Cdk1 is required for the self-renewal of mouse embryonic stem cells.
ABSTRACT Cyclin-dependent kinase 1 (Cdk1) is indispensible for the early development of the embryo. However, its role in maintaining the undifferentiated state of the embryonic stem (ES) cells remains unknown. In this study, we dissected the function of Cdk1 in mouse ES cells by RNA-interference and gene expression analyses. Cdk1 expression is tightly correlated with the undifferentiated state of the ES cells. Upon differentiation, Cdk1 expression reduced drastically. Cdk1 knock-down by RNA interference resulted in the loss of proliferation and colony formation potential of the ES cells. Consequentially, expression of self-renewal genes was reduced while differentiation markers such as Cdx2 were induced. Our results suggest a role for Cdk1 in maintaining the unique undifferentiated and self-renewing state of the mouse ES cells. © 2010 Wiley-Liss, Inc.
- SourceAvailable from: Thomas Lufkin[Show abstract] [Hide abstract]
ABSTRACT: Octamer-binding transcription factor 4 (Oct4) is a master regulator of early mammalian development. Its expression begins from the oocyte stage, becomes restricted to the inner cell mass of the blastocyst and eventually remains only in primordial germ cells. Unearthing the interactions of Oct4 would provide insight into how this transcription factor is central to cell fate and stem cell pluripotency. In the present study, affinity-tagged endogenous Oct4 cell lines were established via homologous recombination gene targeting in embryonic stem (ES) cells to express tagged Oct4. This allows tagged Oct4 to be expressed without altering the total Oct4 levels from their physiological levels. Modified ES cells remained pluripotent. However, when modified ES cells were tested for their functionality, cells with a large tag failed to produce viable homozygous mice. Use of a smaller tag resulted in mice with normal development, viability and fertility. This indicated that the choice of tags can affect the performance of Oct4. Also, different tags produce a different repertoire of Oct4 interactors. Using a total of four different tags, we found 33 potential Oct4 interactors, of which 30 are novel. In addition to transcriptional regulation, the molecular function associated with these Oct4-associated proteins includes various other catalytic activities, suggesting that, aside from chromosome remodeling and transcriptional regulation, Oct4 function extends more widely to other essential cellular mechanisms. Our findings show that multiple purification approaches are needed to uncover a comprehensive Oct4 protein interaction network.Stem Cell Research & Therapy 05/2011; 2(3):26. · 3.65 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: It is a general concern that the success of regenerative medicine-based applications is based on the ability to recapitulate the molecular events that allow stem cells to repair the damaged tissue/organ. To this end biomaterials are designed to display properties that, in a precise and physiological-like fashion, could drive stem cell fate both in vitro and in vivo. The rationale is that stem cells are highly sensitive to forces and that they may convert mechanical stimuli into a chemical response. In this review, we describe novelties on stem cells and biomaterials interactions with more focus on the implication of the mechanical stimulation named mechanotransduction.01/2011; 2:67-87.
Cdk1 Is Required for the Self-Renewal of Mouse Embryonic
Wei Wei Zhang,1*Xiao Jie Zhang,1Hui Xian Liu,1Jie Chen,1Yong Hong Ren,2
Deng Gao Huang,1Xiang Hong Zou,1,3**and Wei Xiao1,4
1College of Life Sciences, Capital Normal University, Beijing, China
2Microarray Laboratory, CapitalBio Corporation, Beijing, China
3Department of Pathology, Arthur G. James Comprehensive Cancer Center, The Ohio State University, Columbus, OH
4Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Cyclin-dependent kinase 1 (Cdk1) is indispensible for the early development of the embryo. However, its role in maintaining the
undifferentiated state of the embryonic stem (ES) cells remains unknown. In this study, we dissected the function of Cdk1 in mouse ES
cells by RNA-interference and gene expression analyses. Cdk1 expression is tightly correlated with the undifferentiated state of the ES cells.
