research article maintenance of self-renewal and pluripotency in...

Research ArticleMaintenance of Self-Renewal and Pluripotency in J1 MouseEmbryonic Stem Cells through Regulating Transcription Factorand MicroRNA Expression Induced by PD0325901

Zhiying Ai12 Jingjing Shao12 Xinglong Shi12 Mengying Yu23 Yongyan Wu23

Juan Du12 Yong Zhang23 and Zekun Guo23

1College of Life Sciences Northwest AampF University Yangling Shaanxi 712100 China2Key Laboratory of Animal Biotechnology Ministry of Agriculture Northwest AampF University Yangling Shaanxi 712100 China3College of Veterinary Medicine Northwest AampF University Yangling Shaanxi 712100 China

Correspondence should be addressed to Zekun Guo zekun guohotmailcom

Received 27 May 2015 Revised 2 August 2015 Accepted 10 August 2015

Academic Editor Chia-Lin Wei

Copyright copy 2016 Zhiying Ai et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Embryonic stem cells (ESCs) have the ability to grow indefinitely and retain their pluripotency in culture and this self-renewalcapacity is governed by several crucial molecular pathways controlled by specific regulatory genes and epigenetic modifications Itis reported that multiple epigenetic regulators such as miRNA and pluripotency factors can be tightly integrated into molecularpathways and cooperate to maintain self-renewal of ESCs However mouse ESCs in serum-containing medium seem to beheterogeneous due to the self-activating differentiation signal ofMEKERKThus to seek for the crucial miRNA and key regulatorygenes that establish ESC properties in MEKERK pathway we performed microarray analysis and small RNA deep-sequencing ofJ1 mESCs treated with or without PD0325901 (PD) a well-known inhibitor of MEKERK signal pathway followed by verificationof western blot analysis and quantitative real-time PCR verification we found that PD regulated the transcript expressions relatedto self-renewal and differentiation and antagonized the action of retinoic acid- (RA-) induced differentiation Moreover PD cansignificantly modulate the expressions of multiple miRNAs that have crucial functions in ESC development Overall our resultsdemonstrate that PD could enhance ESC self-renewal capacity both by key regulatory genes and ES cell-specific miRNA which inturn influences ESC self-renewal and cellular differentiation

1 Introduction

Embryonic stem cells (ESCs) derived from the inner cell massofmammalian embryos have the unique ability to grow indef-initely in culture while retaining their pluripotency [1] Thisself-renewal capacity is established through the integrationof several molecular pathways controlled by key regulatorygenes and complex epigenetic modifications Oct4 Sox2 andNanog [2 3] recognized as fundamental regulatory genescooperate with additional core transcriptional regulatorssuch as Stat3 Esrrb Klf4 Myc and Sall4 to maintain mouseESC properties [3] DNA methylation as one of the keymechanisms of epigenetic regulations is important to theestablishment of pluripotency in ESCs [4] Moreover func-tional studies have shown that inhibition of de novo DNA

methyltransferase by PRDM14 was able to block ESC fromnaive inner cell mass- (ICM-) like state to a primed epiblast-like state [5 6] Meanwhile microRNAs (miRNAs) as animportant mechanism of epigenetic regulation play crucialroles in normal ESC self-renewal and cellular differentiationby tightly controlling ESC self-renewal and differentiationpathways [7 8] These multiple epigenetic regulators andpluripotency factors can be tightly integrated into one orseveral molecular pathways and cooperate to maintain self-renewal of ESCs [9 10]

Mouse ESCs (mESCs) can be maintained in serum-con-taining medium with the presence of leukemia inhibitor fac-tor (LIF) or serum-free N2B27 medium supplemented withtwo small molecule inhibitors (2i) of CHIR99021 (CHIR)

Hindawi Publishing CorporationStem Cells InternationalVolume 2016 Article ID 1792573 12 pageshttpdxdoiorg10115520161792573

2 Stem Cells International

and PD0325901 [11 12] It has been discovered that sev-eral molecular pathways including JAKSTAT BMPSMADWnt120573-catenin and MEKERK are the underlying basis ofthese two ESC media for supporting mESC pluripotency inculture However mESCs in serum-containing medium areheterogeneous which is different from a homogeneous stateof ESC in serum-free N2B27 medium supplemented with2i This is due to the self-activating differentiation signal ofMEKERK that triggers differentiation of ESCs which mightresult in the heterogeneous state of ESCs Recent studieshave identified that PD is one of the inhibitors of MEKERKpathway stimulated by fibroblast growth factor-4 (Fgf4) inmESCs [11 13] Inactivation of MEKERK by PD restrictsthe differentiation of ESCs [14] and this effect is majorlymediated by enhancing Nanog expression [15ndash17] Howeververy little is known about the other possible mechanisms thatfunction in this process For example the role of miRNAshas not been investigated so far when MEKERK signalingcascade was blocked Clarification of miRNAs functionsin MEKERK signaling will provide further insight intomechanisms that ESCs maintain their intrinsic properties

The miRNAs are small noncoding RNAs that regulatemRNA stability andor translational efficiency [18] MostmiRNA genes are transcribed from either miRNA genesor intronic sequences of protein coding genes by RNApolymerase II to generate a stem-loop containing primarymiRNA (pri-miRNA) [19] The hairpin embedded in pri-miRNA is recognized by the RNA-binding protein Dgcr8which directs the RNase III enzymeDrosha to cleave the baseof the hairpin [20 21] Following cleavage by the Drosha-Dgcr8 complex the released short hairpin called precursormiRNA (pre-miRNA) is then transported by the Exportin-5Ran-GTP complex to the cytoplasm where Dicer togetherwith Trbp2 cleaves it into a single short 18ndash25 nt dsRNA[22] Each of them can be recruited into RNA-inducedsilencing complex (RISC) This complex targets mRNAs viabase pairing between the miRNA and mRNA resultingin the regulation of various aspects of stem cell functionsincluding the maintenance and induction of pluripotencyfor reprogramming [7 23] Several lines of evidence furtherindicated the global function ofmiRNAs inDicer orDGCR8-deficient mESCs [24ndash27] Additionally individual miRNAfunction has also been revealed in ESCs [28ndash30] Thus inorder to dissect how epigenetic regulator including miRNAand key regulatory genes establish J1 mouse ESC propertiesin a defined molecular pathway we identified MEKERKsignal-related miRNAs and genome-wide regulation profilesof J1 mESCs stimulated by PD using small RNA deep-sequencing and microarray analysis followed by subsequentverification We demonstrated that PD enhances ESC self-renewal capacity by not only key regulatory genes but alsoESC-specific miRNA which in turn mediates ESC self-renewal and cellular differentiation

2 Materials and Methods

21 ESC Culture The mouse J1 ESC line purchased fromthe American Type Culture Collection (Manassas VA USA)

was cultured in 01 (wv) gelatin coated tissue cultureplates without feeders in ESC media [knockout Dulbeccorsquosmodified Eaglersquos medium supplemented with 15 (vv)knockout serum replacement 01mM 120573-mercaptoethanol 1xnonessential amino acids 2mM GlutaMax 50UmL peni-cillin 50 120583gmL streptomycin (Life Technologies Inc GrandIsland NY USA) and 1000UmL LIF (ESGRO MilliporeUSA)] 293T cell line was cultured at 37∘Chumidified air with5CO2 inDulbeccorsquosmodified Eaglemedium supplementedwith 10 fetal bovine serum

22 Reagents andAntibodies PD0325901 DMSO andmouseanti-GAPDH were purchased from Sigma-Aldrich The pri-mary antibodies used were rabbit anti-Nanog (CST DanversMA USA) rabbit anti-Klf4 (Boster Wuhan China) mouse-anti-c-Myc (Santa Cruz CA USA) goat anti-Tet1 (SantaCruz) rabbit anti-5hmC (Active Motif Carlsbad CA USA)rabbit anti-Ezh2 (Abcam Cambridge UK) rabbit anti-H3K27me3 (Abcam) mouse anti-Oct34 (Santa Cruz) andmouse anti-Sox2 (Santa Cruz) Alexa Fluor 555-labeled goatanti-rabbitmouse IgG and anti-rabbitmouse horseradishperoxidase-conjugated secondary antibody were obtainedfrom the Beyotime Institute of Biotechnology (NantongJiangsu China)

23 RT-qPCR The total RNA was isolated from culturedcells using the Trizol reagent (Life Technologies) First-strand cDNA synthesis was performed using the SYBRPrimeScript RT reagent Kit (Perfect Real Time) (TakaraDalian China) according to the manufacturerrsquos instruc-tions qPCR was performed using SYBR Premix Ex Taq II(Takara) RT-qPCR was performed in an ABI StepOne PlusPCR System (Applied Biosystems California USA) withSYBR Premix Ex TaqTM (Takara) The forward and reverseprimers used for real-time PCR were shown in Supple-mentary Table 4 in Supplementary Material available onlineat httpdxdoiorg10115520161792573 The expression ofeach gene was defined from the threshold cycle (Ct) andrelative expression levels were calculated by using the 2minusΔΔCTmethod after normalization with reference to expression ofthe housekeeping gene GapdhThe gene expression ratio wasshown as mean plusmn SD from three independent experiments

