Current Biology24,347–350,February3,2014a2014Elsevier Ltd All rights reserved https://www.doczj.com/doc/9518162501.html,/10.1016/j.cub.2013.12.050
Report Nanog Is Dispensable for the Generation
of Induced Pluripotent Stem Cells
Benjamin A.Schwarz,1,2,3,4Ori Bar-Nur,1,2,4
Jose′C.R.Silva,5,6and Konrad Hochedlinger1,2,4,7,*
1Cancer Center,Massachusetts General Hospital,
185Cambridge Street,Boston,MA02114,USA
2Center for Regenerative Medicine,Massachusetts General Hospital,185Cambridge Street,Boston,MA02114,USA
3Department of Pathology,Massachusetts General Hospital, 185Cambridge Street,Boston,MA02114,USA
4Harvard Stem Cell Institute,1350Massachusetts Avenue, Cambridge,MA02138,USA
5Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute,University of Cambridge,Tennis Court Road, Cambridge CB21QR,UK
6Department of Biochemistry,University of Cambridge, Tennis Court Road,Cambridge CB21QR,UK
7Howard Hughes Medical Institute and Department of Stem Cell and Regenerative Biology,7Divinity Avenue,Cambridge, MA02138,USA
Summary
Cellular reprogramming from somatic cells to induced pluripotent stem cells(iPSCs)can be achieved through forced expression of the transcription factors Oct4,Klf4, Sox2,and c-Myc(OKSM)[1–4].These factors,in com-bination with environmental cues,induce a stable intrinsic pluripotency network that confers inde?nite self-renewal capacity on iPSCs.In addition to Oct4and Sox2,the homeodomain-containing transcription factor Nanog is an integral part of the pluripotency network[5–11].Although Nanog expression is not required for the maintenance of pluripotent stem cells,it has been reported to be essential for the establishment of both embryonic stem cells(ESCs) from blastocysts and iPSCs from somatic cells[10,12]. Here we revisit the role of Nanog in direct reprogramming. Surprisingly,we?nd that Nanog is dispensable for iPSC formation under optimized culture conditions.We further document that Nanog-de?cient iPSCs are trans-criptionally highly similar to wild-type iPSCs and support the generation of teratomas and chimeric https://www.doczj.com/doc/9518162501.html,stly, we provide evidence that the presence of ascorbic acid in the culture media is critical for overcoming the previ-ously observed reprogramming block of Nanog knockout cells.
Results
Endogenous Nanog Is Not Required for Induced Pluripotency
In order to test whether Nanog is required for direct reprog-ramming,we derived Nanog–/–mouse embryonic?broblasts (MEFs)from chimeric embryos[13]since complete deletion of Nanog is embryonic lethal[10,12].Nanog–/–MEFs could be distinguished from host blastocyst-derived wild-type cells based on constitutive CAG-green?uorescent protein(GFP) expression as well as Nanog promoter-driven neomycin resis-tance.Fluorescence-activated cell sorting(FACS)of GFP+ cells yielded a starting population of89%purity.The re-maining GFP2cells were expected to be wild-type MEFs or Nanog2/2MEFs that had silenced the GFP transgene.The GFP-enriched MEFs were transduced with lentiviral vectors expressing OKSM from a doxycycline(dox)-inducible poly-cistronic construct(also referred to as STEMCCA)and rtTA (reverse tetracycline transactivator)[14].After12days of dox induction,we recovered GFP+and GFP2iPSC-like colonies at a ratio similar to that in the starting MEF population. Moreover,GFP+and GFP2colonies could be maintained in the absence of dox,indicating autonomous self-renewal capacity without the continuous need for exogenous factor expression(Figures1A and1B).
