of pES
C3P10. (B): Teratoma formed in
a nude mouse from injected fertilized embryo-derived ESCs
(F2P11). (C): No teratoma
formed from mouse embryonic
fibroblast cells. Histology shows
gut and intestinal epithelium
indicated by arrow (D), gliacytes
in neuronal tissues (E), neurocoele (F), smooth muscle (G),
hair follicle and skin with massive keratin (H), and cartilage
(I). (J): Chimera of pESCs
(C3P9) derived from B6C3F1
(agouti) mice, injected into ICR
(albino) recipient blastocysts,
showing low integration of
pESCs by coat color. (K): High
integration of pESCs (C3P9) in
a chimera. (L): Microsatellite
gel image, showing contribution
of various tissues from pESC
C3P7 in female chimera 5. (M):
Contribution of pESC C3P9 in
male chimera 1. (N): A chimera
with poor contribution of pESC
C3 at passage 30. Arrow indicates contribution of pESCs.
Table 1. Production of pESC chimeras identified by coat colora
Method
Passage
no.
No.
transferred
No.
recipients
No.
born
Cannibalized
by foster mother
No.
chimeras
Chimera
percentage
Contribution rate
by coat (average)
Aggregation
Blastocyst
Injection
5-10
6-9
30
225
89
162
13
8
8
8
17
31
0
0
8
1
10
8
13%
59%
35% (8/23)
20%
40%
10%
a
pESC3 cell lines were used for aggregation with four to eight cell embryos or injection into blastocysts from ICR mice.
Abbreviation: pESC, parthenogenetic ESC.
markedly greater than those previously reported for chimeras
produced with either parthenogenetic embryos or pESCs [6,
14, 24]. Notably, a high contribution of pESCs was found in
gonads of chimeras, like chimeras produced from fESC.
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Although some chimeras produced from pESC C3 lines were
bitten to death by surrogate mothers and the survived pups
failed to produce agouti pups after limited breeding, other
pESC lines did show a high capacity through germline in the
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Table 2. Contribution by ratio of pESCs in the chimera tissues analyzed by microsatellites
Cell line
Chimera no.
Sex
Coat color
Brain
Coat
Lung
Muscle
Bone
Gonad
Heart
Liver
Spleen
Kidney
Pancreas
Intestine
C3P7-9
1
2
5
8
11
18
1
2
3
F1-1
F1-2
M
F
F
F
M
F
F
F
F
M
M
30%
50%
40%
30%
5%
<5%
<5%
30%
50%
40%
30%
45.3
13.1
14.5
12.0
6.0
0
0
14.5
25.7
24.9
20.3
28.5
33.9
15.6
16.9
11.2
0
0
3.6
13.9
21.2
25.2
38.6
63.9
33.9
23.3
7.8
0
0
8.0
20.7
29.9
36.7
16.2
30.8
21.3
12.0
13.9
0
0
8.1
21.7
17.8
34.2
20.0
39.6
27.8
34.8
29.7
0
0
10.2
19.0
30.0
45.2
34.9
38.8
22.7
13.8
1.3
0
0
7.9
11.3
31.2
40.2
35.4
18.5
11.9
30.6
22.4
0
0
6.0
28.5
43.2
51.7
29.0
31.8
47.6
4.9
7.8
0
0
0.9
6.8
39.7
52.3
35.5
74.3
23.8
10.9
32.2
0
0
2.7
24.7
38.9
55.3
2.05
21.3
6.9
11.4
12.0
0
0
22.0
7.9
25.4
32.4
11.0
56.8
67.4
48.0
67.4
30.1
10.3
39.0
50.6
32.9
63.2
17.8
49.3
17.6
34.1
24.4
0
0
4.7
12.4
34.7
59.8
C3P30
fES
Abbreviations: F, female; M, male; pESC, parthenogenetic ESC.
resultant chimeras after extensive breeding, as reported in
details elsewhere (unpublished data). Our results also showed
that the age of chimeras did not affect the proportions of tissues contributed by pESCs. However, pESCs at later passages
(P30) resulted in lower frequencies of chimera production
(Table 1) and decreased contributions to chimeras compared
with early passage pESCs (Table 2; Fig. 3N), implying that,
just as with fESC lines, in vitro culture conditions and longterm passaging may alter the differentiation potential of
pESCs in vivo.
