LETTERS
Derivation of pluripotent epiblast stem cells from mammalian embryos
I.Gabrielle M.Brons 1,Lucy E.Smithers 2,Matthew W.B.Trotter 2,Peter Rugg-Gunn 1{,Bowen Sun 1,Susana M.Chuva de Sousa Lopes 3,Sarah K.Howlett 4,Amanda Clarkson 5,Lars Ahrlund-Richter 6,Roger A.Pedersen 1&Ludovic Vallier 1
Although the first mouse embryonic stem (ES)cell lines were derived 25years ago 1,2using feeder-layer-based blastocyst cul-tures,subsequent efforts to extend the approach to other mam-mals,including both laboratory and domestic species,have been relatively unsuccessful.The most notable exceptions were the derivation of non-human primate ES cell lines 3followed shortly thereafter by their derivation of human ES cells 4.Despite the apparent common origin and the similar pluripotency of mouse and human embryonic stem cells,recent studies have revealed that they use different signalling pathways to maintain their pluripo-tent status.Mouse ES cells depend on leukaemia inhibitory factor and bone morphogenetic protein,whereas their human counter-parts rely on activin (INHBA)/nodal (NODAL)and fibroblast growth factor (FGF).Here we show that pluripotent stem cells can be derived from the late epiblast layer of post-implantation mouse and rat embryos using chemically defined,activin-containing culture medium that is sufficient for long-term main-tenance of human embryonic stem cells.Our results demonstrate that activin/Nodal signalling has an evolutionarily conserved role in the derivation and the maintenance of pluripotency in these novel stem cells.Epiblast stem cells provide a valuable experi-mental system for determining whether distinctions between mouse and human embryonic stem cells reflect species differences or diverse temporal origins.
We initially determined that prolonged culture of human ES cells in chemically defined medium 5(CDM)containing activin A and FGF2(CDM/AF)maintained their fundamental characteristics (See Supplementary Data).We then tested similar conditions for derivation of pluripotent cells from pre-and post-implantation rodent embryos.Isolated inner cell masses (ICM)grown in CDM/AF never gave rise to pluripotent cell lines,but underwent rapid differentiation (Table 1and Supplementary Fig.2).In contrast,when
late epiblast layers were dissected from pre-gastrulation stages (5.75days post coitum (d.p.c.)/embryonic day (E)5.75)for a B63CBA F 1genetic background or 6.5d.p.c.for a NOD genetic background)and cultured for 24h in CDM/AF,they formed colonies of compact cells with high nucleo–cytoplasmic ratios (Fig.1c),a characteristic trait of pluripotent stem cells.Cells on the periphery started to differentiate the following day,producing a broad ring of stromal cells surround-ing smaller aggregates of compact cells,which grew to form larger colonies of compact cells 4–5days later (Fig.1a–j).Such colonies were picked,fragmented into smaller clumps using collagenase and mechanical dissociation,and similarly passaged at 5-day intervals.Immunostaining after 20passages showed that these colonies of compact cells expressed the pluripotency markers Oct-4(Pou5f1),Nanog and SSEA-1(Fut4).They were designated epiblast stem cells (EpiSCs)on the basis of their origin from pure late epiblast cell layers.The high rate of derivation (83%for B63CBA F 1background and 93%for the NOD genetic background;Table 1)was striking for strains considered ‘non-permissive’for mouse ES cell derivation 6.Expression of Oct-4,Nanog and SSEA-1approached 95%and was maintained for more than 40passages (Fig.1k;Supplementary Fig.3),suggesting that EpiSCs represent a near-homogenous population of pluripotent cells.
EpiSCs grew as flat,compact colonies and were thus morphologi-cally distinct from mouse ES cells,which form rounded colonies (Fig.1k).Unlike mouse ES cells,passaging of EpiSCs using trypsin or other single-cell dissociation methods induced widespread cell death.No EpiSC lines could be derived in the presence of LIF and/or BMP4(Supplementary Fig.4a),the two factors required for mouse ES cell derivation and self-renewal.On the other hand,the activin receptor inhibitor SB431542induced rapid differentiation of EpiSCs (Supple-mentary Fig.3and Supplementary Fig.4b),showing that EpiSC plur-ipotency depends strictly on activin/Nodal signalling.Finally,mouse
1
Department of Surgery and Cambridge Institute for Medical Research,Addenbrooke’s Hospital,University of Cambridge,Cambridge CB20XY,UK.2CR-UK Viral Oncology Group,Wolfson Institute for Biomedical Research,UCL,Cruciform Building Gower Street,London WC1E 6BT,UK.3Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology and Department of Physiology,University of Cambridge,Tennis Court Road,Cambridge CB2,1QR,UK.4Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory,University of Cambridge,Cambridge CB20XY,UK.5Medical Genetics Department Cambridge University Hospital NHS Foundation Trust,Kefford House Maris Lane Cambridge CB22FF,UK.6Dept of Laboratory Medicine Clinical Research Centre,Karolinska University Hospital Karolinska Institutet 14157Stockholm,Sweden.{Present address:Hospital for Sick Children,Toronto Medical Discovery Tower 101College Street,Toronto,Ontario M5G 1L7,TMDT,Canada.
Table 1|Efficiency of derivation of EpiSCs from mouse and rat embryos
Number of epiblasts
d.p.c.Temperature
Matrix
Growth factors added to CDM
Lines derived
Rate (%)
Mouse Rat
Mouse Rat
B 63CBA NOD
Wistar
Sprague-Dawley
B 63CBA
NOD
Wistar
Sprague-Dawley
323.537u C Fibro Activin 1FGF 2006
5.7537u C Fibro Activin 1FGF 25
8396.537u C Fibro Activin 1FGF 2
91005
6.537u C Fibro Activin 1FGF 21noggin 4
8014
7.7538u C FCS Activin 4
28.520
7.7537u C Fibro Activin
15
Vol 448|12July 2007|doi:10.1038/nature05950
191
EpiSCs could be derived in CDM supplemented with activin alone (data not shown),indicating that FGF was not strictly required during this process.However,FGF improved the overall quality of the cul-tures,suggesting that it reinforces the efficiency of activin signalling,as in human ES cells 7.Taken together,these observations show that,despite their expression of standard markers of pluripotency,mouse EpiSCs differ significantly from mouse ES cells in their growth factor dependence,their colony morphology,and in being averse to pas-saging as single cells.Importantly,EpiSCs were also derived from rat embryos (which have not previously yielded ES cells through conven-tional derivation methods)at pre-gastrulation stages (E7.5–7.75)(Fig.1a–j,Fig.1k and Supplementary Information),suggesting that activin/Nodal signalling may have a broader role than that of LIF and BMP4in maintenance of pluripotency in embryonic stem cells.