24 Western Blot Analysis Cultured cells were lysed in RIPAbuffer Equal amounts of proteins were separated by 10polyacrylamide gels and transferred to PVDF membranes(Millipore MA USA) for 2 h at 100V After blocking non-specific binding by soaking the filters in 5 skim milk thedesired proteins were immunodetected with the respectiveantibodies that followed autography using SuperSignal WestPico substrate (Thermo Scientific IL USA) according to themanufacturerrsquos instructions

25 Immunofluorescence Staining Cells were fixed in 4paraformaldehyde for 20min and incubated at 37∘C inblocking buffer (PBS containing 5 BSA and 02 TritonX-100) Cells were incubated in the presence of primaryantibodies at 4∘C overnight and then washed three times

Stem Cells International 3

in PBS Cells were then incubated with Alexa Fluor 555secondary antibody for 1 h at 37∘C Nuclei were stainedwith DAPI Immunofluorescence staining was visualized andimaged by a confocal microscope (Nikon Tokyo Japan)

26 Microarray-Based Gene Expression Profiling and SmallRNADeep-Sequencing ESCs were cultured on gelatin coated6-well plates then PD was added to medium at a finalconcentration of 1120583M and an equal volume of DMSO wasadded to medium for control cells For each treatment threeindependent experiments were conducted to prepare thesamples At 24 h after treatment total RNA was extractedusing Trizol reagent (Life Technologies) following the man-ufacturerrsquos instructions RNA integrity was checked by anAgilent Bioanalyzer 2100 system (Agilent Technologies SantaClara CA USA) Qualified total RNA of each sample wasdivided into two copies one for microarray experiment andthe other for small RNA deep-sequencing The microarrayexperiment was performed as described previously [31 32]

For small RNA sequencing the total RNA from threeindependent experiments of each treatment was pooledrespectively Small RNA library construction and sequencingwere performed by Beijing Genomics Institute (ShenzhenChina) Briefly sRNA (18 to 30 nt) was gel purified and ligatedto the 39 and 59 adaptor The ligated products were reverse-transcribed followed by acrylamide gel purification and PCRamplification to generate sRNA libraries The library wasloaded on an Agilent 2100 Bioanalyzer system to check sizepurity and concentration Libraries were sequenced on anIlluminaHiSeq 2000 sequencing system (Illumina SanDiegoCA USA) Sequencing data has been submitted to the GeneExpression Omnibus (GEO) (accession ID GSE67570)

27 Gene Ontology (GO) and KEGG Pathway Analysis Datascreening was carried out based on a gene expression foldchange of gt15 and statistical significance of 119901 lt 005 Bio-logical themes of the differentially expressed genes wereidentified by the biological processes of GO categories usingthe online tool of the Database for Annotation Visualizationand Integrated Discovery (DAVID) [33] KEGG pathwayanalysis was performed using the SAS online program (httpsasebioservicecomportalrootmolnet shbhindexjsp)withthe thresholds of count gt 10

28 Dual-Luciferase Reporter Assay Pathway reporter vec-tors pAP1-TA-luc pAP1 (PMA)-TA-luc pISRE-TA-luc pP53-TA-luc and the negative control pTA-luc were purchasedfrom Clontech Laboratories Inc (Mountain View CAUSA) Other signaling transduction reporter vectors includ-ing pCRE-TA-luc and pGRE-TA-luc were constructed inour laboratory by inserting their cis-acting DNA bindingsequence into the multiple cloning sites of pTA-luc [31]Luciferase assays were performed with the dual-luciferasereporter assay system (Promega) according to the manu-facturerrsquos instructions Briefly pathway reporter vectors andpRL-SV40 were cotransfected into ESCs by Lipofectamine2000 (Invitrogen) according to the manufacturerrsquos protocolAt 24 h after transfection 1 120583M PD or an equal volume of

DMSO was added to culture medium for another 24 h Cellswere then lysed in passive lysis buffer and luciferase activitywas measured on a VICTOR X5 Multilabel Plate Reader(PerkinElmer Norwalk CT USA)

29 MicroRNA and qPCR Analysis The miRNAs expres-sion was validated by poly(A)-tailed qPCR Total RNA wasextracted from PD-treated or control sample using Trizolreagent and 2 120583g of RNA was reverse-transcribed to cDNAusing miScript II RT Kit (Qiagen GmbH Hilden Germany)according to the manufacturerrsquos instructions qPCR was per-formed using SYBR Premix Ex Taq II (Takara) on a StepOnePlus PCR System (Applied Biosystems) All reactions wereperformed at 95∘C for 15min to activate theHotStarTaqDNAPolymeraseThis processwas followed by 40 cycles of 95∘C for5 s and 60∘C for 30 sThe specificity of the primer applicationwas examined by the analysis of a melting curve The relativeexpression of miRNA was normalized to small nuclear RNA(Rnu6) expression and relative to the control Data wereexpressed as the fold change = 2minusΔΔCT

210 Plasmid Constructs The coding sequence (CDS) ofNanog that contain putative miRNA binding site was ampli-fied from J1 ESC cDNA by PCR The PCR primers wereas follows forward primer 51015840-CCGCTCGAGATGAGT-GTGGGTCTTCCTGGTCC-31015840 (underlined letters indicateXhoI restriction site) and reverse primer 51015840-ATAAGAATG-CGGCCGCTCATATTTCACCTGGTGGAG TCACAG-31015840(underlined letters indicate NotI restriction site) It was thencloned into the psiCHECK-2 vector (Promega) yieldingpsiCHECK-2-Nanog The miR-296-5p mimics were pur-chased from Shanghai GenePharma (Shanghai China) Formimics interference experiments J1 ESCs were transfectedwith the indicated mimics (50 nM final concentrations) for24 h using Lipofectamine 2000 (Invitrogen)

211 Statistical Analysis Numerical data were presented asmean plusmn standard deviation (SD) and statistical significancewas analyzed with a two-tailed Studentrsquos 119905-test A value of119901 lt 005 was considered significant

3 Results

31 Suppression ofMEKERK Signaling Promotes Self-Renewaland Colony Morphology of mESCs Mouse ESCs are derivedand maintained by using a combination of the cytokine LIFto activate STAT3 and either serum or bone morphogeneticprotein (BMP) to induce inhibitor of differentiation pro-teins [34] However in these processes their differentiationinvolves autoinductive stimulation of theMEKERK pathwayby Fgf4 [13 14] To determine the exact contribution of thesuppression of MEKERK signaling to the undifferentiatedstates of mESCs J1 mESCs cultured in gelatin coated disheswith LIF (1000UmL) were treated with 1120583M PD for 24 h Inthe presence of LIF PD significantly promoted the formationof typical J1 mouse ESC morphology as cultured on feeder-free plates which was smooth and tightly protuberant whenPD was added (Figure 1(a)) However after being cultured


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Figure 1 Suppression ofMEKERK signaling promotes self-renewal and colonymorphology ofmESCs (a) PD promotes colonymorphologyofmESCs J1mESCswere treatedwith 1120583MPDor equal volume ofDMSO for 24 hMorphological changes were observed and recorded undera phase contrast microscope Scale bar = 50 120583m (b) PD influences the expression pluripotent factors ESCs were treated with or without 1120583MPD for 24 h then the expression levels ofNanogTfcp2l1Klf4 c-Myc and Egr1were analyzed by RT-qPCR Gapdhwas used as a normalizationcontrol Error bars indicate mean plusmn SD of three independent experiments lowast119901 lt 005 compared with controls (c) PD antagonizes RA-induceddifferentiation of ESCs ESCs were treated with the indicated concentration of PD andor together with 1120583M RA for 24 h equal volume ofDMSOwas added for control samplesThen the protein expression levels ofNanog Klf4 and c-Mycwere analyzed bywestern blot Gapdhwasused as a normalization control (d) PD do not influence epigenetic regulation of ESCs ESCs were treated with the 1 120583MPD or equal volumeof DMSO for 24 h Immunofluorescence staining assay was used for analysis of the expression level of 5hmC Tet1 Ezh2 and H3K27me3Nuclei were stained with DAPI scale bar = 50 120583m

under the feeder-free condition for 3ndash5 passages most J1mESCs colonies lost typical morphology (Figure S1A right)We then detected pluripotency of J1 mESCs cultured inthese conditions for 3 passages by alkaline phosphatase (AP)activity and western blot assays and found that J1 mESCsshowed AP activity in contrast to 3T3 cells which were usedfor negative control (Figure S1A) and expressed high levels ofNanog and Oct4 (Figure S1B) Thus J1 mESCs were pluripo-tent in these conditions when adding PD Furthermore

the addition of PD and the expression levels of pluripotentfactors Tfcp2l1 and Nanog were promoted as measured byquantitative real-time PCR (RT-qPCR) (Figure 1(b)) Egr1a target of the MEKERK signaling pathway was repressedby MEK inhibitor PD (Figure 1(b)) Next we treated J1mESCs with PD or equal volume of DMSO for 24 h andthen assessed the protein induction of pluripotent factorsby PD Western blot showed that Nanog and Klf4 proteinexpression levels were upregulated in contrast to control


sample (Figure 1(c)) another small molecule SC1 a well-known inhibitor ofMEKERK signal pathway also confirmedthese results (Figure S2A) However Myc was repressedsignificantly (Figure 1(c)) Previous studies demonstrate thatmESCs treated with 1120583M retinoic acid (RA) can be inducedto differentiate As indicated in Figure 1(c) Nanog Klf4and Myc were significantly repressed by RA However theexpression levels of these two pluripotent factors were able tobe rescued by the addition of 1 120583M or 3 120583M PD respectivelyWe also confirmed these results by immunostaining andRT-qPCR (Figure S3) These results indicate that PD ispositive for the maintenance of the undifferentiated state ofESCs PD could promote self-renewal of mESCs by inducingthe expression of pluripotency genes Moreover PD couldantagonize RA-induced differentiation of ESCs

