© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 3842-3847 doi:10.1242/dev.110726
RESEARCH REPORT
STEM CELLS AND REGENERATION
Induction of the G2/M transition stabilizes haploid embryonic
stem cells
Saori Takahashi1, *, Jiyoung Lee1,2, *, Takashi Kohda1, Ayumi Matsuzawa1, Miyuri Kawasumi3,
Masami Kanai-Azuma3, Tomoko Kaneko-Ishino4,‡ and Fumitoshi Ishino1,2,‡
The recent successful establishment of mouse parthenogenetic
haploid embryonic stem cells ( phESCs) and androgenetic haploid
ESCs (ahESCs) has stimulated genetic research not only in vitro but
also in vivo because of the germline competence of these cell lines.
However, it is difficult to maintain the haploid status over time without
a frequent sorting of the G1 phase haploid ESCs by fluorescenceactivated cell sorting (FACS) at short intervals, because haploid cells
tend to readily self-diploidize. To overcome this spontaneous diploid
conversion, we developed a phESC culture condition using a small
molecular inhibitor of Wee1 kinase to regulate the cell cycle by
accelerating the G2/M phase transition and preventing re-entry into
extra G1/S phase. Here, we demonstrate that, under this condition,
phESCs maintained the haploid status for at least 4 weeks without the
need for FACS. This method will greatly enhance the availability of
these cells for genetic screening.
KEY WORDS: Parthenogenesis, Haploid embryonic stem cells,
Cell cycle, Mouse
INTRODUCTION
The remarkable progress of forward genetics has largely depended
on the use of haploid organisms with a single genome, such as
bacteria and yeast. This is because a clearly distinguishable
phenotype is evident, even in the case of recessive mutations of
essential genes, due to the absence of an alternate gene copy to
compensate for the modified or missing function. Therefore,
researchers have endeavored to establish haploid cell lines from
diploid organisms, such as human KBM-7 leukemia cell lines
(Carette et al., 2009; Kotecki et al., 1999) and medaka ES cells
(Yi et al., 2009).
Since 2011, several groups have successfully established
parthenogenetic and androgenetic haploid ES cell lines ( phESCs
and ahESCs) from mouse haploid preimplantation embryos (Leeb
and Wutz, 2011; Elling et al., 2011; Yang et al., 2012; Li et al.,
2012) using the 2i condition (Ying et al., 2008). The 2i culture
condition provides a serum-free environment that blocks the activity
of both ERK and GSK3 by the inhibitors PD0325901 and
1
Department of Epigenetics, Medical Research Institute, Tokyo Medical and Dental
2
University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. Global
Center of Excellence Program for International Research Center for Molecular
Science in Tooth and Bone Diseases, Tokyo Medical and Dental University
3
(TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. Center for
Experimental Animals, Tokyo Medical and Dental University (TMDU), 1-5-45
4
Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. School of Health Sciences, Tokai
University, Bohseidai, Isehara, Kanagawa 259-1193, Japan.
*These authors contributed equally to this work
‡
Authors for correspondence ([email protected];
[email protected])
Received 28 March 2014; Accepted 20 August 2014
3842
CHIR99021, respectively, and is used to establish diploid ES
cells with high germline transmissibility. The haploid ES cell lines
obtained using the 2i condition exhibit stemness and differentiation
capacity, as well as germline transmission via chimeric mice
(Leeb et al., 2012). However, it is difficult to maintain haploid ES
cells in the haploid state over time, as they tend to diploidize
spontaneously. Therefore, frequent G1 phase sorting of the haploid
ES cells by fluorescence-activated cell sorting (FACS) is an
inevitable requirement.
It remains unclear how these so readily diploidize, although the
diploid state is congenitally more stable than the haploid state. One
conceivable mechanism is that the diploidization occurs due to
abnormal cell cycle regulation in the haploid cells; for example, by
skipping cell division once and re-entering into an extra G1/S phase.
In this study, we attempted to develop culture conditions suitable for
the long-term maintenance of haploid ESCs by regulating their cell
cycle using an inhibitor of Wee1 kinase in order to induce the
occurrence of a normal transition at the G2/M phase.