Upon differentiation, Cdk1 expression reduced drastically. Cdk1 knock-down by RNA interference resulted in the loss of proliferation and
colony formation potential of the ES cells. Consequentially, expression of self-renewal genes was reduced while differentiation markers such
as Cdx2 were induced. Our results suggest a role for Cdk1 in maintaining the unique undifferentiated and self-renewing state of the mouse ES
cells. J. Cell. Biochem. 112: 942–948, 2011.
? 2010 Wiley-Liss, Inc.
KEY WORDS: EMBRYONIC STEM CELLS; GENE EXPRESSION; SELF-RENEWAL
Kaufman, 1981; Martin, 1981] are good model for the study of early
embryogenesis and development. Differentiation of ES cells into
multiple cell types in culture is a potential source for cell
replacement therapy [Li et al., 1998; Fujikura et al., 2002; Kyba
et al., 2002]. ES cells are also invaluable tools for the study of gene
functions [Nichols et al., 1998; Avilion et al., 2003; Mitsui et al.,
2003]. Understanding the molecular mechanisms underlying the
unique properties of self-renewal and pluripotency of the ES cells is
necessary to realize their clinical and scientific potentials.
Extensive studies have shown that transcription factors Oct4 and
Nanog play critical roles in maintaining the undifferentiated state of
mouse ES cells. Oct4 also known as Pou5f1, belongs to the POU (Pit-
Oct-Unc) transcription factor family. Oct4-null embryo dies at
luripotent mouse ES cells derived from the inner cell mass
(ICM) of the early pre-implantation embryo [Evans and
embryonic day 3.5 and the blastocyst are composed mainly of
trophectodermal cells without the ICM. Interestingly, Oct4 controls
pluripotency of ES cells in a dose-dependent manner [Niwa et al.,
2000]. A twofold induction of Oct4 led to ES cell differentiation into
primitive endoderm and mesoderm. Loss of Oct4, on the other hand,
triggers differentiation into trophectoderm lineages. These observa-
tions indicate that the appropriate level of Oct4 is critical for the
maintenance of ES cells. Nanog was first identified as ‘‘ENK’’ (early
embryo specific NK) based on its homology with members of the NK
gene family [Wang et al., 2003]. Two independent studies confirmed
the essential roles of Nanog in ES cell maintenance and embryonic
development [Chambers et al., 2003; Mitsui et al., 2003]. Nanog-null
embryo fails in the formation of primitive ectoderm, and dies at
embryonic day 4.5. Hence, Nanog is required for the ICM formation
and primitive ectoderm development [Mitsui et al., 2003]. ES cells
Journal of Cellular
Journal of Cellular Biochemistry 112:942–948 (2011)
Xiao Jie Zhang and Hui Xian Liu contributed equally to this work.
Wei Wei Zhang, Xiao Jie Zhang and Hui Xian Liu contributed equally to this work.
Additional Supporting Information may be found in the online version of this article.
*Correspondence to: Wei Wei Zhang, College of Life Sciences, The Capital Normal University, 105 Xi San Huan Bei
Road, Hai Dian District, #732, Lab Building, Beijing 100048, China. E-mail: firstname.lastname@example.org
*Correspondence to: Xiang Hong Zou, Department of Pathology, Arthur G. James Comprehensive Cancer Center, The
Ohio State University, 140 Hamilton Hall, 1645 Neil Ave Avenue, Columbus, OH 43210. E-mail: email@example.com
Received 2 September 2010; Accepted 20 December 2010 ? DOI 10.1002/jcb.23010 ? ? 2010 Wiley-Liss, Inc.
Published online 29 December 2010 in Wiley Online Library (wileyonlinelibrary.com).
derived from Nanog?/? ICM differentiate into endoderm lineage
cells [Chambers et al., 2003; Mitsui et al., 2003; Hatano et al., 2005].
Interestingly, over-expression of Nanog allows ES cells to bypass its
dependence on the LIF and BMP signaling pathway [Chambers
et al., 2003; Mitsui et al., 2003]. These results demonstrate the
indispensable roles of Nanog in early embryonic development and
ES cell maintenance.