To investigate alterations of global epigenetic modifi-cations that were involved in DNA methylation in PD-treated ESCs we performed immunofluorescence staining toexamine epigenetic changes (Figure 1(d)) Previous studiesindicate that 5-hydroxymethyl cytosine (5hmC) exists at highlevels in mESCs and its level significantly decreases aftermESC differentiation [35] However the 5hmC modificationlevel in J1 ESCs was unchanged although a slight reduction ofTet1 was caused after PD treatment (Figure 1(d) upper panel)Moreover the global histone H3 lysine 27 trimethylation(H3K27me3) modification level and Ezh2 expression levelwere also unchanged after PD treatment (Figure 1(d) lowerpanel)

32 Transcripts Involved in Self-Renewal and DifferentiationWere Regulated by PD To investigate how PD affects the EScell fate we performed genome-wide expression microarrayanalysis of J1 ES cells cultured with or without PD for 24 h(GEO ID number GSE67534) Messenger RNAs with foldchanges greater than 15 and 119901 values less than 005 werepresented in Supplementary Table 1 A total of 1206 differen-tially expressed genes were identified in PD-treated J1 mESCscompared with control-treated cells of which 763 genes wereupregulated and 443 were downregulated From Table S1 wefound that beside the well-known pluripotency-associatedgenes identified above (Nanog Tfcp2l1 Figure 1(b)) otherpluripotency-related genes such as Pramel7 and Prdm14were also upregulated in J1 ES cells after 1120583M PD treat-ment Ectopic expression of Pramel7 inhibits differentiationand enhances ESC self-renewal while Pramel7 knockdowninduces differentiation and depresses lineage-specific mark-ers [36 37] Prdm14 ensures naive pluripotency by recruitingPRC2 [5 38] On the other hand genes associated with devel-opment or tissue formation such as Gata6 Cdx2 Wnt8aand Dusp4 were significantly downregulated Cooperatingwith Brachyury Cdx2 is reported to induce ESCs to formmesoderm through BMP-induced differentiation [39] TheRT-qPCR was performed for the part of the indicated genesto confirm the objective reliability of the gene expressionchanges (Figure 2(a)) and consistent results were obtained

Based on expression profiling Oct4 Sox2 and Klf4had no significant expression changes while Myc (c-Myc)transcript was downregulated which were further verifiedby qPCR examination (Figure 1(b) Oct4 and Sox2 data

not shown) We then reevaluated the expression of Oct4and Sox2 using immunofluorescence assay and found thatOct4 and Sox2 were not affected by 1 120583M PD treatment for24 h (Figure 2(b)) Although Klf4 mRNA did not respondto PD signal (Figure 1(b)) Klf4 protein expression levelwas promoted by PD (Figure 1(c)) Myc gene examinationsshowed the consistent results (Figures 1(b) and 1(c)) Thusthese results demonstrate that suppression of ERK12 sig-naling pathway by 1 120583M PD can promote expressions ofkey pluripotency-related gene including Nanog Klf4 andTfcp2l1 and suppress expressions of differentiation-inducinggenes These findings also indicate that PD contributes to theundifferentiated state of ESCs

Functional annotation of differentially expressed genesby Gene Ontology (GO) revealed that PD-upregulated geneswere significantly enriched for terms linked to developmentalprocesses cell adhesion regulation of transcription andmorphogenesis (Figure 2(c)) PD-downregulated genes werehighly enriched for terms associated with developmentalprocesses metabolic processes transcriptional regulationand biosynthetic processes (Figure 2(d)) Kyoto Encyclopediaof Genes and Genomes (KEGG) pathway analysis showedthat PD-regulated genes are involved in the ECM-receptorinteraction the focal adhesion and metabolic processes(Figure 2(e)) To investigate the observed effects of PD onmESCs that were not solely the result ofMEKERK signalingwe performed luciferase reporter assays using signal trans-duction reporter plasmids [31 32] J1 mESCs were transfectedwith reporter plasmids that represented the signal trans-duction pathways of JAK-STAT (pISRE-TA-luc) JNKp38and PKA (pAP1-TA-luc and pCRE-TA-luc) PKCMAPK(pAP2-TA-luc) GlucocorticoidHSP90 (pGRE-TA-luc) andp53 (pP53-TA-luc) 24 h after transfection 1120583M PD or anequal volume ofDMSOwas added to cellmedium for another24 h As shown in Figure 2(f) PD treatment was able todecrease the luciferase activity of JNKp38 PKCMAPK andp53 significantly confirming that PD inhibits these threesignaling pathways in J1 mESCs another small molecule SC1also confirmed these results (Figure S2B) However PD wasable to increase the luciferase activity of JAK-STAT indicatingthat PD promotes JAK-STAT signaling pathway CollectivelyPD treatment alters the expression of transcription factors inJ1 mESCs and fine-tunes the signaling pathways to maintainthe characteristics of stem cells

33 Small RNA Deep-Sequencing of PD-Treated mESCsAlthough key regulatory genes have been well disclosedin ERK12 signaling cascade pathway in mESCs ERK12-related miRNAs have not been investigated so far To identifythe ERK12 signal-related miRNAs in ESCs we performedsmall RNA sequencing using small RNA deep-sequencingtechnology in J1 mESCs treated with 1120583M PD or equalvolume of DMSO for 24 h (Figure 3(a)) Totally 18870345clean reads for control-treated cells (control) and 20944808clean reads for the PD-treated sample (PD)were respectivelyextracted after removal of low-quality sequences and the 51015840and 31015840 adapters pollution reads and reads smaller than 18nucleotides Scatter plots showed the general trend ofmiRNAexpression changes after PD stimulation (Figure 3(b)) After


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Figure 2 Transcripts involved in self-renewal and differentiation were regulated by PD (a) qPCR validation of the microarray data Cellswere treated with 1 120583M PD or equal volume of DMSO for 24 h The expression levels of Prdm14 pramle7 Gata6 Cdx2 Wnt8a and Dusp4were detected by RT-qPCR Error bars indicate mean plusmn SD of three independent experiments lowast119901 lt 005 compared with controls (b) Theexpression of Oct4 and Sox2 Cells were treated with 1120583M PD or equal volume of DMSO for 24 h Immunofluorescence staining assay wasused for analysis of the expression level of Oct4 and Sox2 Nuclei were stained with DAPI scale bar = 50120583m (c and d) GO annotation of PD-regulated genes GO term enrichment of the ldquobiological processrdquo category of PD-regulated genes GO terms ranked according to the minusLog2Pof upregulated genes (count gt 10) (c) or downregulated genes (d) were plotted (e) KEGG pathway analysis of differentially expressed genesKEGG pathway analysis of differentially expressed genes in PD-treated J1 mESCs This result was ranked according to the minusLog2P of PD-regulated genes (count gt 10) (f) Dual-luciferase reporter assay to identify signaling transduction pathways regulated by PD Pathway reportervectors (including negative control) and internal control pRL-SV40 were cotransfected by Lipofectamine 2000 24 h after transfection 1120583MPD or an equal volume of DMSO was added to cell medium for another 24 h Luciferase activity is presented relative to negative controlpTA-luc Data are presented as mean plusmn SD of three independent experiments lowast119901 lt 005


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Figure 3 Experimental scheme for sample preparation of small RNA deep-sequencing andmicroarray analysis (a) Experimental scheme forsmall RNA deep-sequencing andmicroarray analysis J1 mESCs cultured in LIF containingmedia were treated with 1120583MPD or equal volumeofDMSO (control) for 24 h and then the total RNAswere extracted and qualifiedRNAswere analyzed bymicroarray gene expression profilingand small RNA deep-sequencing to identify differentially expressed mRNAs and miRNAs (b) Comparison of the knownmiRNA expressionbetween DMSO- and PD-treated samplesThe scatter plots show the distribution of the detectedmiRNAs with or without 1 120583MPD treatmentin mESC medium for 24 h Significant regulated miRNAs with 15-fold change are marked red (upregulated) and green (downregulated)

mapping the clean reads against the GenBank noncodingRNA database and the Rfam database we found noncodingRNAs (ncRNAs) such as rRNA scRNA tRNA snRNAsnoRNA and other ncRNAs Then small RNA reads weremapped against introns and exons of mRNAs to find andexcise the degraded fragments of mRNA in the small RNAtags Finally the clean readswere aligned tomiRBase (Release18) allowing only perfect matches