We characterized EpiSCs and their differentiated progeny using both quantitative (Q-PCR)and qualitative (PCR,immunostaining)methods.(Because of the apparent similarity of mouse and rat EpiSCs,we focused this further characterization on mouse EpiSCs,except where noted).EpiSCs expressed higher levels of Oct-4and Nanog and similar levels of Sox2as compared with mouse ES cells (Fig.2a).Conversely,EpiSCs did not express Rex1(Zfp42),a specific marker of the ICM 8that is silenced in early epiblast cells just after implantation but is expressed in mouse ES cells (Fig.2a).EpiSC transcript levels for Gbx2,a marker of mouse ICM and ES cells,was one fourth that of mouse ES cells (Supplementary Fig.5a).On
the other hand,transcripts coding for FGF5and Nodal,two genes specifically expressed in the late epiblast layer 8after implantation,were clearly detected in EpiSCs,in contrast to their low levels in mouse ES cells (Fig.2a).Interestingly,early primitive ectoderm-like (EPL)cells that were established by growing mouse ES cells in HepG2-conditioned medium in the absence of LIF 9also express FGF5,but not Rex1.However,EPL cells revert to ES cells in the presence of LIF—which is not possible with EpiSCs (data not shown)—and EPL cells are unable to differentiate in vitro into extra-embryonic tissues,which can be generated from EpiSCs grown in the presence of BMP4(see below),thus excluding the possibility that EpiSCs are EPL cells.Curiously,alkaline phosphatase activity,which generally marks pluripotent cells (including human ES cells and mouse ES cells),was present in the late epiblast layer itself,but disappeared abruptly on culture and was undetectable in EpiSCs (Fig.2b;Supplementary Fig.5b).The lack of alkaline phosphatase activity also distinguishes EpiSCs from germ cells because the activity persists in primordial germ cells 10,11in the gastrulating embryo 12and in their in vitro derivatives,the embryonic germ cells.In addition,EpiSCs did not express germ cell markers such Blimp1and Stella (Supplementary Information),thereby confirming that EpiSCs are not derivatives of primordial germ cells.
To define further the molecular properties of EpiSCs,we performed a global analysis of their expression profile using gene expression microarrays.Expression profiles of EpiSCs were compared to those
Mouse embryos
5.75 dpc
Dissected mouse
epiblast
Rat epiblast colony
day 1Rat epiblast colony
day 2
Rat EpiSCs p7
Rat embryos at 7.5 dpc Dissected rat epiblast
Mouse epiblast colony
day 1
Mouse epiblast colony
day 2
Mouse EpiSCs
p32
a b c d e
j
i h g f k
Mouse EpiSCs
Rat EpiSCs
mESCs
SSEA1
Nanog Oct-4Figure 1|Derivation of pluripotent epiblast stem cells (EpiSCs)from the late epiblast layer of embryos at post-implantation stages.a –j ,Successive stages in EpiSC derivation from mouse (top)and rat (bottom)embryos.a ,f ,Mouse and rat embryos at pre-gastrula stages (respectively,5.75d.p.c and 7.75d.p.c).b ,g ,Pure epiblast layer obtained after removal of the extra-embryonic tissues.c ,h ,Epiblast layer grown for 24h in CDM supplemented with activin (and FGF2for mouse embryos).d ,i ,Epiblast outgrowth after 48h of culture.Arrows indicate compact colonies of undifferentiated cells surrounded by stromal cells.e ,j ,EpiSC colony after prolonged culture (32passages or 9months for mouse EpiSCs;7passages or 2months for rat EpiSCs).Scale bar,100m m.k ,Expression of pluripotency markers in mouse ES cells,in mouse EpiSCs and in rat EpiSCs.Oct-4,Nanog and SSEA-1expression were analysed by immunofluorescence in the mouse ES cell (mESC)line R1of 129strain at passage (p)35,in mouse EpiSCs of the NOD genetic background at p20,and in rat EpiSCs of the Wistar strain at p15.Scale bar,100m m.Nuclei are shown by Hoechst staining.
LETTERS
NATURE |Vol 448|12July 2007
192
of ICMs from mouse blastocysts,late epiblast cells from post-implantation mouse embryos,and mouse ES cells using multidimen-sional scaling(Fig.2c).This showed that EpiSCs had fewer differences with dissected late epiblast than with ICM or mouse ES cells,suggest-ing that EpiSCs are transcriptionally similar to their embryonic tissue source.Despite sharing expression of core transcription factor circuitry with mouse ES cells,the EpiSCs established by culturing late epiblast layers in CDM/AF differ from mouse ES cells in other key attributes,suggesting that EpiSCs are a novel type of embryonic stem cell that closely resembles the late epiblast layer of the intact post-implantation mouse embryo from which they have been derived.
To assess the extent of pluripotency of EpiSCs,we used a com-bination of in vivo and in vitro assessments involving ectopic transplantation,chimaera production,and embryoid body forma-tion to examine their differentiative potential.EpiSC colonies injected into the testis capsule of immunodeficient mice were capable of forming teratomas containing a wide variety of tissues,including muscle,cartilage,neuronal rosettes,liver and gut(Fig.3a–d),thereby reaffirming the multi-lineage pluripotency of EpiSCs.Next,
their R
e
l
a
t
i
v
e
g
e
n
e
e
x
p
r
e
s
s
i
o
n
Oct-4
Pou5f1
0.0
M
D
S
1
MDS2
0.5
1.0
R1 p14
a
CGR8 p25
B6.10a p38
NOD21 p26
1.5
2.0
2.5
Nanog Sox2Rex1
Zfp42
FGF5Nodal
EpiSCs hESCs
Epiblast colony
b
c
Figure2|Embryonic identity of EpiSCs.a,Expression of pluripotency
markers specific for pre-and post-implantation embryonic stages in mouse ES
cells and in mouse EpiSCs.Q-PCR analyses were performed to detect the genes
denoted in two mouse ES cell lines(R1p14and CGR8p25),and in2mouse
EpiSC lines(B6.10a p38,NOD21p26).Expression for each denoted gene was
normalized with B2m(beta-2microglobulin)and the resulting comparative
levels,calculated from cycle thresholds,are indicated.Similar results were
obtained in three independent experiments and error bars indicate their
standard deviation.b,Absence of alkaline phosphatase activity in EpiSCs.