To determine whether iPSC-like colonies exhibit molecular hallmarks of authentic iPSCs,we evaluated endogenous pluripotency factor expression by immunostaining for OCT4, SOX2,and NANOG(Figure1C).We found that GFP+colonies expressed both OCT4and SOX2after dox withdrawal,indi-cating that they had induced the endogenous pluripotency network.GFP+iPSC-like colonies also expressed PECAM1,a marker of undifferentiated ESCs and iPSCs that is absent from more mature epiblast stem cells[15,16](Figure S1A avail-able online).Importantly,NANOG expression was absent from GFP+colonies,whereas it was detectable in GFP2(wild-type) colonies,con?rming that GFP expression indeed identi?es Nanog-de?cient cells.
We next performed global gene expression analysis using microarrays to determine how similar Nanog–/–iPSC-like cells are to wild-type ESCs and iPSCs.Unsupervised clus-tering of these samples revealed that Nanog–/–iPSCs are highly similar to wild-type pluripotent cells but different from the MEFs from which they were derived(Figure1D). Importantly,Nanog–/–MEFs clustered closely with indepen-dently derived wild-type MEFs,indicating that the starting cell populations for reprogramming were differentiated?bro-blasts.Of note,the two Nanog–/–iPSC lines were more similar to each other than they were to wild-type iPSC and ESC lines,suggesting that the loss of Nanog results in mild gene expression differences as has been reported previously for Nanog–/–ESCs[10,17].Alternatively,differences in genetic background between Nanog-de?cient iPSCs and wild-type ESCs and iPSCs might account for the differential clustering[18].The microarray data also con?rmed that Nanog–/–colonies express endogenous pluripotency genes [5–9]at ESC-like levels including Oct4(Pou5f1),Sox2,Klf4, Sall4,Rex1(Zfp42),and Dppa2(Figure1E).However,Esrrb levels were reduced in Nanog–/–cells,which is in agreement with the previous?nding that Esrrb is a direct NANOG target [17].Lin28a and Utf1levels were also reduced whereas Nanog transcripts were undetectable in Nanog-de?cient iPSC-like cells.Bisul?te sequencing of the Nanog and Oct4 promoter regions showed extensive demethylation relative to?broblasts(Figure S1B),indicating that both loci are in an accessible ESC-like epigenetic state.Together,these results show that Nanog–/–MEFs can generate iPSC-like
*Correspondence:
khochedlinger@https://www.doczj.com/doc/9518162501.html,
cells that are phenotypically and molecularly highly similar to bona ?de iPSCs.
Nanog-De?cient iPSCs Give Rise to Teratomas and Chimeras
At a functional level,iPSCs are de?ned by the capacity to self-renew inde?nitely in culture and pluripotency,the ability to give rise to cell types of all three germ layers.Indeed,we were able to maintain GFP +iPSC-like cells in culture for multiple pas-sages,regardless of culture conditions (ESC media supple-mented with serum/LIF or serum-free 2i/LIF conditions)[19].However,we noticed that GFP +cells had a propensity to differ-entiate in culture,in accord with the reported phenotype of Nanog –/–ESCs [10].Of note,exposure of Nanog -de?cient iPSC-like cells to neomycin eliminated differentiated cells and maintained phenotypically undifferentiated
colonies
Figure 1.Nanog –/–MEFs Can Be Reprogrammed to iPSCs
(A)Experimental outline.
(B)Fluorescent image of Nanog –/–iPSCs main-tained in 2i/LIF and neomycin (left,phase;right,GFP).
(C)Immuno?uorescence for OCT4,SOX2,and NANOG on a mixed culture of GFP +Nanog –/–(KO)and GFP 2wild-type (WT)iPSCs.
(D)Global gene expression microarrays were performed on RNA puri?ed from the indicated cell lines.Shown is the hierarchical clustering of one WT ESC line,two WT iPSC lines,two Nanog –/–iPSC lines,and WT and KO MEFs (left),as well as scatter plot analyses comparing the indicated populations (right).
(E)Expression data from the microarray for selected pluripotency markers.Results are shown normalized to WT ESC expression levels.(F)Fluorescent images of E13.5chimeras gener-ated from injecting Nanog –/–iPSCs into wild-type blastocysts (top,phase;bottom,GFP).