After performing fold change analysis we identified 89differentially expressedmiRNAs in J1 mESCs treated with PDcompared with the control library in which 26 miRNAs wereupregulated and 63 miRNAs were downregulated by 15-foldor greater (Table S2) We noted that sim70 of miRNAs (63out of 89) in the PD-treated samples were downregulatedand many miRNAs have been studied in pluripotent cellsThe miR-302-367 miR-290-295 miR-17-92b miR-106a-363and miR-106b-25 cluster of miRNAs belong to the ESC-specific cell cycle (ESCC) family of miRNAs The miR-302-367 cluster is expressed specifically in pluripotent ESCs andits overexpression promotes iPS cell generation efficiency inmouse fibroblasts using three exogenous factors (Oct4 Klf4and Sox2) The miR-290-295 cluster promotes pluripotencymaintenance via regulating cell cycle phase distribution Oursequencing data showed that the expression of miR-302aand miR-302d was upregulated by 1 120583M PD (Figure 4(a))but the other ESCC miRNAs were downregulated followingPD treatment (Figures 4(b)ndash4(e))The differential expression

levels of severalmiRNAswere confirmed by quantitative real-time PCR (RT-qPCR) (Figure 4(f))

34 ERK12 Signal-Related miRNAs Regulate Nanog Expres-sion and Promote Homogeneous ESC We found that PDtreatment inhibited the expression of most miRNAs in ESCsespecially those related to ESCC family of miRNAs Morerecently we reported that sim98 of miRNAs (367 of 373)were downregulated in the CHIR-treated ESCs (GSE54145)(Table S3) This phenomenon attracted our attention andwe thought that miRNAs could be almost totally inhibitedin serum-free medium containing two small moleculesCHIR and PD (N2B272i) We then analyzed the globaldifference of miRNAs in these two small molecule-treatedESCs After comparing the expression of miRNAs in CHIR-and PD-treated ESCs we found that sim925 of differentialmiRNAs (368 of 398) were downregulated in PD and CHIR-treatedESCsVenndiagram showed the upregulatedmiRNAs(Figure 5(a)) and the downregulated miRNAs (Figure 5(b))in PD- and CHIR-treated ESCs respectively and the globaldifferential miRNAs between CHIR- and PD-treated ESC areshown in Figure 5(c)

Recent reports indicate that DGCR8 can be phospho-rylated by MEKERK which increases its intracellular sta-bility and induces a progrowth miRNA profile [22] whileglycogen synthase kinase 3 beta phosphorylates the Droshaand increases its nuclear localization [40ndash42] because


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DMSO PD03

miR-17-92b cluster

mmu-miR-17-5pmmu-miR-17-3pmmu-miR-18a-3pmmu-miR-20a-5pmmu-miR-92a-1-5p

Relat

ive m

iRN

A fo

ld ch

ange

(c)

06

065

07

075

08

085

09

095

1

105

DMSO PD03

miR-106a-363 cluster

Relat

ive m

iRN

A fo

ld ch

ange

mmu-miR-106a-5pmmu-miR-18b-3pmmu-miR-20b-5pmmu-miR-92a-2-5pmmu-miR-363-3p

(d)

08

085

09

095

1

105

DMSO PD03

miR-106b-25 cluster

mmu-miR-106b-5pmmu-miR-106b-3pmmu-miR-25-5pmmu-miR-93-5p

Relat

ive m

iRN

A fo

ld ch

ange

(e)

0 1 2 3 4 5

mmu-miR-331-3pmmu-miR-124-3p

mmu-miR-467a-5pmmu-miR-181d-3p


mmu-miR-101b-3pmmu-miR-92a-3pmmu-miR-382-5pmmu-miR-222-3pmmu-miR-296-3p

PD03DMSO

Relative miRNA expression

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

(f)

Figure 4 PD regulate the expression of the ESCC family ofmiRNAs inmESCs (andashe) Relative fold change ofmature ESCC family ofmiRNAsJ1 mESCs were treated with 1120583MPDor equal volume of DMSO (control) for 24 h and then the total RNAs were extracted and qualified RNAswere analyzed by small RNA deep-sequencing to identify differentially expressed miRNA miR-302-367 cluster miR-290-295 cluster miR-17-92b cluster miR-106a-363 cluster and miR-106b-25 cluster in control and PD-treated J1 mESCs detected by small RNA deep-sequencing (f)RT-qPCR validation of differentially expressed miRNA in PD-treated J1 mESCs J1 mESCs were treated with 1120583M PD for 24 h and then theexpression of miRNAs was determined by RT-qPCR Error bars indicate mean plusmn SD of three independent experiments lowast119901 lt 005 comparedwith controls

the phosphorylation of DGCR8 and Drosha can be repressedby PD and CHIR (Figure 5(d)) which could result in the lossof miRNAs So most of miRNAs were inhibited in N2B272iESC medium and this result was very similar to the effectcaused by the Dgcr8 knockout in ESCs Moreover Dgcr8knockout ESCs were defective in differentiation even understringent differentiation conditions (Figure 5(d)) [26] Thismight be the reason that ESCs in N2B272i ESC medium are

highly homogeneous yet fully pluripotent even in the absenceof feeder while ESCs without feeder and in the presenceof LIF are flattened and heterogeneous (Figure 1(a)) [12]Moreover Nanog reporters are heterogeneously expressed inESCs cultured in serum and LIF without feeder [12] andthe underlying mechanism is the monoallelic expression ofNanog demonstrated by RNA fish [15] We showed that 1120583MPD treatment can change the expression of Nanog from


5 2 24

Up in PD03

Up in CHIR

miRNA

(a)

305 57 6

Down in PD03

Down in CHIR

miRNA

(b)

309 64 25PD03CHIR

miRNA

(c)

Pri-miRNA

Mature miRNA

Pre-miRNA

ESC self-renewal

DGCR8 knockoutESCs

ESC differentiation

Mature miRNA

ESC self-renewal

DGCR8 knockoutESCs

ESC differentiation

Drosha

DGCR8

N2B27

PD03CHIR

(d)

0

02

04

06

08

1

12

Relat

ive l

ucife

rase

activ

ity lowast

Mimics NCmiR-296 mimics

Inhibitor NCmiR-296 inhibitor

+

+

minus

+

+

+

minus

minus

minus +

+

minus

TK hluc+ SV40 hRluc Poly(A)

psiCHECK2NanogCDS

(e)

0

02

04

06

08

1

12

Relat

ive m

RNA

expr

essio

n lowast


DMSOPD03

+

+

+

+

+

minus

minus minus

minus +

+

minus

(f)

Nanog

Gapdh

Mimics NC miR-296 mimics

(g)

Figure 5 MEKERK signal-related miRNAs promote homogeneous ESC (andashc) Venn diagram shows the differential expression of miRNAin PD- and CHIR-treated ESCs Venn diagram showed the upregulated miRNAs (a) and the downregulated miRNAs (b) in PD- and CHIR-treated ESCs respectively The global differential miRNAs between CHIR- and PD-treated ESCs are shown in (c) (d) Schematic diagram ofthe miRNA biosynthesis and functions in maintaining the undifferentiated state of mESCs PD and CHIR influence Dgcr8-Drosha complexactivity rarr means active and perpmeans inactive (e) miR-296 mimics regulated Nanog expression in a posttranscriptional regulation mannerSchematic representation of the 31015840-UTR reporter constructs in the upper panel TK hluc+ SV40 and hRluc represent HSV-TK promoterfirefly luciferase gene SV40 early enhancerpromoter and Renilla luciferase gene respectively In the lower panel psiCHECK2-Nanog-CDSor psiCHECK2 control plasmid was cotransfected with mimics NC or miR-296 mimicsinhibitor into 293T cells At 24 h after incubation1 120583MPD or an equal volume of DMSO was added to cell medium for another 24 h Luciferase activity is presented relative to negative controlpTA-luc Data are presented as mean plusmn SD of three independent experiments lowast119901 lt 005 (f) miR-296 mimics regulated Nanog expressionJ1 mESCs were transfected with mimics NC or miR-296 mimics At 5 h after transfection fresh medium was added and 1120583MPD or an equalvolume of DMSO was added to the transfected cells for another 24 h The expression level of Nanog was detected by RT-qPCR Error barsindicate mean plusmn SD of three independent experiments lowast119901 lt 005 compared with controls (g) miR-296 regulates Nanog expression ESCswere transfected with mimics NC or miR-296 mimics for 24 h then the protein expression level of Nanog was analyzed by western blotGapdh was used as a normalization control


low to high states (Figures 1(b) and 1(c)) In the meantimemiRNAs that targeted Nanog were also inhibited in PD-treated cells For instance RT-qPCR showed that miR-296was significantly downregulated (Figure 4(f))

To examine miR-296 function in PD-induced Nanogexpression we subcloned coding sequence (CDS) fragmentof Nanog downstream the reporter gene in the psiCHECK-2 vector (Figure 5(e) upper panel) Luciferase assays wereperformed by cotransfection of the reporter vector and miR-296 mimics into 293T cells for 24 h As shown in Figure 5(e)the reporter that harbored the CDS fragment of Nanogwas significantly repressed whereas miR-296 inhibitor couldrescue luciferase activity Furthermore western blot and RT-qPCR showed that transfection of miR-296 mimics sup-pressed Nanog levels in J1 mESCs (Figures 5(f) and 5(g))however PD can compromise miR-296 reduction on Nanog(Figure 5(f)) These results strongly suggest that PD treat-ment could promote Nanog expression by inhibiting the levelof miRNA that targets Nanog