Cyclin-dependent kinase 1 (Cdk1) along with cyclin B are
leadstoearly embryoniclethality, suggesting itscritical role inearly
embryonic development [Santamaria et al., 2007]. Repression of
Cdk1 resulted in the differentiation of trophoblast stem cells into
giant cells. Furthermore, inhibition of Cdk1 caused rapid apoptosis
of the ES cells [Ullah et al., 2008]. These findings suggest that Cdk1
could be involved in the maintenance of the unique undifferentiated
stateofearlystemcells.Itwas recentlyshownthatCdk1is amember
of the Oct4 interactome in mouse ES cells [Wang et al., 2006].
Interestingly, many members of the Oct4 interactome network are
necessary for the maintenance of self-renewal and pluripotency
[Wang et al., 2006].
Here we employed several strategies to dissect the functional role
of Cdk1 in ES cells. Upon differentiation of the ES cells by LIF
withdrawal or retinoic acid induction, Cdk1 expression was
Nanog reduced the expression of Cdk1. Using RNAi-mediated
knockdown, Cdk1-depleted ES cells resulted in cell death. Moreover,
mRNA levels of self-renewal genes such as Nanog, Tcl1, and Esrrb
were greatly reduced. On the other hand, trophectodermal genes
such as Cdx2, Hand1, and Mash2 increased significantly. This
suggests a role of Cdk1 in regulating the self-renewal state of the
MATERIALS AND METHODS
Mouse E14 ES cells (ATCC) were cultured under a feeder-free
condition at 378C with 5% CO2. The cells were maintained on
gelatin-coated dishes in Dulbecco’s modified Eagle medium
(DMEM; GIBCO), supplemented with 15% heat-inactivated fetal
bovine serum (FBS; GIBCO), 0.1mM b-mercaptoethanol (GIBCO),
L-glutamine,0.1mM MEM nonessential amino
5,000units/ml penicillin/streptomycin and 1,000units/ml of LIF
KNOCKDOWN PLASMIDS AND CELL TRANSFECTION
Oct4 and Nanog shRNA plasmids were constructed according to the
previous reports [Chew et al., 2005; Loh et al., 2006]. For Cdk1
shRNAs, 19 base pair (bp) gene-specific oligonucleotides for RNA
interference (RNAi) were designed and cloned into pSuperpuro
(Oligoengine) which carries a puromycin resistant. To ensure
the specificity of the oligonucleotides for RNAi, all sequences
were analyzed by BLAST search to remove any cross effect with
other genes. The sequences for shRNAs of different genes are listed
Transfection of shRNA plasmids was performed using Lipofecta-
mine 2000 (Invitrogen). For knockdown, 2mg of shRNA plasmids
were transfected into ES cells on 35mm plates. Puromycin (Sigma)
selection at 1.0mg/ml was introduced 24h after transfection, and
maintained for 2–6 days prior to RNA harvesting or Alkaline
phosphatase staining (Sigma).
RNA ISOLATION, REVERSE TRANSCRIPTION AND REAL-TIME PCR
Total RNA was extracted using Trizol reagent (Invitrogen). cDNA
synthesis was performed with 500ng of total RNA using
RevertAidTMFirst Strand cDNA Synthesis kit (Fermentas) according
to the manufacturer’s instructions. Endogenous mRNA levels were
measured by real-time PCR analysis based on SYBR Green detection
(Fermentas) with the BioRad real-time PCR machine. Results were
normalized with b-actin. All the primers used in the study gave rise
to single product of the right size in agarose gel analysis.
PROTEIN EXTRACTION AND WESTERN BLOTTING
Total protein was extracted by lysing cells with the whole cell
extraction buffer (Tris, 50mM; Nacl, 150mM; NP40, 1%; Glycerol,
10%; EDTA, 1mM; PMSF, 1mM). Thirty micrograms of the total
protein were separated by SDS–PAGE and transferred to PVDF 45
membrane. The membrane was blocked with 5% milk and probed
were developed with ECL Advance Western Blotting Detection Kit
(Amersham). Anti-Cdk1 antibodies (Bioworld, BS1820; Cell Signal-
ing, Y15; Abcam, E161) and mouse anti-b actin antibody (Boster,
BM0627) were used.