(G)Adult chimeric mice generated from Nanog –/–iPSCs.Agouti coat color indicates chimerism derived from the Nanog –/–donor cells,whereas the black coat color indicates chimerism derived from the recipient wild-type blastocyst cells.See also Figure S1.
(Figure 1B).To assess the differentia-tion potential of Nanog –/–colonies,we sorted GFP +and GFP 2cells and in-jected them separately into the ?anks of SCID mice.Both Nanog –/–and wild-type cells gave rise to well-differentiated teratomas,characterized by ecto-dermal,endodermal,and mesodermal derivatives,hence meeting one criteria of pluripotency (Figure S1C).
A more stringent assay of pluripo-tency is the ability of cells to contribute to chimeras.We therefore injected GFP +Nanog –/–iPSC-like cells into E3.5wild-type blastocysts,transplanted them into the uterus of pseudopregnant recipient females,and isolated resultant fetuses at midgestation.We obtained 14viable E13.5embryos from 53implanted blastocyts,of which 11embryos had variable contributions of GFP chimerism
(Figure 1F).These embryos gave rise to GFP +MEFs and GFP +neural progenitor cells (NPCs)in vitro,corroborating that the reprogrammed Nanog –/–cells had the potential to differentiate into mesodermal and a de?ned ectodermal lineage,respec-tively (Figure S1D).Additionally,immunohistochemistry of the chimeric embryos for GFP demonstrated that the Nanog –/–iPSC-like cells contributed to all three germ layers,including the neuroectoderm of the brain,the endoderm-derived lining of the gastrointestinal tract,and the mesoderm-derived smooth muscle layers of the gastrointestinal tract (Figure S1E).We also found that Nanog –/–iPSC-like cells could contribute to adult chimeric mice (Figure 1G),indicating that these progeni-tors have the capacity to fully mature and contribute to adult tissues.Collectively,these data demonstrate that the reprog-rammed Nanog –/–cells are pluripotent iPSCs and are thus functionally equivalent to Nanog –/–ESCs.
Current Biology Vol 24No 3348
Ascorbic Acid Rescues the Reprogramming Potential of Nanog -De?cient Cells
Our results differ from a previous report,which documented that Nanog is required for the generation of iPSCs [12].A number of experimental differences between our studies may account for this discrepancy,including the selection of starting cell type (NPCs versus the MEFs used here)and iPSC derivation conditions.We found that Nanog –/–NPCs derived from our chimeras could be reprogrammed into iPSCs (data not shown),thus excluding the possibility that the use of distinct cell types can explain the observed difference in reprogramming potential.We therefore focused on the possible effect of environmental cues on the reprogramming potential of Nanog –/–cells (Figure 1A).Our reprogramming media contained serum/LIF and ascorbic acid (AA)[20],whereas the previous study initiated reprogramming experi-ments in serum/LIF and then switched to 2i (two inhibitors;a combination of GSK3b and MEK inhibitors)/LIF media [19].Given these differences in reprogramming conditions,we tested the individual effects of 2i and AA on the reprogramming ability of Nanog –/–MEFs.Whereas the addition of 2i had only a
minor effect on reprogramming ef?ciency,the removal of AA signi?cantly impaired the reprogramming potential of Nanog –/–MEFs (Figure 2A).Together,these data suggest that a lack of AA impedes the formation of iPSCs in serum/LIF or serum/2i/LIF conditions and thus may account for the previous failure to derive or detect Nanog -de?cient iPSCs.To gain mechanistic insights into the effect Nanog and AA may have on reprogramming,we analyzed nascent iPSCs based on surface markers that distinguish refractory (THY1+SSEA-12)from progressing (THY12SSEA-1+)intermediates [21–23].Nanog de?ciency appears to impact only mid-to-late stages of reprogramming,as suggested by the relative decrease