Enzymatic activity for alkaline phosphatase was analysed in mouse epiblast
cultured for12h after dissection and mouse EpiSCs of B63CBA F1genetic
background(left and middle panels).Human(h)ESCs were used as positive
controls(right panel).Scale bar,100m m.c,Comparison of expression profiles
of ICM from embryos at the blastocyst stage,late epiblast from embryos at
post-implantation stages,mouse ES cells and mouse EpiSCs.Microarray
analysis of the transcriptional profile of ICM
(mICM),epiblast
(mEpi),
mouse
ES
cells
(mESCs)
and
mouse
EpiSCs(EpiSC)were compared using an MDS
plot(see Supplementary Information for full details of analysis).
a b
c
e
d
βIII tubulin
(Tubb3)
Sox1
Nestin
(Nes)
Brachyury
(T)
Sox17
M
e
s
o
e
n
d
o
d
e
r
m
N
e
u
r
o
e
c
t
o
d
e
r
m
Figure3|EpiSCs are capable of differentiating into the three primary germ
layers in vitro and in vivo.a,Teratomas from mouse EpiSCs.
a–d,Differentiated derivatives of all three embryonic germ layers.Areas
enclosed in boxes in a are enlarged in panels b(neuronal ganglion),
c(striated muscle)and d(gut epithelium with Goblet cells).Scale bar,
100m m.e,Expression of mesendoderm and neuroectoderm markers in
differentiated derivatives of EpiSCs.EpiSCs(NOD21p20)were grown in
culture conditions inducing neuroectoderm or mesendoderm
differentiation.Expression of brachyury,Sox17,b III tubulin,Sox1and
nestin was then analysed by immunostaining.Scale bar,50m m. NATURE|Vol448|12July2007LETTERS
193
capacity for integration into pre-implantation-stage mouse embryos was examined by injecting EpiSCs into mouse blastocysts or by aggregating mouse embryos at the8cells or morula stage with EpiSC clumps(see Supplementary Information).Only two chimaeras were obtained out of385injected blastocysts,and germline transmis-sion was not observed.Together these results suggest that,unlike the case for mouse ES cells,the pre-implantation embryo does not rep-resent a compatible environment for EpiSCs,possibly owing to a developmental asynchrony that limits the ability of EpiSCs to colonise the host embryos.Alternatively,sparse chimaerism could be explained by a limited capacity of EpiSCs for development into early cell lineages.
Accordingly,to examine their differentiative capacity in greater detail,we determined whether EpiSCs could differentiate into a wide variety of cell types in vitro.Differentiation was achieved either by growing EpiSC colonies as embryoid bodies(Supplementary Information)or by differentiating them as monolayers into meso-derm,endoderm and ectoderm cells using a protocol devised for human ES cells(L.V.,Touboul T.,Chng,Z.,Millan,E.,Trotter, M.,Weber,A.&R.A.P.,unpublished observations).Generation of derivatives of the three germ layers was confirmed by the expression of early and late markers(Supplementary Fig.7a)including brachy-ury(mesoderm),Sox17(endoderm),Sox1,nestin and b2III tubulin (neuroectoderm)(Fig.3e).As a further assessment of their in vitro differentiative capacity,EpiSCs were exposed to BMP4,which has been shown to induce differentiation of human ES cells into prim-itive endoderm and trophectoderm13.EpiSCs grown in CDM sup-plemented with BMP4in the absence of activin signalling rapidly differentiated into cells expressing markers of primitive endoderm (Sox7,Gata4,Gata6)and trophectoderm(Cdx2,Hand1,Eomes, H19,a CG/Cga)(Supplementary Fig.7b and7c).Moreover,brachy-ury,Sox17and Mixl1were not detected in these conditions(Sup-plementary Fig.7b),suggesting that BMP4is insufficient to drive differentiation of EpiSCs into mesendoderm.
Finally,similar results were obtained with clonal sublines of mouse EpiSCs(Supplementary Information),demonstrating that single EpiSC cells were capable of recapitulating the entire range of differ-entiative outcomes and thereby ruling out the possibility of a mixed cell population with diverse capabilities.All together,these results demonstrate that EpiSCs were capable of differentiating into deriva-tives of all three primary germ layers and into extra-embryonic tissues in vitro.Such broad differentiative capacity reaffirms the pluripotent status of EpiSCs,thus leading to the conclusion that limited EpiSC contribution in chimaeras is attributable to develop-mental asynchrony.Taken together,these results demonstrate that pluripotent stem cells can be cultured from widely separated mam-malian species using chemically defined culture medium supple-mented with activin.On this basis,we conclude that the activin/ Nodal pathway has a central role in pluripotency of a new type of embryonic stem cells derived from mammalian embryos,specifically representing the late epiblast cell population just before gastrulation. EpiSCs differ from mouse ES cells not only in their embryonic tissue of origin but also in the signalling pathway maintaining their pluripotent status(activin/Nodal versus LIF).This dependence of EpiSC pluripotency on activin/Nodal signalling also highlights their resemblance to late epiblast in vivo.Indeed,recent publications have established that embryos mutant for Nodal lack the expression of the pluripotency markers Oct-4and Nanog,instead ectopically expres-sing markers of anterior neuroectoderm14,15.Our results extend these recent findings by showing that activin/Nodal signalling not only is necessary for blocking neuroectoderm specification in vivo,but also that it is sufficient for maintenance of pluripotency in stem cells cultured from the late epiblast.The dependency of mouse ES cells on LIF signalling has focused attention on the early epiblast(E4.5or equivalent)as their likely tissue of origin16.This reflects the narrow temporal role of LIF/GP130signalling,which is required for mouse ICM viability only during delayed implantation.Moreover,mouse embryos that are homozygous mutants for Nodal17or its type I and II receptors18,19,progress well beyond the blastocyst stage,arresting only at pre-gastrula stages,consistent with the hypothesis that LIF and activin/Nodal signalling immortalize stem cell populations at distinct developmental stages,namely,early and late epiblast,respectively. Intriguingly,EpiSCs share with human ES cells not only a depend-ence on activin/Nodal signalling but also other key differences from mouse ES cells that have previously been attributed to species diver-gence(flattened colony morphology,inefficient clonal propagation and a limited capacity for colonising pre-implantation embryos20). Moreover,despite having similar core transcription factor circuitry, human ES cells and mouse ES cells have substantially different target genes for Oct-4and Nanog21,22.Also,the epigenetic stability of human ES cells seems to be greater than mouse ES cells,as shown here and previously by their extent of monoallelic imprinted gene expression23,24.Finally,EpiSCs(like human ES cells13)have been shown to differentiate into trophectoderm in the presence of BMP4,whereas mouse ES cells have little or no capacity for contri-bution to either primitive endoderm or trophectoderm lineages in chimaeric embryos25and differentiate into trophectoderm only when their Oct-4gene is mutated by homologous recombination26.In summary,EpiSCs are temporally defined,embryonic stem cells that add a strategically important experimental system for under-standing the development of pluripotency.It has not escaped our attention that the unique properties of human ES cells,now seen to be shared with EpiSCs,could reflect a similar late epiblast origin. Understanding the relationships between cell-surface-mediated growth factor activities,core transcription factor circuitry and the epigenetic modifications accompanying pluripotency in EpiSCs could contribute significant insights to this issue.