RESULTS AND DISCUSSION
Derivation of phESCs and effect of the Wee1 inhibitor
PD166285 on stabilizing the haploid state of phESCs
We attempted to generate phESCs from the C57BL/6 (B6) strain
because of its known advantages for genetic research. The
developmental rate of haploid blastocysts from the B6 oocytes
was actually lower than those from F1 hybrid mice, such as BDF1
and DBF1 (18%, 35% and 50%, respectively; Table 1). Their
morphology at the blastocyst stage was also poor compared with
diploid embryos as well as with BDF1 or DBF1 haploid embryos;
nevertheless, the morphology was still good until the morula stage
(supplementary material Fig. S1A). Therefore, we tried to establish
ES cells from embryos at the morula stage or earlier and
successfully obtained 64 ES cell lines, including 27 haploid ES
cell lines (Table 1, Fig. 1A).
In present study, phESCs (which exhibit the haploid karyotype;
Fig. 1B, left and inset) established by existing culture condition,
also exhibited the previously reported self-diploidization (Fig. 1B,
middle and right) (Leeb and Wutz, 2011; Elling et al., 2011), and
frequent sorting of the G1 phase phESCs was inevitably required at
short intervals to maintain their haploid status (supplementary
material Fig. S1B). Therefore, it should be an important issue to
elucidate the mechanism of spontaneous diploidization in haploid
ES cells.
Polyploid cells in a diploid organism are formed by cell
fusion, endoreplication and a variety of defects that result in an
abnormal cell cycling (Storchova and Pellman, 2004). During
endoreplication, cells skip mitosis (e.g. polytene chromosomes) or
omit cytokinesis (e.g. megakaryocytes), and proceed through
several rounds of DNA replication, resulting in polyploid cells,
which generally do not proliferate further (Edgar and Orr-Weaver,
DEVELOPMENT
ABSTRACT
RESEARCH REPORT
Development (2014) 141, 3842-3847 doi:10.1242/dev.110726
Table 1. Establishment of parthenogenetic haploid ES cell lines
Experiment
number
Genetic background
Oocyte
activated
Embryo
status
Number of
embryos
ES cell lines
obtained
ES cell lines with the
haploid contribution
1
C57BL/6
174
2
3
4
5
C57BL/6 (CAG-EGFP)
C57BL/6
BDF1
DBF1
180
137
120
68
8 cell
16 cell
Morula
Blastocyst
Morula
Morula
Morula
Morula
17
20
48
20
63
38
72
21
0
8
25
2
21
18
60
12
0
3 (15%)
13 (27%)
0
7 (16%)
7 (18%)
21 (35%)
6 (50%)
evidence from previous reports also suggested that cell fusion and
endoreplication cannot be the cause of increasing ploidy in haploid
ES cells (Taylor, 2002; Vignery, 2000; Leeb et al., 2012; Lanni and
Jacks, 1998; Andreassen et al., 2001).
Fig. 1. Spontaneous diploidization of phESCs. (A) Morphology of parthenogenetic haploid morula and phESCs before FACS sorting. (B) Flow cytometry
analysis of DNA contents by Hoechst 33342 staining and karyotyping of phESCs using DAPI staining (inset). (C) Hypothetical mechanism of self-diploidization of
haploid ES cells. Spontaneous diploidization may occur by inhibition of cyclin B1/Cdc2 complex by Wee1, which inhibits Cdc2 dephosphorylation for inactivation
of Cdc2. (D) Flow cytometry analysis of DNA contents. (E) Morphology of the phESCs-B6 cultured with 300 nM PD166285 appeared healthy under a feeder
condition. (F) Growth curve of PD166285-treated and untreated phESCs during short-term culture (up to 12 days). Scale bars: 50 μm.