For the Cdk1-depleted ES cells, NimbleGene microarray platform
(NimbleGene mouse Gene Expression 12?135K Array) was used.
The mRNAs derived from Cdk1 shRNA 2 and vector-treated ES cells
were reverse-transcribed, labeled and analyzed. Arrays were
processed following the manufacturer’s instructions. Three bio-
logical replicates of the profiles were performed for both Cdk1-
depleted ES cells and vector control. RMA normalization was use to
normalize the microarrays. Significance analysis of microarrays
Cdk1 shRNA1 Sense strand: 50-GATCCCCGTATAAGGGTAGACAC
Sense strand: 50-GATCCCCGTACTTACGGTGTGGTGTATT
Sense strand: 50-GATCCCCGGACCATATTTGCAGA
Antisense strand: 50-AGCTTAAAAAGGACCATA
Sense strand: 50-GATCCCCGAAGTGGAAACTCA
Sense strand: 50-GATCCCCGTGGTCTTATCGTAGGA
Sense strand: 50-GATCCCCGCAAACATTAGCGAGTT
JOURNAL OF CELLULAR BIOCHEMISTRY
ROLE OF Cdk1 IN ES CELLS
(SAM)was usedto selectdifferentially expressed genes(Fold change
(FC) >1.5 for up-regulated, FC <0.6 for down-regulated; FDR <5%).
(GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG)
pathways through the Molecule Annotation System (MAS) platform
(http://bioinfo.capitalbio.com/mas). The GO terms and KEGG path-
ways with q values <0.01 were considered statistically significant.
FLOW CYTOMETRY ASSAY
ES cellswere platedat 3?106cells/well density in60mmplatesand
transfected with control and Cdk1 shRNA vectors. After 3 days of
1mg/ml puromycin selection, cells were collected for Flow
cytometry analysis. Briefly, cells were washed twice with PBS
and re-suspended in 75% ice-cold ethanol at 48C overnight. Cells
were then stained with 40,6-diamidino-2-phenylindole (DAPI,
Sigma) at a concentration of 1mg/ml at room temperature for
5min in dark. Cell cycle was analyzed using the FACSort flow
cytometer (Becton Dickinson, USA). For some analysis, ES cells were
stained with Annexin V FITC kit (Biosea).
EXPRESSION OF CDK1 CORRELATES WITH THE UNDIFFERENTIATED
STATE OF ES CELLS
We were interested to know whether the expression level of Cdk1
correlates with the undifferentiated state of ES cells. To achieve this,
we induced ES cells differentiation by the addition of retinoic acid
(RA) or the removal of LIF from the culture medium. Both the RA
induction and LIF withdrawal led to the reduction in the expression
levels of pluripotency genes Oct4, Nanog, and Sox2 (Fig. 1A).
Interestingly, differentiation of ES cells induced the suppression of
Cdk1 mRNA level. Next, we performed knockdown of Oct4 or Nanog
using RNA interference. Both the Nanog and Oct4 shRNA constructs
were effective inreducing the mRNAlevel of Nanog and Oct4 mRNA
respectively (Fig. 1B,C). Strikingly, either Oct4 or Nanog depletion
resulted in the dramatic down-regulation of Cdk1 level (Fig. 1D).
These findings suggested that the expression of Cdk1 is highly
correlated with the pluripotent ES cell state, and key regulators such
undifferentiated ES cells (Fig. 1A).