METHODS
For derivation of EpiSCs,the late epiblast layer was dissected intact from pre-gastrula stage mouse[E5.75,(B6XCBA)F1mated inter se;E6.5,NO D mated inter se]or rat[E7.5-7.75Wistar or Sprague Dawley]embryos after15-20min treatment at4u C in Cell Dissociation Buffer(Gibco).Dissection was accomp-lished in Calcium and Magnesium free Flushing and Handling Medium using glass needles to completely separate the epiblast layer from the extraembryonic tissues.Epiblast layers were then placed in chemically defined medium(CDM)5, supplemented with Activin A(20ng/ml,RandD systems)and FGF2(12ng/ml, RandD systems).Every4-5days,cells were harvested using5mg/ml collagenase IV(Invitrogen)then plated into plates(Costar)pre-coated either with fetal bovine serum(FBS)or with15m g/ml of human Fibronectin(Chemicon)for 20min at37C and then washed twice in PBS.(Additional methods can be found in Supplementary Information).
Full Methods and any associated references are available in the online version of the paper at https://www.doczj.com/doc/9c11744833.html,/nature.
Received15January;accepted24May2007.
Published online27June2007.
1.Evans,M.J.&Kaufman,M.H.Establishment in culture of pluripotential cells from
mouse embryos.Nature292,154–156(1981).
2.Martin,G.R.Isolation of a pluripotent cell line from early mouse embryos cultured
in medium conditioned by teratocarcinoma stem cells.Proc.Natl https://www.doczj.com/doc/9c11744833.html,A 78,7634–7638(1981).
3.Thomson,J.A.et al.Isolation of a primate embryonic stem cell line.Proc.Natl
https://www.doczj.com/doc/9c11744833.html,A92,7844–7848(1995).
4.Thomson,J.A.et al.Embryonic stem cell lines derived from human blastocysts.
Science282,1145–1147(1998).
5.Johansson,B.M.&Wiles,M.V.Evidence for involvement of activin A and bone
morphogenetic protein4in mammalian mesoderm and hematopoietic
development.Mol.Cell.Biol.15,141–151(1995).
6.Brook,F.A.et al.The derivation of highly germline-competent embryonic stem
cells containing NOD-derived genome.Diabetes52,205–208(2003).
7.Vallier,L.,Alexander,M.&Pedersen,R.A.Activin/Nodal and FGF pathways
cooperate to maintain pluripotency of human embryonic stem cells.J.Cell Sci.118, 4495–4509(2005).
8.Pelton,T.A.,Sharma,S.,Schulz,T.C.,Rathjen,J.&Rathjen,P.D.Transient
pluripotent cell populations during primitive ectoderm formation:correlation of in vivo and in vitro pluripotent cell development.J.Cell Sci.115,329–339(2002).
LETTERS NATURE|Vol448|12July2007 194
9.Rathjen,J.et al.Formation of a primitive ectoderm like cell population,EPL cells,
from ES cells in response to biologically derived factors.J.Cell Sci.112,601–612 (1999).
10.Resnick,J.L.,Bixler,L.S.,Cheng,L.&Donovan,P.J.Long-term proliferation of
mouse primordial germ cells in culture.Nature359,550–551(1992).
11.Matsui,Y.,Zsebo,K.&Hogan,B.L.Derivation of pluripotential embryonic stem
cells from murine primordial germ cells in culture.Cell70,841–847(1992). 12.De Felici,M.&McLaren,A.Isolation of mouse primordial germ cells.Exp.Cell Res.
142,476–482(1982).
13.Xu,R.H.et al.BMP4initiates human embryonic stem cell differentiation to
trophoblast.Nature Biotechnol.20,1261–1264(2002).
14.Camus,A.,Perea-Gomez,A.,Moreau,A.&Collignon,J.Absence of Nodal
signaling promotes precocious neural differentiation in the mouse embryo.Dev.
Biol.295,743–755(2006).
15.Mesnard,D.,Guzman-Ayala,M.&Constam,D.B.Nodal specifies embryonic
visceral endoderm and sustains pluripotent cells in the epiblast before overt axial patterning.Development133,2497–2505(2006).
16.Nichols,J.,Chambers,I.,Taga,T.&Smith,A.Physiological rationale for
responsiveness of mouse embryonic stem cells to gp130cytokines.Development 128,2333–2339(2001).
17.Conlon,F.L.et al.A primary requirement for nodal in the formation and
maintenance of the primitive streak in the mouse.Development120,1919–1928 (1994).
18.Song,J.et al.The type II activin receptors are essential for egg cylinder growth,
gastrulation,and rostral head development in mice.Dev.Biol.213,157–169(1999).
19.Gu,Z.et al.The type I serine/threonine kinase receptor ActRIA(ALK2)is required
for gastrulation of the mouse embryo.Development126,2551–2561(1999). 20.James,D.,Noggle,S.A.,Swigut,T.&Brivanlou,A.H.Contribution of human
embryonic stem cells to mouse blastocysts.Dev.Biol.295,90–102(2006). 21.Boyer,L.A.et al.Core transcriptional regulatory circuitry in human embryonic
stem cells.Cell122,947–956(2005).
22.Loh,Y.H.et al.The Oct4and Nanog transcription network regulates pluripotency
in mouse embryonic stem cells.Nature Genet.38,431–440(2006).
23.Rugg-Gunn,P.J.,Ferguson-Smith,A.C.&Pedersen,R.A.Epigenetic status of
human embryonic stem cells.Nature Genet.37,585–587(2005).24.Sun,B.W.et al.Temporal and parental-specific expression of imprinted genes in a
newly derived Chinese human embryonic stem cell line and embryoid bodies.
Hum.Mol.Genet.15,65–75(2006).
25.Beddington,R.S.&Robertson,E.J.An assessment of the developmental potential
of embryonic stem cells in the midgestation mouse embryo.Development105, 733–737(1989).
26.Niwa,H.,Miyazaki,J.&Smith,A.G.Quantitative expression of Oct-3/4defines
differentiation,dedifferentiation or self-renewal of ES cells.Nature Genet.24, 372–376(2000).
Supplementary Information is linked to the online version of the paper at https://www.doczj.com/doc/9c11744833.html,/nature.
Acknowledgements We thank A.McLaren for her support.We thank A.Smith for CGR8cells,A.Nagy for R1cells,P.Andrew for the SSEA-1antibody,L.Wicker for access to NOD mice,and S.Thiru for advice in the teratoma analysis.This work was supported by an MRC International Appointments Initiative grant(R.A.P),MRC/ Juvenile Diabetes Research Foundation Centre funding(R.A.P.,I.G.M.B.),Remedi (I.G.M.B.),the Wellcome Trust Functional Genomics Initiative on Stem Cells(L.E.S, M.W.B.T.),the Addenbrookes National Institute for Health Research Biomedical Research Centre,and a Diabetes UK Career Development fellowship(L.V.).We dedicate this paper to the memory of our colleague Isabelle Bouhon.
Author Contributions L.V.conceived the experiment and did the molecular analysis;I.G.M.B.derived and cultured the EpiSCs;R.A.P.performed the epiblast dissections and together with I.G.M.B.carried out the aggregation,chimaera and clonal assays;L.E.S.and M.T.obtained the microarray data;P.R-G.performed the epigenetic analysis;B.S.performed the blastocyst immunosurgery and ICM cultures;S.M.C.d.S.L.did the Stella–GFP dissection and Blimp1staining;S.K.H. provided PCR analysis of chimaeras;A.C.did karyotyping of human ES cell lines; L.A-R.carried out the teratoma work;L.V.,R.A.P.and I.G.M.B.analysed the data and co-wrote the paper.