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DEVELOPMENT
2001; Ravid et al., 2002). In this study, we assumed that
spontaneous diploidization of haploid cells occurs both in vivo
and in vitro as the result of an abnormal cell cycle progression,
because haploid ES cells exhibited rapid proliferating ability and
RESEARCH REPORT
Development (2014) 141, 3842-3847 doi:10.1242/dev.110726
In the normal cell cycle, the cyclin A/B-Cdc2 complex actively
promotes cell cycle progression from the G2 to M phase via Cdc2
dephosphorylation by Cdc25, while Wee1 and Myt1 inhibit the
activity of this complex via Cdc2 phosphorylation, causing G2 arrest
at a DNA damage check point (Perry and Kornbluth, 2007; top
panel of Fig. 1C showing the supposed normal cell cycle in haploid
cells). We hypothesized that the diploid conversion of phESCs
occurs by transient G2 arrest and the abrupt insertion of an extra
G1/S phase (Fig. 1C, bottom), because the diploid state is more
stable for the cells. Therefore, we attempted to regulate the haploid
cell cycle by adding the small molecule Wee1 inhibitor PD166285
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to facilitate a normal transition from the G2 to M phase without entry
into the extra G1/S phase. We first employed culture conditions
of 300, 500 or 1000 nM of PD166285 (supplementary material
Fig. S2A) using phESCs-B6 cells harvested after the sorting of G1
phase haploid cells (Fig. 1D). In accordance with a previous report
(Hashimoto et al., 2006), phESCs-B6 cultured with 500 or 1000 nM
of PD166285 exhibited a growth-retarded morphology, suggesting
an anti-proliferative effect of PD166285. However, in the case of
treatment with 300 nM PD166285, phESCs-B6 formed more
compact colonies than those without PD166285 under the usual
culture condition with feeder cells, as well as under a feeder-free
DEVELOPMENT
Fig. 2. Maintenance of haploidy in phESCs cultured with the Wee1 inhibitors PD166285 and MK1775. (A) Top panel: experimental design. Morphology
(middle) and flow cytometry analysis (bottom) of phESCs cultured for 33 days with or without PD166285 under feeder-free conditions after sorting. Scale bar:
50 μm. (B) Stringent test for the effectiveness of PD166285. Top panel: 24 independent single phESC colonies were isolated from two phESC lines and cultured
with or without PD166285. After 33 days, each of the surviving colonies was analyzed for the 1N cell ratio by PI staining and plotted. Red lines indicate 20% of 1N
cells (reference; 0% of 1N cells in diploid ESCs). Middle panel: average 1N cell ratios of the colonies with or without PD166285. Bottom panel: total FACS
histograms and photographs of representative single clones. Scale bar: 100 μm. (C) Wee1 inhibition by PD166285 as well as MK1775 enabled maintenance of
the haploid status in phESCs for 4 weeks of culture after sorting (top) and did not have any negative effect on the proliferation of phESCs (bottom).
RESEARCH REPORT
Development (2014) 141, 3842-3847 doi:10.1242/dev.110726
condition (Fig. 1E; supplementary material Fig. S2A). No
difference was observed in the doubling time of phESCs-B6 with
and without PD166285 over a period of 12 days (Fig. 1F). As shown
in Fig. 2A and supplementary material Fig. S2B, the cells in the
300 nM PD166285 group retained a haploid state for at least
4 weeks (long term culture). In detail, the ratio of the haploid G1
phase (1N) cells in the phESCs-B6 cells with 300 nM PD166285
was evidently higher than that without PD166285, even after
33 days under the feeder-free condition (26.8 and 12.1%,
respectively), and the ratio of the diploid G2 phase (4N) cells was
lower than that without PD166285 (8.5% and 12.3%).