KNOCKDOWN OF CDK1 RESULTED IN THE REDUCED EXPRESSION
OF SELF-RENEWAL GENES AND THE INDUCTION OF
To investigate the role of Cdk1 in ES cell maintenance, we depleted
its expression using two shRNA constructs that targeted different
sites of the Cdk1 mRNA. After 2 days of puromycin selection, both
constructs efficiently reduced endogenous Cdk1 mRNA by 80% and
70%, respectively (Fig. 2). By western blotting analysis, we
confirmed that both Cdk1 shRNAs resulted in dramatic decrease
of Cdk1 protein (Supplementary Fig. S2). To further characterize the
role of Cdk1, we measured the expression levels of marker genes
cultured in medium with RA for 3 days or withdrawn of LIF for 5 days. The expression of Cdk1 was measured by quantitative real-time PCR analysis. Data are presented as the
mean?SEM. B,C: Oct4 and Nanog mRNA were depleted by their respective shRNA. The mRNA levels of Oct4 and Nanog were determined by real-time PCR after 4 days of
puromycin selection. Data are presented as the mean?SEM. D: The expression of Cdk1 was measured by real-time PCR in both Oct4- and Nanog-depleted ES cells. Data are
presented as the mean?SEM.
Undifferentiated mouse ES cells express high level of Cdk1. A: Reduction of Cdk1 expression in ES cells cultured in differentiation-inducing conditions. ES cells were
ROLE OF Cdk1 IN ES CELLS
JOURNAL OF CELLULAR BIOCHEMISTRY
important for self-renewal and differentiation. The expressions of
Sox2, Esrrb, and Tcl1 were significantly reduced to 30–40%, while
the expressions of Nanog and Tdgf1 were reduced to 50–60%
(Fig. 3A). Oct4 expression, however, did not change with the Cdk1
depletion. Conversely, the expressions of trophectoderm genes, such
as Cdx2, Hand1, and Mash2, were induced significantly upon the
knockdown of Cdk1. Changes in the expression of other lineage
specific genes such as Fgf5 and Msx1 were also observed (Fig. 3B). A
third Cdk1 shRNA construct targeting different site of Cdk1 gene
also show similar maker gene expression profiles (Supplementary
To further confirm the specificity of the knockdown, three shRNA
constructs with scrambled sequences were used. As expected, the
scrambled shRNA constructs did not reduce Cdk1 level (Supple-
mentary Fig. S3A), nor result in perturbation of the marker gene
expressions as seen in the Cdk1 knockdown (Supplementary Fig.
S3B). Taken together, the Cdk1 shRNA constructs were specific and
the cellular effects induced by the knockdown were not due to
aberrant off-targeting effects.
Next, we performed global gene expression profiling of the Cdk1-
knockdown ES cells using the NimbleGene microarray. We found
(q value?0.05) (Supplementary Table I). To further examine the
functional roles of Cdk1 in ES cell biology, we performed gene
ontology (GO) analysis. Notably, we found significant representa-
tion of various cellular processes, such as transcription regulation
and developmental processes (Supplementary Table II). Pathway
mapping of the genes regulated by Cdk1 identified their involve-
ment in various signaling pathways including the MAPK pathway
and Wnt signaling pathway (Supplementary Table III).
CDK1 DEPLETION REPRESSED ES CELL PROLIFERATION AND
RESULTED IN INCREASED APOPTOSIS
Next, we performed alkaline phosphatase (AP) staining of the Cdk1-
depleted ES cells. Cdk1 knockdown cells maintained positive signals
for the AP staining (Fig. 4A). However the cells grew in smaller
colonies as compared with the mock RNAi control. In cell
proliferation assay, Cdk1 knockdown significantly repressed the
ES cell growth and proliferation (Fig. 4B). Moreover, in the colony
re-plating assay, Cdk1 depleted ES cells did not form any colony
Furthermore, we analyzed the cell cycle profile of the Cdk1-
depleted ES cells. Using flow cytometry, we found that 4 days after
Cdk1 knockdown, ES cells were arrested at G2 phase (Fig. 5).
Together with the earlier findings demonstrating the reduction in
cell proliferation (Fig. 4), we speculated that the Cdk1 depletion in
ES cells may result in apoptosis. To demonstrate the possibility, we
performed Annexin V staining followed by flow cytometry.