Author Information Reprints and permissions information is available at https://www.doczj.com/doc/9c11744833.html,/reprints.The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to L.V.
(lv225@https://www.doczj.com/doc/9c11744833.html,).
NATURE|Vol448|12July2007LETTERS
195
doi:10.1038/nature05950
METHODS
EpiSC and human ES cell culture in feeder-free and serum-free conditions. For feeder-and serum-free culture,human ES cells(H9and H1;WiCell, Madison),hSF-6(UCSF,San Francisco)and EpiSCs were grown in chemically defined medium(CDM)5,supplemented with activin(10ng ml21for human ES cells;20ng ml21for mouse and rat EpiSCs;RandD systems)and FGF2 (12ng ml21,RandD systems for human ESCs and mouse EpiSCs).The com-position of CDM was50%IMDM(Gibco)plus50%F12NUT-MIX(Gibco), supplemented with7m g ml21of insulin(Roche),15m g ml21of transferrin (Roche),450m M of monothioglycerol(Sigma)and5mg ml21bovine serum albumin fraction V(Europabioproducts).Every4–5days,cells were harvested using5mg ml21collagenase IV(Invitrogen)or Accutase(BioWest)and then plated into plates(Costar)pre-coated either with fetal bovine serum(FBS)or with15m g ml21of human Fibronectin(Chemicon)for20min at37u C and then washed twice in PBS.EpiSCs and human ES cells were regularly frozen in a medium containing90%Serum Replacer(Invitrogen)and10%DMSO(Sigma). ICM immuno-surgery and epiblast dissection.For derivation at the blastocyst stage,ICMs were isolated from3.5d.p.c.blastocysts obtained from B63CBA F1 mice mated inter se using immuno-surgery and then placed in CDM/AF on fibronectin.For derivation at post-implantation stages,the late epiblast layer was dissected intact from pre-gastrula stage mouse(E5.75,B63CBA F1mated inter se;E6.5,NO D mated inter se)or rat(E7.5–7.75Wistar or Sprague Dawley) embryos after15–20min treatment at4u C in Cell Dissociation Buffer(Gibco). Dissection was accomplished in calcium-and magnesium-free Flushing and Handling Medium using glass needles to completely separate the epiblast layer from the extra-embryonic tissues.Epiblast layers were then placed in CDM supplemented with growth factors(activin(20ng ml21)and FGF2(12ng ml21)) for culture as above.
Subcloning of EpiSCs.EpiSC colonies were dissociated into single cells after 5min of treatment in0.05%Trypsin-EDTA(Gibco)at37u C.Individual cells were then seeded in three different culture conditions.Subcloning in CDM/AF on fibronectin never gave any EpiSC colonies,confirming that clonal propaga-tion efficiency of EpiSCs in chemically defined medium is very low.Two other approaches based on feeder cells were then used to generate EpiSC sublines.The first approach consisted of seeding individual NOD background EpiSCs on green fluorescent protein(GFP)-expressing EpiSC colonies that had been irradiated to block their https://www.doczj.com/doc/9c11744833.html,ing this approach,the clonal efficiency of EpiSCs was 5%;three non-fluorescent colonies were picked and amplified to study their property of differentiation.Another method of subcloning consisted of seeding individual NOD background EpiSCs on a layer of irradiated mouse embryonic fibroblasts in CDM/AF.The clonal efficiency of EpiSCs in these culture condi-tions was,15%with single cells seeded either by limiting dilution or using glass pipettes;11EpiSC sublines derived on mouse feeders were picked and amplified in CDM/AF to study their capacity to differentiate into the three germ layers and into extra-embryonic tissues.
如何写先进个人事迹 篇一：如何写先进事迹材料 如何写先进事迹材料 一般有两种情况：一是先进个人，如先进工作者、优秀党员、劳动模范等；一是先进集体或先进单位，如先进党支部、先进车间或科室，抗洪抢险先进集体等。无论是先进个人还是先进集体，他们的先进事迹，内容各不相同，因此要整理材料，不可能固定一个模式。一般来说，可大体从以下方面进行整理。 (1)要拟定恰当的标题。先进事迹材料的标题，有两部分内容必不可少，一是要写明先进个人姓名和先进集体的名称，使人一眼便看出是哪个人或哪个集体、哪个单位的先进事迹。二是要概括标明先进事迹的主要内容或材料的用途。例如《王鬃同志端正党风的先进事迹》、《关于评选张鬃同志为全国新长征突击手的材料》、《关于评选鬃处党支部为省直机关先进党支部的材料》等。 (2)正文。正文的开头，要写明先进个人的简要情况，包括：姓名、性别、年龄、工作单位、职务、是否党团员等。此外，还要写明有关单位准备授予他(她)什么荣誉称号，或给予哪种形式的奖励。对先进集体、先进单位，要根据其先进事迹的主要内容，寥寥数语即应写明，不须用更多的文字。 然后，要写先进人物或先进集体的主要事迹。这部分内容是全篇材料
的主体，要下功夫写好，关键是要写得既具体，又不繁琐；既概括，又不抽象；既生动形象，又很实在。总之，就是要写得很有说服力，让人一看便可得出够得上先进的结论。比如，写一位端正党风先进人物的事迹材料，就应当着重写这位同志在发扬党的优良传统和作风方面都有哪些突出的先进事迹，在同不正之风作斗争中有哪些突出的表现。又如，写一位搞改革的先进人物的事迹材料，就应当着力写这位同志是从哪些方面进行改革的，已经取得了哪些突出的成果，特别是改革前后的．经济效益或社会效益都有了哪些明显的变化。在写这些先进事迹时，无论是先进个人还是先进集体的，都应选取那些具有代表性的具体事实来说明。必要时还可运用一些数字，以增强先进事迹材料的说服力。 为了使先进事迹的内容眉目清晰、更加条理化，在文字表述上还可分成若干自然段来写，特别是对那些涉及较多方面的先进事迹材料，采取这种写法尤为必要。如果将各方面内容材料都混在一起，是不易写明的。在分段写时，最好在每段之前根据内容标出小标题，或以明确的观点加以概括，使标题或观点与内容浑然一体。 最后，是先进事迹材料的署名。一般说，整理先进个人和先进集体的材料，都是以本级组织或上级组织的名义；是代表组织意见的。因此，材料整理完后，应经有关领导同志审定，以相应一级组织正式署名上报。这类材料不宜以个人名义署名。 写作典型经验材料-般包括以下几部分： (1)标题。有多种写法，通常是把典型经验高度集中地概括出来，一
How to manage time Time treats everyone fairly that we all have 24 hours per day. Some of us are capable to make good use of time while some find it hard to do so. Knowing how to manage them is essential in our life. Take myself as an example. When I was still a senior high student, I was fully occupied with my studies. Therefore, I hardly had spare time to have fun or develop my hobbies. But things were changed after I entered university. I got more free time than ever before. But ironically, I found it difficult to adjust this kind of brand-new school life and there was no such thing called time management on my mind. It was not until the second year that I realized I had wasted my whole year doing nothing. I could have taken up a Spanish course. I could have read ten books about the stories of successful people. I could have applied for a part-time job to earn some working experiences. B ut I didn’t spend my time on any of them. I felt guilty whenever I looked back to the moments that I just sat around doing nothing. It’s said that better late than never. At least I had the consciousness that I should stop wasting my time. Making up my mind is the first step for me to learn to manage my time. Next, I wrote a timetable, setting some targets that I had to finish each day. For instance, on Monday, I must read two pieces of news and review all the lessons that I have learnt on that day. By the way, the daily plan that I made was flexible. If there’s something unexpected that I had to finish first, I would reduce the time for resting or delay my target to the next day. Also, I would try to achieve those targets ahead of time that I planed so that I could reserve some more time to relax or do something out of my plan. At the beginning, it’s kind of difficult to s tick to the plan. But as time went by, having a plan for time in advance became a part of my life. At the same time, I gradually became a well-organized person. Now I’ve grasped the time management skill and I’m able to use my time efficiently.