To determine the effectiveness of Wee1 inhibition, a stringent test
was performed using 24 independent phESC colonies derived from
two phESC lines and culturing them for 33 days with and without
PD166285. The number of clones that survived out of the 24
colonies after 33 days of culture were 21 (line 1) and 20 (line 2) with
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DEVELOPMENT
Fig. 3. Characterization of phESCs treated with PD166285. (A) Array CGH analysis of phESCs. No major genomic amplifications or losses in phESCs
compared with a reference genome from the C57BL/6NCr liver. (B) Scatter plot between phESCs and mESCs. (C) Alkaline phosphatase activity and the
expression of some phESC markers (SSEA1, Nanog and Oct3/4). (D) Teratoma formation in vivo 30 days after the injection of phESCs. Histological analysis
showed the involvement of all three germ layers. (E) DNA methylation analysis of imprinted DMR in phESCs. The open circles indicate unmethylated sites and the
closed circles indicate methylated sites. (F) G1 phase phESCs (1N peak) were used for the blastocyst injection (left). EGFP fluorescence indicated chimeric
contribution of phESCs at E14.5 chimeric embryo and gonads (middle). The ratio of GFP-positive cells in each of the chimeric embryos (right). (G) EGFPexpressing newborn offspring from female chimeric mice. (H) Lentiviral infection of phESCs-GFP by hKO1 and HRasV12-hKO1 under Wee1 inhibition. Scale bar:
100 μm.
RESEARCH REPORT
Characterization of phESCs treated with PD166285
Most of the cells in the two phESCs-B6 lines had a complete
haploid set of 20 chromosomes, with the identified diploid
cells below 10% and no abnormal chromosomes detected by
G-band analysis (supplementary material Fig. S2C). Detailed array
comparative genomic hybridization (aCGH) analysis of GFPexpressing phESCs-B6 revealed that the genome stability of the
phESCs was well maintained in this culture system (Fig. 3A).
To compare the gene expression profiles of phESCs-B6 and
mESCs-B6 (natural mating ESCs), we performed DNA microarray
analysis using G1 phase cells collected by FACS. They exhibited
very similar global gene expression profiles (Fig. 3B) and expressed
several stem cell marker genes as normal diploid mESCs
(supplementary material Fig. S2D), although a slight difference in
the gene expression between pharthenogenetic ESCs (both haploid
and diploid) and mESCs was detected (supplementary material
Fig. S2E). In terms of pluripotency, phESCs also had alkaline
phosphatase activity (Fig. 3C, left) in addition to the typical ES cell
markers, including SSEA1, Nanog and Oct3/4, as shown by
immunostaining analysis (Fig. 3C, right). We further investigated
the in vivo differentiation capacity of phESCs-B6 by subcutaneous
injection and the formation of a teratoma comprising three germ
layers was confirmed (Fig. 3D). These results suggest that phESCs
treated with PD166285 have normal capacity of pluripotency as well
as differentiation in vitro and in vivo.
Epigenetic properties and germline competence of phESCs
treated with PD166285
DNA methylation analyses of the differentially methylated regions
(DMRs) in several of the imprinted regions were performed by
COBRA and bisulfite sequencing. The paternally imprinted regions,
such as IG and H19 DMRs were unmethylated in phESCs-B6, while
methylation of maternally imprinted regions, such as Snrpn and
Igf2r DMRs remained, confirming their parthenogenetic origin.
However, maternal DMRs were not fully methylated, especially in
the later passages (around passage 60), they were completely
unmethylated (Fig. 3E, supplementary material Fig. S3A).
Furthermore, we analyzed phESCs-B6 cells in passage three and
found that even these cells were unable to maintain a complete
maternal imprinting status, suggesting methylation was perturbed at
the initial stage of the establishment of haploid cells (supplementary
material Fig. S3B). It has also been reported that diploid
parthenogenetic ES cell lines from B6C3F1 hybrid mouse are not
3846
able to maintain a maternally imprinted status (Li et al., 2009) and
that the in vitro culture environment was capable of introducing
alterations into the imprinting status of mouse diploid ES cell lines
(Humpherys et al., 2001), especially in the case of prolonged
passage (Dean et al., 1998). For future applications of these phESCs
to artificial reproductive technology (ART), the problem of the
imprinting issue needs to be overcome, as in the previous report for
semi-cloning experiment using phESCs (Wan et al., 2013).