pESCs Can Produce Term Pups by Tetraploid
Embryo Complementation
To determine whether pESCs have the capacity to differentiate
into all tissue types in the body, we took advantage of the
TEC method, which was considered the most stringent test of
pluripotency and genomic stability of ESCs and has been used
previously to test term development of fESCs, ntESCs, and
induced pluripotent stem cells [25–27]. In a series of experiments, 10-20 agouti pESCs were injected into albino tetraploid
blastocysts (also see research plan in supporting information
Fig. 1). Surprisingly, one B6C3F1 pESC-derived pup (designated as T1) was obtained by natural delivery after transfer of
420 tetraploid embryos injected with C3 pESCs into 26
pseudo-pregnant recipients (Fig. 4A). The newborn T1 was
alive, appeared anatomically normal, and had normal birth
weight, similar to other newborns (Fig. 4B), although T1 died
shortly after birth. We dissected uteri of nine recipient mice
that had received 161 C3 pESC-tetraploid embryos and found
23 implantation sites (14.3%). No pups were produced from
the transfer of 148 C2 pESC-tetraploid embryos, although implantation did occur. In our B6C3F1 fESCs by TEC as controls, only one fES (F1)-derived pup was obtained by cesarean
from the transfer of 247 tetraploid embryos injected with
fESCs into 15 pseudo-pregnant recipients. However, when
using 129B6F1 hybrid fESCs for tetraploid complementation,
we obtained five pups at a much higher efficiency (14%, n ¼
37; Table 3). The differential efficiency in obtaining pups
from fESCs is likely caused by strain differences of donor
ESCs and recipient embryos, as shown previously in the literature [25, 28]. Together, these data suggest that our pESC lines
are comparable to fESCs for B6C3F1 strain in terms of TEC
efficiency.
Microsatellite analysis at D12mit136 and Dxmit1 showed
that the alleles of various tissues from the pup T1 were identical to those of C3 pESC lines but completely different from
the tetraploid blastocysts KM17 and KM18 (Fig. 4C). The
identity of the fESCs and pESC-derived pups confirmed by
DNA microsatellite profiling is shown in supporting informa-
tion Tables S2 and S3, respectively. We indicated by microsatellite profiling that the tetraploid embryos made no contribution to the tissues tested from the derived pup (Fig. 4C–4E).
The origin of T1 from pESCs was further confirmed by SNP
analysis (supporting information Fig. 3), although we could
not exclude the possibility of contribution of tetraploid
embryos in the pES fetuses, because of limitations of the
assays uses. The pattern of C3P7 pESC differs from that of
B6C3F1, suggesting that pESCs had undergone some loss of
heterozygosity in culture. Together, this is the first demonstration that pESCs can support full-term development, resulting
in a pESC-derived newborn, similar to those reported previously for fESCs [28].
To confirm our surprising observation of term development from the newly established pESC line in this study, we
independently performed experiments using a pESC line constitutively expressing enhanced green fluorescent protein
(EGFP) complemented by TEC in the laboratory of X.Y.
Thus far, three live pups with GFP expression have been produced from pESCs, albeit also with low efficiency (<1%), but
these pups also died shortly after birth. Preliminary data by
microsatellite analysis also showed that these pups were
derived from the pESCs. Further analysis of the tissues and
organs of the pups is expected to be reported shortly.
Epigenetic Reprogramming of Imprinted Genes
During Derivation of pESCs by In Vitro Culture
Genomic imprinting plays critical roles for embryonic and
fetal development and may have been changed during isolation and in vitro culture of pESCs. Our group (data not
shown) and others have found that parthenogenetic hybrid
embryos complemented with tetraploid embryos die abruptly
around 13-14 days of fetal development [29]. Abnormal
imprinting likely contributes to the limited development of
parthenogenetic embryos and the restricted potential of pESCs
[1, 3, 4, 6, 8, 30, 31]. To understand expression and methylation of imprinted genes in our newly derived pESCs reported
in this study, we initially analyzed expression of imprinted
genes in blastocysts using quantitative real-time PCR. Not
unexpectedly, maternally expressed imprinted genes H19 and
Igf2r exhibited remarkably higher expression (p < .01) in
parthenogenetic blastocysts, whereas paternally expressed
imprinted gene Snrpn was expressed at lower levels than in
fertilized blastocysts (p < .01). Expression TSSC3 did not differ between parthenogenetic and fertilized blastocysts (Fig.
4F). To understand whether expression of imprinted genes in
the resultant ESCs differ from their progenitor blastocysts, we
compared expression of the imprinted genes in pESCs with
fESCs. Notably, expression of Igf2r, Tssc3, and Snrpn did not
Chen, Liu, Huang et al.