4 Discussion

ESCs were heterogeneous because of self-activating differ-entiation signal of MEKERK that triggers differentiationof ESCs in serum-containing medium To examine theeffects of the suppression of MEKERK signaling to mESCswe treated ESCs with PD and found that colonies werehomogeneous in ESC morphology GO annotation of differ-entially expressed genes also revealed that PD-upregulatedgenes were enriched for terms linked to the regulationof morphogenesis (Figure 2(c)) These results indicate thatsuppression of self-activating differentiation signal is positivefor the homogeneous ESC morphology in serum-containingmedium Moreover PD promoted the expression of Nanogand Klf4 under this condition (Figures 1(b) and 1(c)) andcould rescue the expression ofNanog andKlf4 induced byRA(Figure 1(c))These results indicate that PD is positive for themaintenance of the undifferentiated state of ESCs by inducingthe expression of pluripotency genes and antagonizing RA-induced differentiation of ESCs

Genome-wide expression microarray analysis confirmedthese results that pluripotency-related genes were unregu-lated and lineage-specific markers were downregulated afterPD treatment in mESCs However the increase of Klf4protein level was not accompanied by that of the Klf4mRNAlevel (Figures 1(b) and 1(c)) This phenomenon indicates thatMEKERK may regulate Klf4 expression at posttranscrip-tional level consistent with previous report that MEKERKcould phosphorylate Klf4 which results in Klf4 ubiquitina-tion and degradation [43] We also noted an unwarrantedside effect of suppressing MEKERK signaling that is thedepression of Myc messenger RNA and Myc protein levels(Figures 1(b) and 1(c)) consistent with previous study thatelevated Myc is not necessary for ESC propagation [11]

The dynamical regulation of DNA methylation is impor-tant for the establishment of pluripotency in mESCs [4]Although ESCs exist at high level of 5-hydroxymethyl cyto-sine (5hmC) [35] the 5hmC modification level in J1 ESCswas unchanged even if a slight reduction (sim25) of Tet1 was

caused after PD treatment (Figure 1(d)) This phenomenonmight be attributed to the change of 5hmC that cannot bedistinguished at the whole genome level In addition PDpromote the expression of Prdm14 which can blockmES cellsfrom naive inner cell mass- (ICM-) like state to a primedepiblast-like state by inhibiting de novo DNA methyltrans-ferase [38] In undifferentiated ESCs the majority of chro-matin appears homogeneous [44] However histone markH3k27me3 commonly associated with repressive chromatinwas not influenced by PD (Figure 1(d))

Recently increasing evidence suggests that miRNAs asan important mechanism of epigenetic regulation are crucialfor normal ESC self-renewal and cellular differentiation bytightly controlling ES cell self-renewal and differentiationpathways [7] We performed small RNA sequencing to studyhowmiRNAs establish ESC properties inMEKERK pathway(Figure 3(a)) After performing fold change analysis wenoted that sim70 of miRNAs were downregulated in PD-treated samples including the ESCC family of miRNAsWe also found that sim925 of differential miRNAs weredownregulated after comparing the expression of miRNAs inCHIR- and PD-treated ESCsThe reduction of most miRNAsstimulated by PD and CHIR might be the reason that ESCsappear to be homogeneous in N2B27 medium supplementedwith PD and CHIR Inhibition of MEKERK represses Dgcr8intracellular stability which in turn influencesmiRNAprofile[22] Meanwhile inhibition of glycogen synthase kinase 3beta by CHIR reduces Drosha nuclear localization whichwill result in the loss of miRNAs (Figure 5(d)) These couldbe the reasons the expression of most of miRNAs wasinhibited in PD- or CHIR-treated ESCs (Figures 5(b) and5(c)) A consistent result was also proved by GO annotationand GO annotation revealed that PD-regulated genes weresignificantly enriched for terms linked to the regulation ofRNA metabolic process and negative regulation of nucleicmetabolic process (Figure 2(d))

Previous study has demonstrated that Dgcr8 knockoutmESCs showed a global loss of miRNAs (Figure 5(d)) andfurther caused proliferation defect [45] Reintroduction ofdeficient canonical miRNAs that suppress inhibitors of G1-S transition can rescue the ESC proliferation defect in Dgcr8knockout mESCs The key factor in this process is Cdkn1a(also known as p21) an inhibitor of G1-S transition whichis inhibited by ESCC miRNAs However the repression ofmiRNAs in PD-treated ESCs did not induce the elevatedp21 (Table S1) which results in proliferation defect [45]consistent with the signal transduction reporter assay that PDtreatment inhibits the signaling pathway of p53 in J1 mESCs(Figure 2(f))Thus ESC proliferation cannot be influenced bythe loss of miRNAs In addition the monoallelic expressionof Nanog that causes ESC heterogeneously in serum andLIF medium without feeder can be promoted by inhibitingthe level of miRNA that targets Nanog after PD treatment(Figure 5(f)) Thus the suppression of MEKERK is quiteimportant for the homogeneous undifferentiated ESCs

RNase III family members play diverse roles in RNAmetabolism [46] Drosha is known to play a critical role inmiRNA maturation [47] and mRNA stability control [48]For instance in Hela cells sim2 genes detected by Affymetrix


chip were upregulated over 2-fold in Drosha-depleted cellsFurthermore those genes are also upregulated in DGCR8-depleted cells Thus sim100 genes are controlled by DGCR8-Drosha complexWe cannot rule out the possibility that someof these genes indirectly influenced by CHIR and PD inN2B272i ESC medium exist which in turn influence ESCpluripotency

Taken together our experiments showed the MEKERKsignal-related regulation profiles and miRNAs in J1 mESCsPD not only regulates the transcript expressions relatedto self-renewal and differentiation but also antagonizes theaction of RA-induced differentiation Moreover PD wasable to significantly modulate the expression of multiplemiRNAs especially those that have crucial functions in EScell development Thus key regulatory genes and complexepigenetic modifications are integrated into the MEKERKmolecular pathway which in turn influence ES cell self-renewal and cellular differentiation

5 Conclusions

ESCs have the unique ability to grow indefinitely in culturewhile retaining their pluripotency This self-renewal capacityis established through the integration of several molecularpathways controlled by key regulatory genes and complexepigenetic modifications It is reported that multiple epige-netic regulators such as miRNA and pluripotency factors canbe tightly integrated into the molecular pathway and cooper-ate together to maintain self-renewal of ESCs However theeffects of miRNA and key regulatory genes that establish ESCproperties in MEKERK pathway are poorly understood Inthis study we found PD-related transcripts and miRNAs thatwere involved in self-renewal and differentiation We alsodemonstrated that PD enhances ESC self-renewal capacitynot only by key regulatory genes but also influences ES cell-specific miRNA which in turn influences ESC self-renewaland cellular differentiation This study also highlights thatERK12 signal-related miRNAs can promote ESC homoge-neous

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Zhiying Ai and Zekun Guo conceived the research ZhiyingAi YongyanWu and ZekunGuo designed the study ZhiyingAi Jingjing Shao Xinglong Shi Mengying Yu Yongyan Wuand Juan Du performed the experiments and collected thedata Zhiying Ai Jingjing Shao and Zekun Guo analyzed thedata and wrote the paper

Acknowledgments

The study was supported by the grants from Natural Sci-ence Foundation of China (no 31172279) and Key Science

and Technology Innovation Team in Shaanxi Province (no2014KCT-26) The authors thank Xiaoyan Shi for reagentsand help with experiments

References

[1] A G Smith ldquoEmbryo-derived stem cells of mice and menrdquoAnnual Review of Cell and Developmental Biology vol 17 pp435ndash462 2001

[2] Y-H Loh Q Wu J-L Chew et al ldquoThe Oct4 and Nanog tran-scription network regulates pluripotency in mouse embryonicstem cellsrdquo Nature Genetics vol 38 no 4 pp 431ndash440 2006

[3] J Kim J Chu X Shen J Wang and S H Orkin ldquoAn extendedtranscriptional network for pluripotency of embryonic stemcellsrdquo Cell vol 132 no 6 pp 1049ndash1061 2008

[4] Y Costa J Ding T W Theunissen et al ldquoNANOG-dependentfunction of TET1 and TET2 in establishment of pluripotencyrdquoNature vol 495 no 7441 pp 370ndash374 2013

[5] M Yamaji J Ueda K Hayashi et al ldquoPRDM14 ensuresnaive pluripotency through dual regulation of signaling andepigenetic pathways in mouse embryonic stem cellsrdquo Cell StemCell vol 12 no 3 pp 368ndash382 2013

[6] N Okashita Y Kumaki K Ebi et al ldquoPRDM14 promotes activeDNA demethylation through the Ten-eleven translocation(TET)-mediated base excision repair pathway in embryonicstem cellsrdquo Development vol 141 no 2 pp 269ndash280 2014

[7] E-M Heinrich and S Dimmeler ldquoMicroRNAs and stem cellscontrol of pluripotency reprogramming and lineage commit-mentrdquo Circulation Research vol 110 no 7 pp 1014ndash1022 2012

[8] A Marson S S Levine M F Cole et al ldquoConnectingmicroRNAgenes to the core transcriptional regulatory circuitryof embryonic stem cellsrdquo Cell vol 134 no 3 pp 521ndash533 2008