Next, to evaluate the ability of the phESCs-B6GFP cells treated
with PD166285 to physically contribute to mice, we injected several
dozen G1 phase phESCs-B6GFP (GFP-positive) cells into C57BL/
6 (GFP-negative) blastocysts (Fig. 3F, left). GFP-positive embryos
at embryonic day (E) 14.5 were smaller than GFP-negative
embryos, but no morphological abnormalities were found in these
tissues (Fig. 3F, middle). Through FACS analysis, ∼50% of the
entire body of the fetus exhibited chimerism, with the brain having
the highest degree but no chimerism detected in the liver, indicating
a different proportion of GFP-positive cells in each tissue and
embryo (Fig. 3F, right). GFP-positive cells were also observed in the
female gonads (Fig. 3F, middle), suggesting probable germline
transmission of phESCs-B6GFP cells. Expectedly, one chimeric
female mouse successfully delivered healthy, GFP-positive mice
after mating with a GFP-negative male (Fig. 3G), demonstrating the
germline competence of the phESCs-B6GFP cells. We next tried to
introduce genetic modifications into phESCs-B6GFP by infecting
them with CSII-EF-HRasV12-IRES2-hKO1 (humanized KusabiraOrange 1) and CSII-EF-IRES2-hKO1 lentivirus. It was revealed that
both were successfully introduced while retaining a haploid state
under Wee1 inhibition (Fig. 3H), although HRasV12 may affect
haploidy negatively at long-term culture.
In summary, phESCs were established from C57BL/6 mice and
the culture conditions were modified so as to maintain the haploid
status over a long period of time. These results should enable a more
useful application of haploid ESCs for forward and reverse genetic
screening. Furthermore, our findings will provide a new insight on
cell cycle regulation in haploid ES cells.
MATERIALS AND METHODS
Animals
All mouse experiments were approved by the Institutional Animal Care and
Use Committee of Tokyo Medical and Dental University (TMDU).
Derivation of phESCs and cell culture
Oocytes (C57BL/6NCr and C57BL/6CAG-EGFP) were activated using
5 mM SrCl2 and 2 mM EGTA (Kishigami and Wakayama, 2007), cultured
in vitro until the morula stage, and the morula embryos were put on feeder
cells. ES cells were cultured in 2i medium (Ying et al., 2008) supplemented
with 5 μM forskolin. The haploid cells were purified by cell sorting after
staining with Hoechst 33342 (37°C, 30 min) on a FACS AriaII (BD), as
described previously (Leeb and Wutz, 2011). After sorting, haploid cells
were cultured in ES medium containing 300 nM PD166285 and MK1775,
respectively. The analysis of the DNA content was performed after fixation
with 70% ethanol and staining with propidium iodide (PI) on a FACS
Calibur (BD). For the purpose of carrying out a stringent test, 24 colonies
derived from each of the two phESC lines were isolated and cultured for
33 days with or without PD166285 under a feeder-free condition. For
karyotype analysis, colcemid-treated (0.1 μg/ml) phESCs were resuspended
0.075 M KCl, and fixed in methanol-acetic acid (3:1). G-banding analysis
was performed at Nihon Gene Research Laboratories.
Array CGH analysis
Genomic DNA was isolated from purified G1 phase phESCs (approximately
2×106 cells) by FACS. As a reference for the array used for CGH analysis,
the C57BL/6NCr liver genome was used. The Cy3-labeled reference and
DEVELOPMENT
PD166285, and 18 (both lines 1 and 2) without PD166285. Among
them, 16 colonies (both lines 1 and 2) maintained a haploid
karyotype with >20% of 1N cells under the Wee1 inhibiting
condition, while only 1 (line 1) and 0 (line 2) colonies were present
without PD166285. The average 1N cell ratios were 27.2 and 24.4
versus 12.7 and 11.3, respectively (Fig. 2B). We also tested the
effect of another Wee1 inhibitor, MK1775, on maintaining
the haploidy of the phESCs during long-term culture (4 weeks).
The ratio of the 1N cells treated with 300 nM MK1775 was also
higher than that of control (Fig. 2C, top). Importantly, the doubling
time during days 11-31 of both the PD166285- and MK1775-treated
cells relatively shorter (1.06, 1.12, line 1 and 0.99, 1.13, line 2) than
that of control (1.18, line 1 and 1.34, line 2; Fig. 2C, bottom). These
results clearly demonstrate that the acceleration of the G2/M
transition by means of these Wee1 inhibitors prevented the
spontaneous diploidization of the haploid cells and efficiently
maintained the haploid status of phESCs-B6 cells, even though
further optimization of this procedure is still needed.