2143
Figure 4. Full-term development generated completely from parthenogenetic ESCs (pESCs)by tetraploid embryo complementation and gene
imprinting of pESCs. (A): A live pup (T1) at day 0.5 from pESC C3P6 delivered by the surrogate KM mother. Inset: the live pup based on
movement. (B): The pESC pup (T1) at day 1 after birth was found dead and had a bite on the neck compared with similar size of KM-B6C3F1
pES chimera and B6C3F1 pups at the same age. (C): Microsatellite analysis shows the alleles of various tissues from pup T1 are identical to
those of pESC C3 but completely different from the tetraploid blastocysts KM17 and another KM18. (D): T1 was the same as B6C3F1
(C57C3H) but heterozygous to pESC3, but again completely different from KM blastocysts. (E): T1 was the same as B6C3F1, but heterozygous to pESC C3, and completely different from KM blastocysts. (F, G): Expression of imprinted genes in mouse parthenogenetic blastocysts
(F) and pESCs (G) compared with fertilized counterparts by real-time polymerase chain reaction analysis. b-actin served as internal controls.
Bars indicate mean SEM (n ¼ 3). *, p < .05 and **, p < .01 compared with fertilized counterparts (Control). (H): Hypomethylation
of imprinted Igf2/H19 ICRs in pESCs. Circles are CpG sites within the regions analyzed: l, methylated cytosines; *, unmethylated cytosines.
Abbreviation: RU, recipient uterus.
Table 3. Production of pESC and fESC pups by tetraploid blastocyst complementationa
Cell
lines
Strain
fES
B6C3F1
129B6F1
pES
B6C3F1
Passage
no.
No.
transferred
No.
recipients
No.
born
ESC
pupa
ESC pup
percentage
F1P4-9
F1P5
F1P9
C3P6-9
C2P4-11
247
37
68
420
148
15
2
2
26
11
2b
5
7
1c
0
1
5
7
1
0
0.4%
14%
10%
0.24%
—
*Identified by microsatellite and/or single nucleotide polymorphism analysis.
Cesarean, died at day 0.5 after birth.
Natural delivery, born alive, but died at 1 day after birth, bitten by surrogate mother.
b
c
www.StemCells.com
Pups Produced from Parthenogenetic ES Cells
2144
differ (p > .05) between pESCs and fESCs (Fig. 4G). H19
showed sixfold increase in parthenogenetic blastocysts, but
only about twofold increase in the derived pESCs, suggesting
reprogramming of H19 to certain degrees. Consistent with
expression data, pESC lines showed a hypomethylated status of
imprinted genes (Igf2/H19; Fig. 4H), which is most consistent
with the parental origin of the hypomethylated oocytes that they
were derived from. Further experiments showed that pESCs and
pESC fetuses complemented by TEC exhibited balanced methylation of Snrpn, Peg1, and U2af1-rs1, and global methylation
increased in parthenogenetic embryos but decreased in pESCs
[32]. These data suggest that culture in vitro modifies the
expression of imprinted genes in pESC after isolation from parthenogenetic blastocysts. Our data are consistent with previous
observations that in vitro culture itself can alter the expression
of imprinted genes in both fESCs and pESCs [33, 34]. Appropriate epigenetic alterations may facilitate parthenogenetic embryonic development from pESCs.
DISCUSSION
In this study, we successfully established hybrid pESCs that
are indistinguishable from fESCs. These pESCs express molecular markers typical of fESCs in vitro, differentiate fully in
vivo, and exhibit unrestricted potential to generate various tissues in vivo, as evidenced by teratoma formation and chimera
production, as well as by the surprising births of parthenote
pups from these pESCs by TEC. By microsatellite and SNP
analysis, the pups were in fact derived from pESCs, and no
detectable contribution, if any, could be found from tetraploid
host blastocyst cells to the tissues or organs of the pups. Any
contribution from tetraploid host blastocysts would have to be
below the detection limits of the assays.
Based on current knowledge of the role of and requirement
for proper genetic imprinting in development, it is not entirely
clear how or why pESC are capable of full-term development.
What is clear is that these in vitro systems offer only a limited
and partial view of the action of imprinted gene regulation and
function in development, and these results open the door to
future studies examining additional imprinting modifications
that may occur at various stages of development complemented by TEC. It is also not clear why term development for
pESC-TEC has such a low success rate, but in future studies,
we plan to examine the effects of different genetic backgrounds on efficiency, as well as early embryonic contribution
and support by tetraploid cells, followed by selection against
them in embryonic tissues as development proceeds. Efficiency
in producing ES pups by TEC generally is very low and
depends on the genetic background of donor ESCs and recipient tetraploid embryos, as well as passage numbers of ESCs
[35, 36]. We show that pESCs could directly produce pups by
TEC albeit at very low frequency, like fESCs of the B6C3F1
strain. This could result from modification of genomic imprinting, particularly activation of paternally expressed imprinted
genes by in vitro culture, such that expression of most
imprinted genes did not differ between pESCs and fESCs [32].