of GFP +SSEA1+intermediates by day 12of reprog-ramming in the absence of AA (Figure 2B).This ?nding is consistent with the late activation of a Nanog -GFP reporter during iPSC formation (Figure S2).Remarkably,exposure of reprogramming cultures to AA entirely rescued this defect.We next analyzed Nanog –/–reprogramming intermediates for EPCAM and PECAM1surface expression,which identify the mid and late stages of reprogramming,respectively [21],in order to delineate the precise step at which Nanog is required (Figure 2C).In wild-type cells undergoing reprogram-ming,EPCAM expression becomes detectable by day 6of OKSM expression and correlates with Nanog transcription.Furthermore,the Epcam locus is bound by NANOG in ESCs,suggesting a direct regulation of Epcam expression by NANOG [21].In contrast,PECAM1expression is activated late (day 9)in iPSC formation and coincides with Oct4expres-sion in wild-type cells.Surprisingly,EPCAM was expressed normally in Nanog –/–nascent iPSC cultures at day 6,indicating that Nanog de?ciency affects neither Epcam transcription nor the mid stages of reprogramming.However,PECAM1expres-sion was absent from Nanog –/–intermediates at day 9under serum/LIF conditions.Importantly,continuous AA treatment of Nanog –/–reprogramming cultures restored normal PECAM expression at day 9.Whereas nearly all SSEA1+cells had turned on PECAM1by day 12of reprogramming in the presence of AA,a minor population of PECAM1+cells was also detectable in the absence of AA,and these cultures gave rise to rare iPSC-like cells.Altogether,these results are consistent with the interpretation that Nanog is important dur-ing late stages of reprogramming by facilitating the transition to a stable self-sustaining pluripotency network (as indicated by PECAM1and hence Oct4positivity).AA treatment facili-tates this step but may not be absolutely required (Figure 2A).Discussion
Our results show that Nanog is dispensable for iPSC induction when directly reprogramming ?broblasts in serum/LIF in the presence of AA.More generally,these results demonstrate that subtle changes in culture conditions can profoundly in?u-ence the genetic requirements for induced pluripotency.We surmise that the previous failure to derive iPSCs from Nanog -de?cient cells was due to alternative derivation condi-tions,which involved the generation of a pre-iPSC intermedi-ate and a switch from serum/LIF to 2i/LIF in the absence of AA [12].A recent study demonstrated that overexpression of NANOG’s target ESRRB can substitute for NANOG during induced pluripotency,suggesting functional redundancy [17].However,iPSC formation in that study also required addi-tion of the global demethylating agent 5-aza-cytidine,whereas we obtained iPSC colonies in conventional culture conditions without the need for 5-aza-cytidine or ectopic expression
of
Figure 2.Ascorbic Acid Rescues the Nanog –/–Reprogramming Defect (A)Nanog –/–MEFs were reprogrammed in serum/LIF with or without 2i and/or AA as indicated.The resulting dox-independent iPSC colonies were stained for alkaline phosphatase and counted.Results are shown as the average percent reprogramming ef?ciency (iPSC colonies /starting number of MEFs)based on four separate replicates 61SD (left).Alkaline phospha-tase staining of a representative plate is shown (right).
(B)MEFs (d0Thy1+)or reprogramming intermediates (Thy12SSEA-1+)were analyzed by ?ow cytometry for average percent GFP positivity 61SD based on three to ?ve replicates per time point.
(C)Reprogramming intermediates at the indicated times after dox induction were analyzed by ?ow cytometry for Thy1,SSEA-1,and EpCAM (left)or PECAM1(right)expression.Plots are gated on Thy12SSEA-1+cells (gray shaded histogram,isotype-matched control antibody).Statistical signi?-cance was determined by the Student’s t test (*p <0.05;**p <0.005).See also Figure S2.