Development (2014) 141, 3842-3847 doi:10.1242/dev.110726
RESEARCH REPORT
Gene expression analysis by microarray
The cells in the G1 phase (approximately 2×105 cells) were collected from
phESCs and pdESCs by FACS, and total RNA was isolated by Trizol
(Invitrogen). Microarray analysis was performed using a whole mouse
genome DNA microarray kit (4×44K ver.2, Agilent).
Alkaline phosphatase activity and immunocytochemistry of
stem cell markers
Alkaline phosphatase activity was detected using a BCIP/NBT alkaline
phosphatase substrate kit (VECTOR). For immunofluorescence staining,
the cells were incubated with goat anti-mouse Oct3/4, rabbit anti-mouse
Nanog and purified mouse anti-SSEA1 (MC480) antibody at 4°C overnight
(1/100; Santa Cruz sc-8629, sc-33760 and BD 560079, respectively). Alexa
Fluor 594-conjugated donkey anti-goat IgG, Alexa Fluor 568-conjugated
goat anti-rabbit IgG and Alexa Fluor 647-conjugated goat anti-mouse IgM
were used as the secondary antibodies (1/750; Invitrogen A11058, A11036
and A21238, respectively).
Teratoma formation
phESCs (2×106) were injected subcutaneously into nude mice. After
30 days, the dissected teratoma was processed for paraffin sectioning after
fixation and stained with Hematoxylin and Eosin (HE).
DNA methylation analysis
Genomic DNA from G1 phase phESCs was treated with sodium bisulfite.
For COBRA, amplified bisulfite PCR products using specific primer sets
(supplementary material Table S1) were digested with restriction enzymes,
including PvuI, AciI, HhaI and TaqI. Bisulfite PCR products were
subcloned and sequenced.
Injection of phESCs into diploid blastocysts
Eight-cell host embryos (C57BL/6) at 2.5 dpc were cultured in KSOM
medium until the blastocyst stage. Only G1 phase phESCs were injected
into host blastocysts and blastocysts were transferred into each uterine
horn at 2.5 dpc in pseudopregnant ICR. Pregnant recipients were
dissected on day 14.5. Analysis of chimerism was performed using a
FACS AriaII.
Lentiviral infection of phESCs
Lentivirus particles were produced by transient transfection of a CSIIEF-HRasV12-IRES2-hKO1 and a CSII-EF-IRES2-hKO1 vector (gifts from
Dr T. Shinohara, Kyoto University) into 293T cells using Fugene HD, and
were infected into phESCs-B6GFP with polybrane.
Accession numbers
The NCBI accession numbers for the gene expression and CGH reported in
this paper are GSE55446 and GSM1132971.
Acknowledgements
We thank Drs T. Wakayama (Yamanashi University) for advice on the ESC culture
methods.
Competing interests
The authors declare no competing financial interests.
Author contributions
F.I. and T.K.-I. conceived of the study and S.T., J.L., T.K. T.K.-I. and F.I. participated
in the experimental design and data analysis. S.T., J.L. and A.M. performed most of
the analyses. M.K. and M.K.-A. performed chimera mice analysis. S.T., J.L., T.K.,
T.K.-I. and F.I. wrote the manuscript. All of the authors read and approved the final
manuscript.
Funding
S.T. is a Research Fellow of Japan Society for the Promotion of Science (JSPS).
This work was supported by grants from Research Fellowships of JSPS for Young
Scientists to S.T.; by Creative Science Research (JSPS) and the Joint Usage/
Research Program of MRI-TMDU to F.I. and T.K.-I.; by Grants-in-Aid for Scientific
Research (S) to F.I.; by GCOE (MEXT) to F.I. and J.L.; and by NEXT (JSPS) to T.K.-I.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.110726/-/DC1
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3847
DEVELOPMENT
Cy5-labeled ES genomes were hybridized to an In situ oligo DNA
microarray kit (4×180K, Agilent).
Development (2014) 141, 3842-3847 doi:10.1242/dev.110726