英语演讲稿：未来的工作 这篇《英语演讲稿范文：未来的工作》，是特地，希望对大家有所帮助！ 热门演讲推荐：竞聘演讲稿 | 国旗下演讲稿 | 英语演讲稿 | 师德师风演讲稿 | 年会主持词 | 领导致辞 everybody good afternoon:. first of all thank the teacher gave me a story in my own future ideal job. everyone has a dream job. my dream is to bee a boss, own a pany. in order to achieve my dreams, i need to find a good job, to accumulate some experience and wealth, it is the necessary things of course, in the school good achievement and rich knowledge is also very important. good achievement and rich experience can let me work to make the right choice, have more opportunities and achievements. at the same time, munication is very important, because it determines whether my pany has a good future development. so i need to exercise their municative ability. i need to use all of the free time to learn
小学生个人读书事迹简介怎么写800字 书,是人类进步的阶梯,苏联作家高尔基的一句话道出了书的重要。书可谓是众多名人的“宠儿”。历来,名人说出关于书的名言数不胜数。今天小编在这给大家整理了小学生个人读书事迹,接下来随着小编一起来看看吧! 小学生个人读书事迹1 “万般皆下品,惟有读书高”、“书中自有颜如玉,书中自有黄金屋”,古往今来,读书的好处为人们所重视,有人“学而优则仕”,有人“满腹经纶”走上“传道授业解惑也”的道路……但是,从长远的角度看,笔者认为读书的好处在于增加了我们做事的成功率,改善了生活的质量。 三国时期的大将吕蒙,行伍出身,不重视文化的学习,行文时,常常要他人捉刀。经过主君孙权的劝导,吕蒙懂得了读书的重要性,从此手不释卷,成为了一代儒将,连东吴的智囊鲁肃都对他“刮目相待”。后来的事实证明,荆州之战的胜利,擒获“武圣”关羽,离不开吕蒙的“运筹帷幄,决胜千里”,而他的韬略离不开平时的读书。由此可见,一个人行事的成功率高低,与他的对读书,对知识的重视程度是密切相关的。 的物理学家牛顿曾近说过,“如果我比别人看得更远,那是因为我站在巨人的肩上”,鲜花和掌声面前,一代伟人没有迷失方向,自始至终对读书保持着热枕。牛顿的话语告诉我们,渊博的知识能让我们站在更高、更理性的角度来看问题,从而少犯错误,少走弯路。
读书的好处是显而易见的,但是,在社会发展日新月异的今天,依然不乏对读书,对知识缺乏认知的人,《今日说法》中我们反复看到农民工没有和用人单位签订劳动合同,最终讨薪无果;屠户不知道往牛肉里掺“巴西疯牛肉”是犯法的;某父母坚持“棍棒底下出孝子”,结果伤害了孩子的身心,也将自己送进了班房……对书本,对知识的零解读让他们付出了惨痛的代价,当他们奔波在讨薪的路上,当他们面对高墙电网时,幸福,从何谈起?高质量的生活,从何谈起? 读书,让我们体会到“锄禾日当午,汗滴禾下土”的艰辛;读书,让我们感知到“四海无闲田,农夫犹饿死”的无奈;读书,让我们感悟到“为报倾城随太守,西北望射天狼”的豪情壮志。 读书的好处在于提高了生活的质量,它填补了我们人生中的空白,让我们不至于在大好的年华里无所事事,从书本中,我们学会提炼出有用的信息,汲取成长所需的营养。所以,我们要认真读书,充分认识到读书对改善生活的重要意义,只有这样,才是一种负责任的生活态度。 小学生个人读书事迹2 所谓读一本好书就是交一个良师益友,但我认为读一本好书就是一次大冒险,大探究。一次体会书的过程,真的很有意思,咯咯的笑声,总是从书香里散发;沉思的目光也总是从书本里透露。是书给了我启示,是书填补了我无聊的夜空,也是书带我遨游整个古今中外。所以人活着就不能没有书,只要爱书你就是一个爱生活的人,只要爱书你就是一个大写的人,只要爱书你就是一个懂得珍惜与否的人。可真所谓
关于坚持的英语演讲稿 Results are not important, but they can persist for many years as a commemoration of. Many years ago, as a result of habits and overeating formed one of obesity, as well as indicators of overall physical disorders, so that affects my work and life. In friends to encourage and supervise, the participated in the team Now considered to have been more than three years, neither the fine rain, regardless of winter heat, a day out with 5:00 time. The beginning, have been discouraged, suffering, and disappointment, but in the end of the urging of friends, to re-get up, stand on the playground. 成绩并不重要，但可以作为坚持多年晨跑的一个纪念。多年前，由于庸懒习惯和暴饮暴食，形成了一身的肥胖，以及体检指标的全盘失常，以致于影响到了我的工作和生活。在好友的鼓励和督促下，参加了晨跑队伍。现在算来，已经三年多了，无论天晴下雨，不管寒冬酷暑，每天五点准时起来出门晨跑。开始时，也曾气馁过、痛苦过、失望过，但最后都在好友们的催促下，重新爬起来，站到了操场上。 In fact, I did not build big, nor strong muscles, not a sport-born people. Over the past few years to adhere to it, because I have a team behind, the strength of a strongteam here, very grateful to our team, for a long time, we encourage each other, and with sweat, enjoying common health happy. For example, Friends of the several run in order to maintain order and unable to attend the 10，000 meters race, and they are always concerned about the brothers and promptly inform the place and time, gives us confidence and courage. At the same time, also came on their own inner desire and pursuit for a good health, who wrote many of their own log in order to refuel for their own, and inspiring. 其实我没有高大身材，也没健壮肌肉，天生不属于运动型的人。几年来能够坚持下来，因为我的背后有一个团队，有着强大团队的力量，在这里，非常感谢我们的晨跑队，长期以来，我们相互鼓励着，一起流汗，共同享受着健康带来的快
语言学名词解释 Define the following terms: 1. design feature:are features that define our human languages,such as arbitrariness,duality,creativity,displacement,cultural transmission,etc. 2. function: the use of language tocommunicate,to think ,https://www.doczj.com/doc/9c11744833.html,nguage functions inclucle imformative function,interpersonal function,performative function,interpersonal function,performative function,emotive function,phatic communion,recreational function and metalingual function. 3. etic: a term in contrast with emic which originates from American linguist Pike’s distinction of phonetics and phonemics.Being etic mans making far too many, as well as behaviously inconsequential,differentiations,just as was ofter the case with phonetic vx.phonemic analysis in linguistics proper. 4. emic: a term in contrast with etic which originates from American linguist Pike’s distinction of phonetics and phonemics.An emic set of speech acts and events must be one that is validated as meaningful via final resource to the native members of a speech communith rather than via qppeal to the investigator’s ingenuith or intuition alone. 5. synchronic: a kind of description which takes a fixed instant(usually,but not necessarily,the present),as its point of observation.Most grammars are of this kind. 6. diachronic:study of a language is carried through the course of its history. 7. prescriptive: the study of a language is carried through the course of its history. 8. prescriptive: a kind of linguistic study in which things are prescribed how ought to be,https://www.doczj.com/doc/9c11744833.html,ying down rules for language use. 9. descriptive: a kind of linguistic study in which things are just described. 10. arbitrariness: one design feature of human language,which refers to the face that the forms of linguistic signs bear no natural relationship to their meaning. 11. duality: one design feature of human language,which refers to the property of having two levels of are composed of elements of the secondary.level and each of the two levels has its own principles of organization. 12. displacement: one design feature of human language,which means human language enable their users to symbolize objects,events and concepts which are not present c in time and space,at the moment of communication. 13. phatic communion: one function of human language,which refers to the social interaction of language. 14. metalanguage: certain kinds of linguistic signs or terms for the analysis and description of particular studies. 15. macrolinguistics: he interacting study between language and language-related disciplines such as psychology,sociology,ethnograph,science of law and artificial intelligence etc.Branches of macrolinguistics include