[9] J Ding H Xu F Faiola A Marsquoayan and J Wang ldquoOct4 linksmultiple epigenetic pathways to the pluripotency networkrdquo CellResearch vol 22 no 1 pp 155ndash167 2012

[10] M Pardo B Lang L Yu et al ldquoAn expanded Oct4 interactionnetwork implications for stem cell biology development anddiseaserdquo Cell Stem Cell vol 6 no 4 pp 382ndash395 2010

[11] Q-L Ying J Wray J Nichols et al ldquoThe ground state ofembryonic stem cell self-renewalrdquoNature vol 453 no 7194 pp519ndash523 2008

[12] J Wray T Kalkan and A G Smith ldquoThe ground state ofpluripotencyrdquo Biochemical Society Transactions vol 38 no 4pp 1027ndash1032 2010

[13] T Kunath M K Saba-El-Leil M Almousailleakh J WrayS Meloche and A Smith ldquoFGF stimulation of the Erk12signalling cascade triggers transition of pluripotent embryonicstem cells from self-renewal to lineage commitmentrdquo Develop-ment vol 134 no 16 pp 2895ndash2902 2007

[14] M P Stavridis J S Lunn B J Collins and K G Storey ldquoAdiscrete period of FGF-induced Erk12 signalling is required forvertebrate neural specificationrdquo Development vol 134 no 16pp 2889ndash2894 2007

[15] Y Miyanari and M-E Torres-Padilla ldquoControl of ground-statepluripotency by allelic regulation of Nanogrdquo Nature vol 483no 7390 pp 470ndash473 2012

[16] T Hamazaki S M Kehoe T Nakano and N Terada ldquoTheGrb2Mek pathway represses nanog in murine embryonic stemcellsrdquoMolecular and Cellular Biology vol 26 no 20 pp 7539ndash7549 2006


[17] KMitsui Y TokuzawaH Itoh et al ldquoThehomeoproteinNanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003

[18] N Bushati and S M Cohen ldquoMicroRNA functionsrdquo AnnualReview of Cell and Developmental Biology vol 23 pp 175ndash2052007

[19] N Suh and R Blelloch ldquoSmall RNAs in early mammaliandevelopment from gametes to gastrulationrdquo Development vol138 no 9 pp 1653ndash1661 2011

[20] A M Denli B B J Tops R H A Plasterk R F Kettingand G J Hannon ldquoProcessing of primary microRNAs by theMicroprocessor complexrdquo Nature vol 432 no 7014 pp 231ndash235 2004

[21] R I Gregory K-P Yan G Amuthan et al ldquoTheMicroprocessorcomplex mediates the genesis of microRNAsrdquo Nature vol 432no 7014 pp 235ndash240 2004

[22] K M Herbert G Pimienta S J DeGregorio A Alexandrovand J A Steitz ldquoPhosphorylation of DGCR8 increases itsintracellular stability and induces a progrowth miRNA profilerdquoCell Reports vol 5 no 4 pp 1070ndash1081 2013

[23] Z Li C-S Yang K Nakashima and T M Rana ldquoSmall RNA-mediated regulation of iPS cell generationrdquoThe EMBO Journalvol 30 no 5 pp 823ndash834 2011

[24] C Kanellopoulou S AMuljo A L Kung et al ldquoDicer-deficientmouse embryonic stem cells are defective in differentiation andcentromeric silencingrdquo Genes amp Development vol 19 no 4 pp489ndash501 2005

[25] E P Murchison J F Partridge O H Tam S Cheloufi andG J Hannon ldquoCharacterization of Dicer-deficient murineembryonic stem cellsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 102 no 34 pp12135ndash12140 2005

[26] Y Wang R Medvid C Melton R Jaenisch and R BlellochldquoDGCR8 is essential for microRNA biogenesis and silencing ofembryonic stem cell self-renewalrdquo Nature Genetics vol 39 no3 pp 380ndash385 2007

[27] J B Tennakoon H Wang C Coarfa A J Cooney and PH Gunaratne ldquoChromatin changes in dicer-deficient mouseembryonic stem cells in response to retinoic acid induceddifferentiationrdquo PLoS ONE vol 8 no 9 Article ID e74556 2013

[28] Y M-S Tay W-L Tam Y-S Ang et al ldquoMicroRNA-134modulates the differentiation of mouse embryonic stem cellswhere it causes post-transcriptional attenuation of Nanog andLRH1rdquo Stem Cells vol 26 no 1 pp 17ndash29 2008

[29] N Xu T Papagiannakopoulos G Pan J AThomson and K SKosik ldquoMicroRNA-145 regulates OCT4 SOX2 and KLF4 andrepresses pluripotency in human embryonic stem cellsrdquo Cellvol 137 no 4 pp 647ndash658 2009

[30] Z Xu J Jiang C Xu et al ldquomicroRNA-181 regulates CARM1and histone aginine methylation to promote differentiation ofhuman embryonic stem cellsrdquo PLoS ONE vol 8 no 1 ArticleID e53146 2013

[31] X Shi Y Wu Z Ai et al ldquoAICAR sustains J1 mouse embryonicstem cell self-renewal and pluripotency by regulating tran-scription factor and epigenetic modulator expressionrdquo CellularPhysiology and Biochemistry vol 32 no 2 pp 459ndash475 2013

[32] Y Wu Z Ai K Yao et al ldquoCHIR99021 promotes self-renewalof mouse embryonic stem cells by modulation of protein-encoding gene and long intergenic non-coding RNA expres-sionrdquo Experimental Cell Research vol 319 no 17 pp 2684ndash26992013

[33] D W Huang B T Sherman and R A Lempicki ldquoSystematicand integrative analysis of large gene lists using DAVID bioin-formatics resourcesrdquo Nature Protocols vol 4 no 1 pp 44ndash572009

[34] Q-L Ying J Nichols I Chambers and A Smith ldquoBMPinduction of Id proteins suppresses differentiation and sustainsembryonic stem cell self-renewal in collaboration with STAT3rdquoCell vol 115 no 3 pp 281ndash292 2003

[35] A Szwagierczak S Bultmann C S Schmidt F Spada and HLeonhardt ldquoSensitive enzymatic quantification of 5-hydroxy-methylcytosine in genomic DNArdquo Nucleic Acids Research vol38 no 19 article e181 2010

[36] A OrsquoLoghlen A M Munoz-Cabello A Gaspar-Maia et alldquoMicroRNA regulation of Cbx7 mediates a switch of polycomborthologs during ESC differentiationrdquoCell Stem Cell vol 10 no1 pp 33ndash46 2012

[37] EACasanovaO Shakhova S S Patel et al ldquoPramel7mediatesLIFSTAT3-dependent self-renewal in embryonic stem cellsrdquoStem Cells vol 29 no 3 pp 474ndash485 2011

[38] Y-S Chan J Goke X Lu et al ldquoA PRC2-dependent repressiverole of PRDM14 in human embryonic stem cells and inducedpluripotent stem cell reprogrammingrdquo Stem Cells vol 31 no 4pp 682ndash692 2013

[39] A S Bernardo T Faial L Gardner et al ldquoBRACHYURYand CDX2mediate BMP-induced differentiation of human andmouse pluripotent stem cells into embryonic and extraembry-onic lineagesrdquo Cell Stem Cell vol 9 no 2 pp 144ndash155 2011

[40] X Tang Y Zhang L Tucker and B Ramratnam ldquoPhosphoryla-tion of the RNase III enzyme Drosha at Serine300 or Serine302is required for its nuclear localizationrdquo Nucleic Acids Researchvol 38 no 19 Article ID gkq547 pp 6610ndash6619 2010

[41] X Tang M Li L Tucker and B Ramratnam ldquoGlycogensynthase kinase 3 beta (GSK3120573) phosphorylates the RNAase IIIenzyme Drosha at S300 and S302rdquo PLoS ONE vol 6 no 6Article ID e20391 2011

[42] Y Wu F Liu Y Liu et al ldquoGSK3 inhibitors CHIR99021 and6-bromoindirubin-31015840-oxime inhibit microRNA maturation inmouse embryonic stem cellsrdquo Scientific Reports vol 5 p 86662015

[43] MOKim S-HKim Y-Y Cho et al ldquoERK1 andERK2 regulateembryonic stem cell self-renewal through phosphorylation ofKlf4rdquoNature Structural andMolecular Biology vol 19 no 3 pp283ndash290 2012

[44] S Efroni R Duttagupta J Cheng et al ldquoGlobal transcriptionin pluripotent embryonic stem cellsrdquo Cell Stem Cell vol 2 no5 pp 437ndash447 2008

[45] YWang S Baskerville A Shenoy J E Babiarz L Baehner andR Blelloch ldquoEmbryonic stem cell-specific microRNAs regulatethe G1-S transition and promote rapid proliferationrdquo NatureGenetics vol 40 no 12 pp 1478ndash1483 2008

[46] I J MacRae and J A Doudna ldquoRibonuclease revisited struc-tural insights into ribonuclease III family enzymesrdquo CurrentOpinion in Structural Biology vol 17 no 1 pp 138ndash145 2007

[47] Y Lee C Ahn J Han et al ldquoThe nuclear RNase III Droshainitiates microRNA processingrdquo Nature vol 425 no 6956 pp415ndash419 2003