Nanog Is Dispensable for Induced Pluripotency 349
Esrrb.Given the enhancing effect of AA on iPSC formation from Nanog null cells,it will be interesting to further dissect the mechanism by which AA compensates for the lack of Nanog expression.One attractive model is that AA acts as a cofactor for TET enzymes,which have been shown to bind to NANOG and induce demethylation of pluripotency targets including Esrrb and Oct4,thus promoting induced pluripo-tency[24,25].
Accession Numbers
The GEO accession number for the microarray data reported in this paper is GSE53832.
Supplemental Information
Supplemental Information includes Supplemental Experimental Procedures and two?gures and can be found with this article online at https://www.doczj.com/doc/9518162501.html,/ 10.1016/j.cub.2013.12.050.
Acknowledgments
We thank members of the Hochedlinger lab for their help and support,as well as the MGH CRM/HSCI?ow cytometry core,the Harvard University Genome Modi?cation Facility,and the Partners Center for Personalized Genetic Medicine core microarray facility.B.A.S.was supported through an MGH Pathology Department grant(NIH5-T32-CA-9216-33),O.B.was supported by the Gruss Lipper postdoctoral fellowship,and K.H.was sup-ported by the NIH(R01-HD-058013).
Received:October15,2013
Revised:December13,2013
Accepted:December19,2013
Published:January23,2014
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Current Biology Vol24No3 350
英语阅读系列·有趣的字母
攀登英语阅读系列·有趣的字母 北京师范大学“认知神经科学与学习”国家重点实验室攀登英语项目组编著 Lesson1 Frank the Rat 大老鼠弗兰克Frank the rat is in a bag. 大老鼠弗兰克在袋子里呢。 Frank is in a hat. 他在圆顶帽子里呢。 Frank is in a pan. 他在平底锅里呢。 Frank is on an apple. 他在苹果上呢。 Frank is on a bat. 他在球棒上呢。 “Oh, no!” Frank is on a cat. “噢，糟了！”弗兰克落在了猫身上！ Lesson 2 The Biscuits 我们来做饼干“I’m hungry!”“我饿了！” A biscuit…一块饼干可以是...... A biscuit can be a bus. 一块饼干可以是公共汽车。 A biscuit can be a bike. 和一块饼干可以是自行车。 A biscuit can be a boat. 一块饼干可以是小船。 A biscuit can be a banana. 一块饼干可以是香蕉。