Compared to the control cells, Cdk1 shRNA treated cells had higher
rate of apoptosis. This indicates that Cdk1 may be involved in the
inhibition of ES cell apoptosis (Fig. 6). For the cell cycle profiling
and Annexin V staining experiments, Cdk1 shRNA 3 showed similar
time PCR analysis of pluripotency associated genes in the Cdk1-depleted ES
cells. Cells were harvested after 4days of puromycin selection. The levels of the
transcripts were normalized against the control vector-transfected cells. Data
marker gene expressions in the Cdk1-depleted ES cells. Cells were harvested
after 4 days of puromycin selection. The levels of the transcripts were
normalized against the control pSuper-transfected cells. Data are presented
as the mean?SEM.
The expressions of marker genes in Cdk1-depleted ES cells. A: Real-
fected with Cdk1 shRNA constructs. Levels of knockdown were determined by
real-time PCR quantification of mRNA harvested after 1, 2, or 4 days of
puromycin selections. Data are presented as the mean?SEM.
shRNA knockdown of Cdk1 in ES cells. Mouse ES cells were trans-
JOURNAL OF CELLULAR BIOCHEMISTRY
ROLE OF Cdk1 IN ES CELLS
results with the shRNA 1 and 2 (Supplementary Figs. S4 and S5).
Together, our results indicate a role for Cdk1 in the regulation of
proliferation and the self-renewal of ES cells.
The cell cycle progression in eukaryotic somatic cells is tightly
regulated. Cdks, together with cyclins, are the major components of
the cell cycle machinery [Morgan, 1997]. Different Cdk-cyclin
complexes are respectively involved in specific cell cycle stages. For
example, the G1/S transition checkpoint is mainly regulated by the
Cdk4/6-cyclin D and Cdk2-cyclin Ecomplexes which phosphorylate
Rb and release E2F [Mittnacht, 1998; Trimarchi and Lees, 2002].
seeded at a density of 60?104cells in 60mm culture plate and transfected
with vector control, Cdk1 shRNA1 or Cdk1 shRNA2. The cells were fixed with
75% ethanol after 4 days of puromycin selection and analyzed by flow
Cdk1-depleted ES cells were arrested at G2 phase. ES cells were
puromycin selection. Pictures were taken of the bright-field magnification (upper panel) and the whole culture plate (lower panel) after AP staining. B: ES cells were seeded
at 104cells/well in a 24-well culture plate and transfected with vector control, Cdk1 shRNA1 and Cdk1 shRNA2, respectively. The cell growth was monitored for 4 days after
transfection. C: Transfected ES cells from B were dissociated after4 days of puromycin selection and re-seeded in a 6-well plate at 3?105cells perwell. The cells were cultured
in the medium with 1mg/ml puromycin and stained on day 7 after seeding.
Cdk1 depletion inhibits self-renewal of ES cells. A: Alkaline phosphatase staining assay was used to define undifferentiated cells. The cells were stained after 4 days of
ROLE OF Cdk1 IN ES CELLS
JOURNAL OF CELLULAR BIOCHEMISTRY
However, in ES cells, the cell cycle is uniquely short, primarily
owing to absence of the G1/S checkpoint [Savatier et al., 1996;
Becker et al., 2006]. And the Cdk4/Cdk6-associated kinase activity is
not present whereas the Cdk2-cyclinA/E activity is constitutively
active throughout the cell cycle in murine ES cells [Savatier et al.,
1996; Stead et al., 2002]. It is of great interest to determine the
relationship between the cell cycle regulation and pluripotency
maintenance of ES cells. Recently, Zhang et al. showed that
direct interaction with CDK6 and CDC25A in human ES cells [Zhang
et al., 2009]. The kinase activity of Cdk6 and its interaction with
Cyclin D can be detected in murine ES cells, and importantly, its
kinase activity decreased significantly upon differentiation [Faast
etal.,2004]. Genome-wide mapping ofthecoretranscription factors
in mouse ES cells had previously identified binding of Nanog
68797328) at the Cdk1 gene locus, indicating that Cdk1 may be
involved in the regulatory network responsible for maintaining the
properties of ES cells [Chen et al., 2008].