1.How to build a business that lasts100years 0:11Imagine that you are a product designer.And you've designed a product,a new type of product,called the human immune system.You're pitching this product to a skeptical,strictly no-nonsense manager.Let's call him Bob.I think we all know at least one Bob,right?How would that go? 0:34Bob,I've got this incredible idea for a completely new type of personal health product.It's called the human immune system.I can see from your face that you're having some problems with this.Don't worry.I know it's very complicated.I don't want to take you through the gory details,I just want to tell you about some of the amazing features of this product.First of all,it cleverly uses redundancy by having millions of copies of each component--leukocytes,white blood cells--before they're actually needed,to create a massive buffer against the unexpected.And it cleverly leverages diversity by having not just leukocytes but B cells,T cells,natural killer cells,antibodies.The components don't really matter.The point is that together,this diversity of different approaches can cope with more or less anything that evolution has been able to throw up.And the design is completely modular.You have the surface barrier of the human skin,you have the very rapidly reacting innate immune system and then you have the highly targeted adaptive immune system.The point is,that if one system fails,another can take over,creating a virtually foolproof system. 1:54I can see I'm losing you,Bob,but stay with me,because here is the really killer feature.The product is completely adaptive.It's able to actually develop targeted antibodies to threats that it's never even met before.It actually also does this with incredible prudence,detecting and reacting to every tiny threat,and furthermore, remembering every previous threat,in case they are ever encountered again.What I'm pitching you today is actually not a stand-alone product.The product is embedded in the larger system of the human body,and it works in complete harmony with that system,to create this unprecedented level of biological protection.So Bob,just tell me honestly,what do you think of my product? 2:47And Bob may say something like,I sincerely appreciate the effort and passion that have gone into your presentation,blah blah blah-- 2:56(Laughter) 2:58But honestly,it's total nonsense.You seem to be saying that the key selling points of your product are that it is inefficient and complex.Didn't they teach you 80-20?And furthermore,you're saying that this product is siloed.It overreacts, makes things up as it goes along and is actually designed for somebody else's benefit. I'm sorry to break it to you,but I don't think this one is a winner.
关于工作的优秀英语演讲稿 Different people have various ambitions. Some want to be engineers or doctors in the future. Some want to be scientists or businessmen. Still some wish to be teachers or lawers when they grow up in the days to come. Unlike other people, I prefer to be a farmer. However, it is not easy to be a farmer for Iwill be looked upon by others. Anyway,what I am trying to do is to make great contributions to agriculture. It is well known that farming is the basic of the country. Above all, farming is not only a challenge but also a good opportunity for the young. We can also make a big profit by growing vegetables and food in a scientific way. Besides we can apply what we have learned in school to farming. Thus our countryside will become more and more properous. I believe that any man with knowledge can do whatever they can so long as this job can meet his or her interest. All the working position can provide him with a good chance to become a talent. 1 ————来源网络整理，仅供供参考
个人先进事迹简介 01 在思想政治方面,xxxx同学积极向上,热爱祖国、热爱中国共产党,拥护中国共产党的领导.利用课余时间和党课机会认真学习政治理论,积极向党组织靠拢. 在学习上,xxxx同学认为只有把学习成绩确实提高才能为将来的实践打下扎实的基础,成为社会有用人才.学习努力、成绩优良. 在生活中,善于与人沟通,乐观向上,乐于助人.有健全的人格意识和良好的心理素质和从容、坦诚、乐观、快乐的生活态度,乐于帮助身边的同学,受到师生的好评. 02 xxx同学认真学习政治理论,积极上进,在校期间获得原院级三好生,和校级三好生,优秀团员称号,并获得三等奖学金. 在学习上遇到不理解的地方也常常向老师请教,还勇于向老师提出质疑.在完成自己学业的同时,能主动帮助其他同学解决学习上的难题,和其他同学共同探讨,共同进步. 在社会实践方面,xxxx同学参与了中国儿童文学精品“悦”读书系,插画绘制工作,xxxx同学在班中担任宣传委员,工作积极主动,认真负责,有较强的组织能力.能够在老师、班主任的指导下独立完成学院、班级布置的各项工作. 03 xxx同学在政治思想方面积极进取,严格要求自己.在学习方面刻苦努力,不断钻研,学习成绩优异,连续两年荣获国家励志奖学金；作
为一名学生干部,她总是充满激情的迎接并完成各项工作,荣获优秀团干部称号.在社会实践和志愿者活动中起到模范带头作用. 04 xxxx同学在思想方面,积极要求进步,为人诚实,尊敬师长.严格 要求自己.在大一期间就积极参加了党课初、高级班的学习,拥护中国共产党的领导,并积极向党组织靠拢. 在工作上,作为班中的学习委员,对待工作兢兢业业、尽职尽责 的完成班集体的各项工作任务.并在班级和系里能够起骨干带头作用.热心为同学服务,工作责任心强. 在学习上,学习目的明确、态度端正、刻苦努力,连续两学年在 班级的综合测评排名中获得第1.并荣获院级二等奖学金、三好生、优秀班干部、优秀团员等奖项. 在社会实践方面,积极参加学校和班级组织的各项政治活动,并 在志愿者活动中起到模范带头作用.积极锻炼身体.能够处理好学习与工作的关系,乐于助人,团结班中每一位同学,谦虚好学,受到师生的好评. 05 在思想方面,xxxx同学积极向上,热爱祖国、热爱中国共产党,拥护中国共产党的领导.作为一名共产党员时刻起到积极的带头作用,利用课余时间和党课机会认真学习政治理论. 在工作上,作为班中的团支部书记,xxxx同学积极策划组织各类 团活动,具有良好的组织能力. 在学习上,xxxx同学学习努力、成绩优良、并热心帮助在学习上有困难的同学,连续两年获得二等奖学金. 在生活中,善于与人沟通,乐观向上,乐于助人.有健全的人格意 识和良好的心理素质.