[48] J Han J S Pedersen S C Kwon et al ldquoPosttranscriptionalcrossregulation betweenDrosha andDGCR8rdquoCell vol 136 no1 pp 75ndash84 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


and PD0325901 [11 12] It has been discovered that sev-eral molecular pathways including JAKSTAT BMPSMADWnt120573-catenin and MEKERK are the underlying basis ofthese two ESC media for supporting mESC pluripotency inculture However mESCs in serum-containing medium areheterogeneous which is different from a homogeneous stateof ESC in serum-free N2B27 medium supplemented with2i This is due to the self-activating differentiation signal ofMEKERK that triggers differentiation of ESCs which mightresult in the heterogeneous state of ESCs Recent studieshave identified that PD is one of the inhibitors of MEKERKpathway stimulated by fibroblast growth factor-4 (Fgf4) inmESCs [11 13] Inactivation of MEKERK by PD restrictsthe differentiation of ESCs [14] and this effect is majorlymediated by enhancing Nanog expression [15ndash17] Howeververy little is known about the other possible mechanisms thatfunction in this process For example the role of miRNAshas not been investigated so far when MEKERK signalingcascade was blocked Clarification of miRNAs functionsin MEKERK signaling will provide further insight intomechanisms that ESCs maintain their intrinsic properties

The miRNAs are small noncoding RNAs that regulatemRNA stability andor translational efficiency [18] MostmiRNA genes are transcribed from either miRNA genesor intronic sequences of protein coding genes by RNApolymerase II to generate a stem-loop containing primarymiRNA (pri-miRNA) [19] The hairpin embedded in pri-miRNA is recognized by the RNA-binding protein Dgcr8which directs the RNase III enzymeDrosha to cleave the baseof the hairpin [20 21] Following cleavage by the Drosha-Dgcr8 complex the released short hairpin called precursormiRNA (pre-miRNA) is then transported by the Exportin-5Ran-GTP complex to the cytoplasm where Dicer togetherwith Trbp2 cleaves it into a single short 18ndash25 nt dsRNA[22] Each of them can be recruited into RNA-inducedsilencing complex (RISC) This complex targets mRNAs viabase pairing between the miRNA and mRNA resultingin the regulation of various aspects of stem cell functionsincluding the maintenance and induction of pluripotencyfor reprogramming [7 23] Several lines of evidence furtherindicated the global function ofmiRNAs inDicer orDGCR8-deficient mESCs [24ndash27] Additionally individual miRNAfunction has also been revealed in ESCs [28ndash30] Thus inorder to dissect how epigenetic regulator including miRNAand key regulatory genes establish J1 mouse ESC propertiesin a defined molecular pathway we identified MEKERKsignal-related miRNAs and genome-wide regulation profilesof J1 mESCs stimulated by PD using small RNA deep-sequencing and microarray analysis followed by subsequentverification We demonstrated that PD enhances ESC self-renewal capacity by not only key regulatory genes but alsoESC-specific miRNA which in turn mediates ESC self-renewal and cellular differentiation

2 Materials and Methods

21 ESC Culture The mouse J1 ESC line purchased fromthe American Type Culture Collection (Manassas VA USA)

was cultured in 01 (wv) gelatin coated tissue cultureplates without feeders in ESC media [knockout Dulbeccorsquosmodified Eaglersquos medium supplemented with 15 (vv)knockout serum replacement 01mM 120573-mercaptoethanol 1xnonessential amino acids 2mM GlutaMax 50UmL peni-cillin 50 120583gmL streptomycin (Life Technologies Inc GrandIsland NY USA) and 1000UmL LIF (ESGRO MilliporeUSA)] 293T cell line was cultured at 37∘Chumidified air with5CO2 inDulbeccorsquosmodified Eaglemedium supplementedwith 10 fetal bovine serum

22 Reagents andAntibodies PD0325901 DMSO andmouseanti-GAPDH were purchased from Sigma-Aldrich The pri-mary antibodies used were rabbit anti-Nanog (CST DanversMA USA) rabbit anti-Klf4 (Boster Wuhan China) mouse-anti-c-Myc (Santa Cruz CA USA) goat anti-Tet1 (SantaCruz) rabbit anti-5hmC (Active Motif Carlsbad CA USA)rabbit anti-Ezh2 (Abcam Cambridge UK) rabbit anti-H3K27me3 (Abcam) mouse anti-Oct34 (Santa Cruz) andmouse anti-Sox2 (Santa Cruz) Alexa Fluor 555-labeled goatanti-rabbitmouse IgG and anti-rabbitmouse horseradishperoxidase-conjugated secondary antibody were obtainedfrom the Beyotime Institute of Biotechnology (NantongJiangsu China)

23 RT-qPCR The total RNA was isolated from culturedcells using the Trizol reagent (Life Technologies) First-strand cDNA synthesis was performed using the SYBRPrimeScript RT reagent Kit (Perfect Real Time) (TakaraDalian China) according to the manufacturerrsquos instruc-tions qPCR was performed using SYBR Premix Ex Taq II(Takara) RT-qPCR was performed in an ABI StepOne PlusPCR System (Applied Biosystems California USA) withSYBR Premix Ex TaqTM (Takara) The forward and reverseprimers used for real-time PCR were shown in Supple-mentary Table 4 in Supplementary Material available onlineat httpdxdoiorg10115520161792573 The expression ofeach gene was defined from the threshold cycle (Ct) andrelative expression levels were calculated by using the 2minusΔΔCTmethod after normalization with reference to expression ofthe housekeeping gene GapdhThe gene expression ratio wasshown as mean plusmn SD from three independent experiments

24 Western Blot Analysis Cultured cells were lysed in RIPAbuffer Equal amounts of proteins were separated by 10polyacrylamide gels and transferred to PVDF membranes(Millipore MA USA) for 2 h at 100V After blocking non-specific binding by soaking the filters in 5 skim milk thedesired proteins were immunodetected with the respectiveantibodies that followed autography using SuperSignal WestPico substrate (Thermo Scientific IL USA) according to themanufacturerrsquos instructions

25 Immunofluorescence Staining Cells were fixed in 4paraformaldehyde for 20min and incubated at 37∘C inblocking buffer (PBS containing 5 BSA and 02 TritonX-100) Cells were incubated in the presence of primaryantibodies at 4∘C overnight and then washed three times











3 Results



DMSO PD03

50120583M50120583M

(a)

0

04

08

12

16

2


Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)

DMSOPD03

lowastlowast

lowastlowast

(b)

RA DMSOPD03

Nanog

Gapdh

RA

Klf4

PD03DMSO

c-Myc

Gapdh

Gapdh

PD03DMSO

1120583M

1120583M

1120583M3120583M

3120583M 1120583M 3120583M

RA1120583M 3120583M 1120583M 3120583M

3120583MPD + RA

PD03 + RA

PD03 + RA(c)

DMSO PD03

Tet1

5hmC

EZH2

H3K27me3

50120583M 50120583M 50120583M 50120583M

50120583M50120583M50120583M50120583M

50120583M 50120583M 50120583M 50120583M

50120583M50120583M50120583M50120583M

(d)













0

05

1

15

2

25

3

DMSOPD03

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


lowast

lowast

lowast lowast

lowastlowast

(a)

Oct4

Sox2

PD03DMSO

50120583M50120583M50120583M50120583M

50120583M 50120583M 50120583M 50120583M

(b)





Cell adhesion

Biological adhesion




Up

minusLog2P

(c)

0 2 4 6 8 10 12







minusLog2P

Dow

n

(d)

0 5 10 15 20


Focal adhesion

Ribosome




way

anal

ysis

minusLog2P

(e)

0

04

08

12

16

2


Relat

ive l

ucife

rase

activ

ity

DMSOPD03

lowast

lowast

lowastlowast

(f)



mESC media (LIF+)

DMSO (control) PD03

RNA extraction


sequencing

Microarrayanalysis


24h24h

(a)

01

1

10

100

1000

10000

100000

01 10 1000 100000


Expr

essio

n le

vel (

PD03

2590

1)



(b)








09

12

15

18

21

24

DMSO PD03


Relat

ive m

iRN

A fo

ld ch

ange

miR-302-367 cluster

(a)

08

085

09

095

1

105

DMSO PD03

miR-290-295 cluster

Relat

ive m

iRN

A fo

ld ch

ange


(b)

05

07

09

11

13

DMSO PD03

miR-17-92b cluster


Relat

ive m

iRN

A fo

ld ch

ange

(c)

06

065

07

075

08

085

09

095

1

105

DMSO PD03


Relat

ive m

iRN

A fo

ld ch

ange


(d)

08

085

09

095

1

105

DMSO PD03

miR-106b-25 cluster


Relat

ive m

iRN

A fo

ld ch

ange

(e)

0 1 2 3 4 5





PD03DMSO



lowastlowast

lowastlowast

lowastlowast

lowast

(f)





5 2 24

Up in PD03

Up in CHIR

miRNA

(a)

305 57 6

Down in PD03

Down in CHIR

miRNA

(b)

309 64 25PD03CHIR

miRNA

(c)

Pri-miRNA

Mature miRNA

Pre-miRNA

ESC self-renewal

DGCR8 knockoutESCs

ESC differentiation

Mature miRNA

ESC self-renewal

DGCR8 knockoutESCs

ESC differentiation

Drosha

DGCR8

N2B27

PD03CHIR

(d)