It was reported that deletion of Cdk1 leads to early embryonic
lethality prior to day E3.5. This indicates an essential role of Cdk1 in
early embryonic development [Santamaria et al., 2007; Satyanar-
ayana et al., 2008]. In our study, we show that Cdk1 depletion
compromised the proliferation and self-renewal of the ES cells. This
is consistent with a previous study where treatment of mouse ES
cells with Cdk1 inhibitor resulted in cell death and increased
apoptosis [Ullah et al., 2008]. In Cdk1 knockdown cells, we found
reduction in the level of Sox2, Esrrb, and Tcl1. Interestingly, we did
not detect a reduction in the Oct4 mRNA level during Cdk1
knockdown. The regulation of Cdk1 on self-renewal genes could be
direct or indirect. Of note, Cdk1 has been previously shown to
interact with Oct4, a key transcription factor that regulates
expression of many genes critical for ES cells [Wang et al.,
2006]. The mechanistic role of Cdk1 and Oct4 interaction remains to
be identified. Cdk1 repression also caused the up-regulation of
marker genes for trophectoderm lineages. Interestingly, a previous
differentiation [Ullah et al., 2008]. How Cdk1 regulates the
repression of ES cell differentiation to the trophectoderm lineage,
and whether it shares similar mechanistic role in ES cells and
trophoblast stem cells will be of great interest for future studies.
Inconclusion, ourstudy demonstrated highexpression ofCdk1 in
undifferentiated state of ES cells. We confirmed the important role
of Cdk1 in maintaining ES cell proliferation and self-renewal.
Furthermore, we uncovered the role for Cdk1 in the inhibition of
trophectoderm differentiation of the ES cells.
This work was supported by special fund for Cell Biology Project
09532310099, 211 special fund (09531971399) and the PhD startup
fund from the Capital Normal University.
Avilion AA, Nicolis SK, Pevny LH, Perez L, Vivian N, Lovell-Badge R. 2003.
Multipotent cell lineages in early mouse development depend on SOX2
function. Genes Dev 17:126–140.
2006. Self-renewal of human embryonic stem cells is supported by a
shortened G1 cell cycle phase. J Cell Physiol 209:883–893.
Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A.
2003. Functional expression cloning of Nanog, a pluripotency sustaining
factor in embryonic stem cells. Cell 113:643–655.
Chen X, Xu H, Yuan P, Fang F, Huss M, Vega VB, Wong E, Orlov YL, Zhang
W, Jiang J, Loh YH, Yeo HC, Yeo ZX, Narang V, Govindarajan KR, Leong B,
Shahab A, Ruan Y, Bourque G, Sung WK, Clarke ND, Wei CL, Ng HH. 2008.
Integration of external signaling pathways with the core transcriptional
network in embryonic stem cells. Cell 133:1106–1117.
Chew JL, Loh YH, Zhang W, Chen X, Tam WL, Yeap LS, Li P, Ang YS, Lim B,
Robson P, Ng HH. 2005. Reciprocal transcriptional regulation of Pou5f1 and
Sox2 via the Oct4/Sox2 complex in embryonic stem cells. Mol Cell Biol
Evans MJ, Kaufman MH. 1981. Establishment in culture of pluripotential
cells from mouse embryos. Nature 292:154–156.
Faast R, White J, Cartwright P, Crocker L, Sarcevic B, Dalton S. 2004. Cdk6-
cyclin D3 activity in murine ES cells is resistant to inhibition by p16(INK4a).
Fujikura J, Yamato E, Yonemura S, Hosoda K, Masui S, Nakao K, Miyazaki Ji
J, Niwa H. 2002. Differentiation of embryonic stem cells is induced by GATA
factors. Genes Dev 16:784–789.
Hatano SY, Tada M, Kimura H, Yamaguchi S, Kono T, Nakano T, Suemori H,
Nakatsuji N, Tada T. 2005. Pluripotential competence of cells associated with
Nanog activity. Mech Dev 122:67–79.
myeloid engraftment potential on embryonic stem cell and yolk sac hema-
topoietic progenitors. Cell 109:29–37.