A biscuit can be a bear. 一块饼干可以是小熊。 A biscuit can be a butterfly. 一块饼干可以是蝴蝶。 But…If birds are nearby…但是…..如果小鸟恰好在旁边的话…… The biscuits can only be birds.饼干就只是小鸟啦。 Lesson 3 Cool Cat 酷猫卡里 Cary is a cool cat. 卡里是只酷酷的小猫。 Cary can cut carrots like this. 卡里能像这样切胡萝卜。 Cary can climb a coconut tree like this. 卡里能像这样爬树。 Cary can clean a crocodile like this. 卡里能像这样清理鳄鱼。 Cary can catch flies like this. 卡里能像这样抓苍蝇。 Can Cary color like this? 卡里能像这样涂色吗？ Yes! Cary is a cool cat. 是的！卡里是只酷酷的小猫。 Lesson 4 Dancing Dad 爸爸爱跳舞
一年级攀登英语一级A教学计划 一年级攀登英语一级A教学计划 1、激发学生学习英语的兴趣,培养他们学习英语的积极态度,使他们初步建立学习英语的自信心。 2、培养学生具有一定的语感和良好的语音、语调及书写基础,以及良好的学习习惯。 3、培养学生的观察、记忆、思维、想象和创造能力。 一年级攀登英语一级A教学计划 (20**学年度第一学期) 本学期我担任一年级4个班的攀登英语教学工作,现我对本学期教学工作做如下计划: 一、学生情况分析 一年级的孩子的年龄多为6周岁,具有好奇、好活动、爱表现、善模仿等特点。他们的学习兴趣很浓,接受能力、模仿能力很强,学习习惯初步养成,因此在本学期应注重培养学生良好的学习习惯,训练学生的听说能力,调动他们的自主能动性、积极性,营造互帮互助,共同学习英语的语境。 二、教学目标 1、激发学生学习英语的兴趣,培养他们学习英语的积极态度,使他们初步建立学习英语的自信心。 2、培养学生具有一定的语感和良好的语音、语调及书写基础,以及良好的学习习惯。
3、培养学生的观察、记忆、思维、想象和创造能力能在图片、手势、情境等非语言提示的帮助下,听懂清晰的话语和录音。 三、教材分析 本书共由五部分构成:本册教材学习内容包括:日常英语(Everyday English)、歌曲童谣(Songs and Chants).动画英语(Cartoon English)等 1、日常英语:与学生日常生活密切相关的情景对话,共12个话题,每个话题包括2-4句情景对话。 2、动画英语:以《迪士尼神奇英语》(上)VCD为基本学习材料。 共6张VCD,12课,每课4个小节,由学生熟悉和喜欢的迪士尼动画片片段组合而成。 3、歌曲童谣:包括歌曲和童谣两大块,其中有精心制作的传统英文经典歌曲,也有攀登英语独创的童谣日常英语替换内容:以日常英语重 点句型为主线,结合歌曲童谣和动画英语中高频出现的词汇进行的开放性交替。 5、节奏英语:将日常英语替换内容编成12段朗朗上口的韵文,并 配以节奏明快的背景音乐形成的独特学习内容。 四、教学措施 1、考虑到小学生好动爱玩的特点,以活动为课堂教学的主要形式,设计丰富多彩的教学活动,让学生在乐中学、学中用,从而保证学生英 语学习的可持续性发展。 2、通过听、说、读、写、唱、游、演、画、做等形式,进行大量 的语言操练和练习。
攀登英语阅读系列·有趣的字母 北京师范大学“认知神经科学与学习”国家重点实验室攀登英语项目组编著 Lesson1Frank the Rat 大老鼠弗兰克 Frank the rat is in a bag. 大老鼠弗兰克在袋子里呢。 Frank is in a hat. 他在圆顶帽子里呢。 Frank is in a pan. 他在平底锅里呢。 Frank is on an apple. 他在苹果上呢。 Frank is on a bat. 他在球棒上呢。 “Oh, no!” Frank is on a cat. “噢，糟了！”弗兰克落在了猫身上！ Lesson 2 The Biscuits 我们来做饼干 “I’m hungry!”“我饿了！” A biscuit…一块饼干可以是...... A biscuit can be a bus.一块饼干可以是公共汽车。 A biscuit canbe a bike. 和一块饼干可以是自行车。 A biscuit can be a boat. 一块饼干可以是小船。 A biscuit can be a banana. 一块饼干可以是香蕉。 A biscuit can be a bear. 一块饼干可以是小熊。 A biscuit can be a butterfly. 一块饼干可以是蝴蝶。 But…If birds are nearby…但是…..如果小鸟恰好在旁边的话…… The biscuits can only be birds.饼干就只是小鸟啦。 Lesson 3 Cool Cat 酷猫卡里 Cary is a cool cat. 卡里是只酷酷的小猫。 Cary can cut carrots like this. 卡里能像这样切胡萝卜。 Cary can climb a coconut tree like this. 卡里能像这样爬树。 Cary can clean a crocodile like this. 卡里能像这样清理鳄鱼。 Cary can catch flies like this.卡里能像这样抓苍蝇。 Can Cary color like this? 