尊敬的领导，老师，亲爱的同学们， 大家好！我是5班的梁浩东。今天早上我坐车来学校的路上，我仔细观察了路上形形色色的人，有开着小车衣着精致的叔叔阿姨，有市场带着倦容的卖各种早点的阿姨，还有偶尔穿梭于人群中衣衫褴褛的乞丐。于是我问自己，十几年后我会成为怎样的自己，想成为社会成功人士还是碌碌无为的人呢，答案肯定是前者。那么十几年后我怎样才能如愿以偿呢，成为一个受人尊重，有价值的人呢？正如我今天演讲的题目是：自主管理。 大家都知道爱玩是我们孩子的天性，学习也是我们的责任和义务。要怎样处理好这些矛盾，提高自主管理呢？ 首先，我们要有小主人翁思想，自己做自己的主人，要认识到我们学习，生活这一切都是我们自己走自己的人生路，并不是为了报答父母，更不是为了敷衍老师。 我认为自主管理又可以理解为自我管理，在学习和生活中无处不在，比如通过老师，小组长来管理约束行为和同学们对自身行为的管理都属于自我管理。比如我们到一个旅游景点，看到一块大石头，有的同学特别兴奋，会想在上面刻上：某某某到此一游话。这时你就需要自我管理，你需要提醒自己，这样做会破坏景点，而且是一种素质低下的表现。你设想一下，如果别人家小孩去你家墙上乱涂乱画，你是何种感受。同样我们把自主管理放到学习上，在我们想偷懒，想逃避，想放弃的时候，我们可以通过自主管理来避免这些，通过他人或者自己的力量来完成。例如我会制定作息时间计划表，里面包括学习，运动，玩耍等内容的完成时间。那些学校学习尖子，他们学习好是智商高于我们吗，其实不然，在我所了解的哪些优秀的学霸传授经验里，就提到要能够自我管理，规范好学习时间的分分秒秒，只有辛勤的付出，才能取得优异成绩。 在现实生活中，无数成功人士告诉我们自主管理的重要性。十几年后我想成为一位优秀的，为国家多做贡献的人。亲爱的同学们，你们们？让我们从现在开始重视和执行自主管理，十几年后成为那个你想成为的人。 谢谢大家！
关于工作的英语演讲稿 【篇一：关于工作的英语演讲稿】 关于工作的英语演讲稿 different people have various ambitions. some want to be engineers or doctors in the future. some want to be scientists or businessmen. still some wish to be teachers or lawers when they grow up in the days to come. unlike other people, i prefer to be a farmer. however, it is not easy to be a farmer for iwill be looked upon by others. anyway,what i am trying to do is to make great contributions to agriculture. it is well known that farming is the basic of the country. above all, farming is not only a challenge but also a good opportunity for the young. we can also make a big profit by growing vegetables and food in a scientific way. besides we can apply what we have learned in school to farming. thus our countryside will become more and more properous. i believe that any man with knowledge can do whatever they can so long as this job can meet his or her interest. all the working position can provide him with a good chance to become a talent. 【篇二：关于责任感的英语演讲稿】 im grateful that ive been given this opportunity to stand here as a spokesman. facing all of you on the stage, i have the exciting feeling of participating in this speech competition. the topic today is what we cannot afford to lose. if you ask me this question, i must tell you that i think the answer is a word---- responsibility. in my elementary years, there was a little girl in the class who worked very hard, however she could never do satisfactorily in her lessons. the teacher asked me to help her, and it was obvious that she expected a lot from me. but as a young boy, i was so restless and thoughtless, i always tried to get more time to play and enjoy myself. so she was always slighted over by me. one day before the final exam, she came up to me and said, could you please explain this to me? i can not understand it. i
Fill in the blanks: 1.An i_________ is pronounced letter by letter, while an a________ is pronounced as a word. 2.Lexicon, in most cases, is synonymous with v_______. 3.All words may be said to contain a root m________. 4.A small set of conjunctions, prepositions and pronouns belongs to c_______ class, while the largest part of nouns, verbs, adjectives and adverbs belongs to o_____ class. 5.B______ is a reverse process of derivation, and therefore is a process of shortening. 6.Words are divided into simple, compound and derived words on the m_______ level. 7.A word formed by derivation is called a d_______, and a word formed by compounding is called a c________. 8.The poor is an example of p______ conversion. 9.The m________ is the smallest linguistic unit that carries meaning. 10. M________ studies the internal structure of words and the rules by which words are formed. 11. C__________ is the combination of two or sometimes more than two words to create new words. 答案： 1. initialism; acronym 2. vocabulary 3. morpheme
Dear teacher and colleagues: my topic is on “spare time”. It is a huge blessing that we can work 996. Jack Ma said at an Ali's internal communication activity, That means we should work at 9am to 9pm, 6 days a week .I question the entire premise of this piece. but I'm always interested in hearing what successful and especially rich people come up with time .So I finally found out Jack Ma also had said :”i f you don’t put out more time and energy than others ,how can you achieve the success you want? If you do not do 996 when you are young ,when will you ?”I quite agree with the idea that young people should fight for success .But there are a lot of survival activities to do in a day ,I want to focus on how much time they take from us and what can we do with the rest of the time. As all we known ,There are 168 hours in a week .We sleep roughly seven-and-a-half and eight hours a day .so around 56 hours a week . maybe it is slightly different for someone . We do our personal things like eating and bathing and maybe looking after kids -about three hours a day .so around 21 hours a week .And if you are working a full time job ,so 40 hours a week , Oh! Maybe it is impossible for us at