Li M, Pevny L, Lovell-Badge R, Smith A. 1998. Generation of purified neural
precursors from embryonic stem cells by lineage selection. Curr Biol 8:971–
Loh YH, Wu Q, Chew JL, Vega VB, Zhang W, Chen X, Bourque G, George J,
Leong B, Liu J, Wong KY, Sung KW, Lee CW, Zhao XD, Chiu KP, Lipovich L,
Kuznetsov VA, Robson P, Stanton LW, Wei CL, Ruan Y, Lim B, Ng HH. 2006.
The Oct4 and Nanog transcription network regulates pluripotency in mouse
embryonic stem cells. Nat Genet 38:431–440.
Martin GR. 1981. Isolation of a pluripotent cell line from early mouse
embryos cultured in medium conditioned by teratocarcinoma stem cells.
Proc Natl Acad Sci USA 78:7634–7638.
Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K,
Maruyama M, Maeda M, Yamanaka S. 2003. The homeoprotein Nanog is
were transfected with vector control, Cdk1 shRNA1 and Cdk1 shRNA2,
respectively. After 4 days, transfected cells were double-stained with
Annexin V and propidium iodide and analyzed by flow cytometry.
Reduction of Cdk1 expression resulted in ES cells apoptosis. ES cells
JOURNAL OF CELLULAR BIOCHEMISTRY
ROLE OF Cdk1 IN ES CELLS
required for maintenance of pluripotency in mouse epiblast and ES cells. Cell
Mittnacht S. 1998. Control of pRB phosphorylation. Curr Opin Genet Dev
Morgan DO. 1997. Cyclin-dependent kinases: Engines, clocks, and micro-
processors. Annu Rev Cell Dev Biol 13:261–291.
Nichols J, Zevnik B,Anastassiadis K,Niwa H, Klewe-Nebenius D, ChambersI,
Scholer H, Smith A. 1998. Formation of pluripotent stem cells in the
mammalian embryo depends on the POU transcription factor Oct4. Cell
Niwa H, Miyazaki J, Smith AG. 2000. Quantitative expression of Oct-3/4
defines differentiation, dedifferentiation or self-renewal of ES cells. Nat
Dubus P, Malumbres M, Barbacid M. 2007. Cdk1 is sufficient to drive the
mammalian cell cycle. Nature 448:811–815.
Satyanarayana A, Berthet C, Lopez-Molina J, Coppola V, Tessarollo L,
Kaldis P. 2008. Genetic substitution of Cdk1 by Cdk2 leads to embryonic
lethality and loss of meiotic function of Cdk2. Development 135:3389–3400.
Savatier P, Lapillonne H, van Grunsven LA, Rudkin BB, Samarut J. 1996.
Withdrawal of differentiation inhibitory activity/leukemia inhibitory factor
embryonic stem cells. Oncogene 12:309–322.
Stead E, White J, Faast R, Conn S, Goldstone S, Rathjen J, Dhingra U, Rathjen
P, Walker D, Dalton S. 2002. Pluripotent cell division cycles are driven by
ectopic Cdk2, cyclin A/E and E2F activities. Oncogene 21:8320–8333.
Trimarchi JM, Lees JA. 2002. Sibling rivalry in the E2F family. Nat Rev Mol
Cell Biol 3:11–20.
CDK1 activity. Genes Dev 22:3024–3036.
Wang SH, Tsai MS, Chiang MF, Li H. 2003. A novel NK-type homeobox gene,
ENK (early embryo specific NK), preferentially expressed in embryonic stem
cells. Gene Expr Patterns 3:99–103.
A protein interaction network for pluripotency of embryonic stem cells.
Zhang X, Neganova I, Przyborski S, Yang C, Cooke M, Atkinson SP, Any-
fantis G, Fenyk S, Keith WN, Hoare SF, Hughes O, Strachan T, Stojkovic M,
Hinds PW, Armstrong L, Lako M. 2009. A role for NANOG in G1 to S
transition in human embryonic stem cells through direct binding of CDK6
and CDC25A. J Cell Biol 184:67–82.
ROLE OF Cdk1 IN ES CELLS
JOURNAL OF CELLULAR BIOCHEMISTRY