卡里能像这样涂色吗？ Yes! Cary is a cool cat. 是的！卡里是只酷酷的小猫。 Lesson 4 Dancing Dad 爸爸爱跳舞 Dad loves dancing! 爸爸爱跳舞！ Dad is dancing with the desk. 他和桌子一起跳。 Dad is dancing with the duck. 他和鸭子一起跳。 Dad is dancing with the door. 他和门一起跳。 Dad is dancing with the dog. 他和小狗一起跳。 Dad is dancing with the deer. 他和小鹿一起跳。 Where is Dad? 爸爸在哪儿呀？ Wow！哇！ What a dancing Dad! 真是个爱跳舞的爸爸！ Lesson 5 Red Ben 小本的红色世界 Ben likes red. 小本喜欢红色。 Ben paints the eggs red. 他把鸡蛋涂成红色。 Ben paints the eggplants red. 他把茄子涂成红色。 Ben paints the lemons red. 他把柠檬涂成红色。
攀登英语——有趣的字母 A: Frank the rat 老鼠弗兰克 Frank the rat is in a bag. 大老鼠弗兰克在袋子里。 Frank is in a hat. 弗兰克在圆顶帽子里。 Frank is in a pan. 弗兰克在平底锅里。 Frank is on an apple. 弗兰克在苹果里。 Frank is on a bat. 弗兰克在球棒上。 “Oh/no!”F r ank is on a cat. “哦?不！”弗兰克落在了猫身上。 B：The Biscuits 饼干 “I’m h ungry!”“我饿了!” A biscuit, —块饼干 A biscuit can be a bus. —块饼干可以是公共汽车。 A biscuit can be a bike. 可以是自行车。 A biscuit can be a boat. 可以是船。 A biscuit can be a banana. 可以是香蕉。 A biscuit can be a bear.可以是小熊。 A biscuit can be a butterfly.可以是蝴蝶。 But，if birds are nearby,但是，如果鸟儿靠近 The biscuits can only be birds.饼干只能是小鸟啦。 C： Cool Cat酷猫卡里 Cary is a cool cat.卡里是只酷酷的小猫。 Cary can cut carrots like this.卡里能像这样切胡萝卜。 Cary can climb a coconut tree like this. 卡里能像这样爬树。Cary can clean a crocodile like this.卡里能像这样淸洁鳄鱼。Cary can catch flies like this.卡里能像这样抓苍蝇。 Can Cary color like this? 卡里能像这样涂色吗？ Yes!是的 Cary is a cool cat.卡里是只酷酷的小猫。 D: Dancing Dad爸爸爱跳舞 Dad loves dancing!爸爸爱跳舞！ Dad is dancing with the desk.他和桌子一起跳。
攀登英语-有趣的字母(中英文_纯文字_26 篇全)
攀登英语——有趣的字母 A: Frank the rat 老鼠弗兰克 Frank the rat is in a bag. 大老鼠弗兰克在袋子里。 Frank is in a hat. 弗兰克在圆顶帽子里。 Frank is in a pan. 弗兰克在平底锅里。 Frank is on an apple. 弗兰克在苹果里。 Frank is on a bat. 弗兰克在球棒上。 “Oh/no!”F r ank is on a cat. “哦?不！”弗兰克落在了猫身上。 B：The Biscuits 饼干 “I’m h ungry!”“我饿了!” A biscuit, —块饼干 A biscuit can be a bus. —块饼干可以是公共汽车。 A biscuit can be a bike. 可以是自行车。 A biscuit can be a boat. 可以是船。 A biscuit can be a banana. 可以是香蕉。 A biscuit can be a bear.可以是小熊。 A biscuit can be a butterfly.可以是蝴蝶。 But，if birds are nearby,但是，如果鸟儿靠近 The biscuits can only be birds.饼干只能是小鸟啦。 C： Cool Cat酷猫卡里 Cary is a cool cat.卡里是只酷酷的小猫。 Cary can cut carrots like this.卡里能像这样切胡萝卜。 Cary can climb a coconut tree like this. 卡里能像这样爬树。Cary can clean a crocodile like this.卡里能像这样淸洁鳄鱼。Cary can catch flies like this.卡里能像这样抓苍蝇。 Can Cary color like this? 卡里能像这样涂色吗？ Yes!是的 Cary is a cool cat.卡里是只酷酷的小猫。 D: Dancing Dad爸爸爱跳舞 Dad loves dancing!爸爸爱跳舞！ Dad is dancing with the desk.他和桌子一起跳。 Dad is dancing with the duck.他和鸭子一起跳