However, pESC pups died within 1 day, but fESC pups survived to adults, and this could be because of relative high
homozygosity of pESCs in contrast to heterozyosity of fESC.
Indeed, ES pups produced from inbred mouse ESCs often died
shortly after birth because of respiratory deficiency, whereas
ES pups produced from hybrid mouse ESCs survived and grew
normally [37]. Genetic manipulation of ICR of H19/Igf2
resulted in normal expression of the genes, leading to live birth
of parthenogenetic pups [5, 30]; thus, incomplete epigenetic
changes in the ICR of H19/Igf2 found in pESCs may also negatively influence survival of pESC pups.
Mammalian parthenogenetic embryos are not viable for
term development and die from defects in genomic imprinting
[1, 2]. pESCs are isolated from the ICM of parthenogenetic
blastocysts through an in vitro derivation and culture process
and thus may escape factors that regulate their development in
vivo, including imprinting. Genomic imprinting thus may have
less influence on those pESCs derived from parthenogenetic
embryos than the parthenogenetic embryos themselves. Szabo
and Mann [38] showed that H19 is not expressed but Igf2r is
expressed in normally fertilized preimplantation mouse embryos
using RT-PCR, as did Hamatani et al. [39] using gene expression microarrays. Interestingly, our study showed that expression levels of Snrpn, Igf2r, and Tssc3 in C3 pESCs were comparable to fESCs controls and that H19 also show significantly
reduced expression; these expression patterns differed from their
progenitor blastocysts. This observation supports the notion that
expression of imprinted genes in embryonic cells can be
changed by in vitro culture [33, 34]. These epigenetic changes
of pESCs in culture may partially explain why those pESCs by
TEC can lead to full-term development. This novel finding is
well supported by the report that parthenogenetic and bi-maternal mouse pups can be successfully produced from reconstructed parthenogenetic embryos after genetic modification of
the key imprinted loci particularly Igf2 and H19 [5].
We further found that derivation of the pESC line is more
efficient than that of fESCs. Favorable signaling involved in
parthenogenesis could explain the efficient derivation of
pESCs. Naturally decreased MAPK signaling in parthenogenetic embryos and ICM outgrowth is associated with derivation
of pESCs at higher efficiency than that of fESCs from B6C3F1
mouse strain. This is consistent with the notion that inhibition
of MAPK signaling promotes derivation of fESC lines from
hybrid embryos [40, 41]. In contrast, the Grb2/Mek pathway
represses Nanog, a key transcription factor that maintains selfrenewal and pluripotency in mESCs [21, 42, 43]. Indeed, inhibition of MAPK pathway by PD98059 also increased derivation of fESCs from fertilized B6C3F1 mouse embryos. It is
also important for the LIF-STAT3 pathway to maintain the
self-renewal and pluripotency of mouse ESCs even though the
MAPK pathway activated by LIF-STAT3 can induce ESC differentiation. Decreased MAPK signaling in parthenogenetic
embryos reduces differentiation of ICM outgrowth, likely contributing to high efficient generation of pESCs.
CONCLUSIONS
Overall, newly established pESCs from activation of oocytes
by strontium reported here are indistinguishable to fESCs for
tissue/organ contribution. Reliable and efficient derivation of
pluripotent pESCs is a critical step toward the feasibility of
female patient-specific ESC therapy in regenerative medicine
[16, 17]. Our findings that parthenogenetic embryo-derived
ESCs can differentiate fully in vivo, exhibit unrestricted
potential to generate various tissues in vivo, and lead to fullterm development via tetraploid embryo complementation
open the door for more epigenetic studies in stem cell biology
and may show promising evidence for future therapeutic
applications of an alternative stem cell resource for regenerative medicine, without involving fertilized embryos or somatic
cell nuclear transfer cloning. More work is needed to understand the molecular dynamics and mechanisms underlying
epigenetic changes of pESCs in vitro and to further explore
the therapeutic potential of pESCs for basic developmental
biology research and for regenerative medicine.
Chen, Liu, Huang et al.
2145
ACKNOWLEDGMENTS
We thank Jingping Yun for histological examination and technical support and Peng Xiong for providing a cell line. This work
was supported in part by National Natural Science Foundation,
Science and Technology Division of Guangdong Province, and
China Ministry of Science and Technology (2009CB941000) to
L.L. and by USDA-ARS Contracts AG 58-1265-2-018 and 581265-2-020 to X.Y.
DISCLOSURE
OF
OF
POTENTIAL CONFLICTS
INTEREST
The authors indicate no potential conflicts of interest.
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