英语阅读系列·有趣的字母 攀登英语阅读系列?有趣的字母 攀登英语阅读系列·有趣的字母 北京师范大学“认知神经科学与学习”国家重点实验室攀登英语项目组编著 Lesson1 Frank the Rat 大老鼠弗兰克 Frank the rat is in a bag. 大老鼠弗兰克在袋子里呢。
Frank is in a hat. 他在圆顶帽子里呢。Frank is in a pan. 他在平底锅里呢。Frank is on an apple. 他在苹果上呢。Frank is on a bat. 他在球棒上呢。“Oh, no!” Frank is on a cat. “噢，糟了！”弗兰克落在了猫身上！ Lesson 2 The Biscuits 我们来做饼干 “I'm hungry!”“我饿了！” A biscuit…一块饼干可以是...... A biscuit can be a bus. 一块饼干可以是公共汽车。 A biscuit can be a bike. 和一块饼干可以是自行车。 A biscuit can be a boat. 一块饼干可以是小船。 A biscuit can be a banana. 一块饼干可以是香蕉。． 攀登英语阅读系列?有趣的字母 A biscuit can be a bear. 一块饼干可以是小熊。 A biscuit can be a butterfly. 一
块饼干可以是蝴蝶。 But…If birds are nearby…但是…..如果小鸟恰好在旁边的话…… The biscuits can only be birds.饼干就只是小鸟啦。 Lesson 3 Cool Cat 酷猫卡里 Cary is a cool cat. 卡里是只酷酷的小猫。 Cary can cut carrots like this. 卡里能像这样切胡萝卜。 Cary can climb a coconut tree like this. 卡里能像这样爬树。 Cary can clean a crocodile like this. 卡里能像这样清理鳄鱼。 Cary can catch flies like this. 卡里能像这样抓苍蝇。 Can Cary color like this? 卡里能像这样涂色吗？ Yes! Cary is a cool cat. 是的！卡里是只酷酷的小猫。 爸爸爱跳舞Dancing Dad Lesson 4 攀登英语阅读系列?有趣的字母
攀登英语——有趣的字母 A:Franktherat老鼠弗兰克 Franktheratisinabag.大老鼠弗兰克在袋子里。Frankisinahat.弗兰克在圆顶帽子里。 Frankisinapan.弗兰克在平底锅里。 Frankisonanapple.弗兰克在苹果里。 Frankisonabat.弗兰克在球棒上。 “Oh/no!”F rankisonacat.“哦?不！”弗兰克落在了猫身上。 B：TheBiscuits饼干 “I’mhungry!”“我饿了!” Abiscuit,—块饼干 Abiscuitcanbeabus.—块饼干可以是公共汽车。Abiscuitcanbeabike.可以是自行车。 Abiscuitcanbeaboat.可以是船。 Abiscuitcanbeabanana.可以是香蕉。 Abiscuitcanbeabear.可以是小熊。Abiscuitcanbeabutterfly.可以是蝴蝶。 But，ifbirdsarenearby,但是，如果鸟儿靠近Thebiscuitscanonlybebirds.饼干只能是小鸟啦。 C：CoolCat酷猫卡里 Caryisacoolcat.卡里是只酷酷的小猫。Carycancutcarrotslikethis.卡里能像这样切胡萝卜。Carycanclimbacoconuttreelikethis.卡里能像这样爬树。Carycancleanacrocodilelikethis.卡里能像这样淸洁鳄鱼。Carycancatchflieslikethis.卡里能像这样抓苍蝇。CanCarycolorlikethis?卡里能像这样涂色吗？ Yes!是的 Caryisacoolcat.卡里是只酷酷的小猫。 D:DancingDad爸爸爱跳舞 Dadlovesdancing!爸爸爱跳舞！Dadisdancingwiththedesk.他和桌子一起跳。