0

02

04

06

08

1

12

Relat

ive l

ucife

rase

activ

ity lowast



+

+

minus

+

+

+

minus

minus

minus +

+

minus


psiCHECK2NanogCDS

(e)

0

02

04

06

08

1

12

Relat

ive m

RNA

expr

essio

n lowast


DMSOPD03

+

+

+

+

+

minus

minus minus

minus +

+

minus

(f)

Nanog

Gapdh


(g)





4 Discussion











5 Conclusions






Acknowledgments



References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











3 Results



DMSO PD03

50120583M50120583M

(a)

0

04

08

12

16

2


Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)

DMSOPD03

lowastlowast

lowastlowast

(b)

RA DMSOPD03

Nanog

Gapdh

RA

Klf4

PD03DMSO

c-Myc

Gapdh

Gapdh

PD03DMSO

1120583M

1120583M

1120583M3120583M

3120583M 1120583M 3120583M

RA1120583M 3120583M 1120583M 3120583M

3120583MPD + RA

PD03 + RA

PD03 + RA(c)

DMSO PD03

Tet1

5hmC

EZH2

H3K27me3

50120583M 50120583M 50120583M 50120583M

50120583M50120583M50120583M50120583M

50120583M 50120583M 50120583M 50120583M

50120583M50120583M50120583M50120583M

(d)













0

05

1

15

2

25

3

DMSOPD03

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


lowast

lowast

lowast lowast

lowastlowast

(a)

Oct4

Sox2

PD03DMSO

50120583M50120583M50120583M50120583M

50120583M 50120583M 50120583M 50120583M

(b)





Cell adhesion

Biological adhesion




Up

minusLog2P

(c)

0 2 4 6 8 10 12







minusLog2P

Dow

n

(d)

0 5 10 15 20


Focal adhesion

Ribosome




way

anal

ysis

minusLog2P

(e)

0

04

08

12

16

2


Relat

ive l

ucife

rase

activ

ity

DMSOPD03

lowast

lowast

lowastlowast

(f)



mESC media (LIF+)

DMSO (control) PD03

RNA extraction


sequencing

Microarrayanalysis


24h24h

(a)

01

1

10

100

1000

10000

100000

01 10 1000 100000


Expr

essio

n le

vel (

PD03

2590

1)



(b)








09

12

15

18

21

24

DMSO PD03


Relat

ive m

iRN

A fo

ld ch

ange

miR-302-367 cluster

(a)

08

085

09

095

1

105

DMSO PD03

miR-290-295 cluster

Relat

ive m

iRN

A fo

ld ch

ange


(b)

05

07

09

11

13

DMSO PD03

miR-17-92b cluster


Relat

ive m

iRN

A fo

ld ch

ange

(c)

06

065

07

075

08

085

09

095

1

105

DMSO PD03


Relat

ive m

iRN

A fo

ld ch

ange


(d)

08

085

09

095

1

105

DMSO PD03

miR-106b-25 cluster


Relat

ive m

iRN

A fo

ld ch

ange

(e)

0 1 2 3 4 5





PD03DMSO



lowastlowast

lowastlowast

lowastlowast

lowast

(f)





5 2 24

Up in PD03

Up in CHIR

miRNA

(a)

305 57 6

Down in PD03

Down in CHIR

miRNA

(b)

309 64 25PD03CHIR

miRNA

(c)

Pri-miRNA

Mature miRNA

Pre-miRNA

ESC self-renewal

DGCR8 knockoutESCs

ESC differentiation

Mature miRNA

ESC self-renewal

DGCR8 knockoutESCs

ESC differentiation

Drosha

DGCR8

N2B27

PD03CHIR

(d)

0

02

04

06

08

1

12

Relat

ive l

ucife

rase

activ

ity lowast



+

+

minus

+

+

+

minus

minus

minus +

+

minus


psiCHECK2NanogCDS

(e)

0

02

04

06

08

1

12

Relat

ive m

RNA

expr

essio

n lowast


DMSOPD03

+

+

+

+

+

minus

minus minus

minus +

+

minus

(f)

Nanog

Gapdh


(g)





4 Discussion











5 Conclusions






Acknowledgments



References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


DMSO PD03

50120583M50120583M

(a)

0

04

08

12

16

2


Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)

DMSOPD03

lowastlowast

lowastlowast

(b)

RA DMSOPD03

Nanog

Gapdh

RA

Klf4

PD03DMSO

c-Myc

Gapdh

Gapdh

PD03DMSO

1120583M

1120583M

1120583M3120583M

3120583M 1120583M 3120583M

RA1120583M 3120583M 1120583M 3120583M

3120583MPD + RA

PD03 + RA

PD03 + RA(c)

DMSO PD03

Tet1

5hmC

EZH2

H3K27me3

50120583M 50120583M 50120583M 50120583M

50120583M50120583M50120583M50120583M

50120583M 50120583M 50120583M 50120583M

50120583M50120583M50120583M50120583M

(d)













0

05

1

15

2

25

3

DMSOPD03

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


lowast

lowast

lowast lowast

lowastlowast

(a)

Oct4

Sox2

PD03DMSO

50120583M50120583M50120583M50120583M

50120583M 50120583M 50120583M 50120583M

(b)





Cell adhesion

Biological adhesion




Up

minusLog2P

(c)

0 2 4 6 8 10 12







minusLog2P

Dow

n

(d)

0 5 10 15 20


Focal adhesion

Ribosome




way

anal

ysis

minusLog2P

(e)

0

04

08

12

16

2


Relat

ive l

ucife

rase

activ

ity

DMSOPD03

lowast

lowast

lowastlowast

(f)



mESC media (LIF+)

DMSO (control) PD03

RNA extraction


sequencing

Microarrayanalysis


24h24h

(a)

01

1

10

100

1000

10000

100000

01 10 1000 100000


Expr

essio

n le

vel (

PD03

2590

1)



(b)








09

12

15

18

21

24

DMSO PD03


Relat

ive m

iRN

A fo

ld ch

ange

miR-302-367 cluster

(a)

08

085

09

095

1

105

DMSO PD03

miR-290-295 cluster

Relat

ive m

iRN

A fo

ld ch

ange


(b)

05

07

09

11

13

DMSO PD03

miR-17-92b cluster


Relat

ive m

iRN

A fo

ld ch

ange

(c)

06

065

07

075

08

085

09

095

1

105

DMSO PD03


Relat

ive m

iRN

A fo

ld ch

ange


(d)

08

085

09

095

1

105

DMSO PD03

miR-106b-25 cluster


Relat

ive m

iRN

A fo

ld ch

ange

(e)

0 1 2 3 4 5





PD03DMSO



lowastlowast

lowastlowast

lowastlowast

lowast

(f)





5 2 24

Up in PD03

Up in CHIR

miRNA

(a)

305 57 6

Down in PD03

Down in CHIR

miRNA

(b)

309 64 25PD03CHIR

miRNA

(c)

Pri-miRNA

Mature miRNA

Pre-miRNA

ESC self-renewal

DGCR8 knockoutESCs

ESC differentiation

Mature miRNA

ESC self-renewal

DGCR8 knockoutESCs

ESC differentiation

Drosha

DGCR8

N2B27

PD03CHIR

(d)

0

02

04

06

08

1

12

Relat

ive l

ucife

rase

activ

ity lowast



+

+

minus

+

+

+

minus

minus

minus +

+

minus


psiCHECK2NanogCDS

(e)

0

02

04

06

08

1

12

Relat

ive m

RNA

expr

essio

n lowast


DMSOPD03

+

+

+

+

+

minus

minus minus

minus +

+

minus

(f)

Nanog

Gapdh


(g)





4 Discussion











5 Conclusions






Acknowledgments



References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










0

05

1

15

2

25

3

DMSOPD03

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


lowast

lowast

lowast lowast

lowastlowast

(a)

Oct4

Sox2

PD03DMSO

50120583M50120583M50120583M50120583M

50120583M 50120583M 50120583M 50120583M

(b)





Cell adhesion

Biological adhesion




Up

minusLog2P

(c)

0 2 4 6 8 10 12







minusLog2P

Dow

n

(d)

0 5 10 15 20


Focal adhesion

Ribosome




way

anal

ysis

minusLog2P

(e)

0

04

08

12

16

2


Relat

ive l

ucife

rase

activ

ity

DMSOPD03

lowast

lowast

lowastlowast

(f)



mESC media (LIF+)

DMSO (control) PD03

RNA extraction


sequencing

Microarrayanalysis


24h24h

(a)

01

1

10

100

1000

10000

100000

01 10 1000 100000


Expr

essio

n le

vel (

PD03

2590

1)



(b)








09

12

15

18

21

24

DMSO PD03


Relat

ive m

iRN

A fo

ld ch

ange

miR-302-367 cluster

(a)

08

085

09

095

1

105

DMSO PD03

miR-290-295 cluster

Relat

ive m

iRN

A fo

ld ch

ange


(b)

05

07

09

11

13

DMSO PD03

miR-17-92b cluster


Relat

ive m

iRN

A fo

ld ch

ange

(c)

06

065

07

075

08

085

09

095

1

105

DMSO PD03


Relat

ive m

iRN

A fo

ld ch

ange


(d)

08

085

09

095

1

105

DMSO PD03

miR-106b-25 cluster


Relat

ive m

iRN

A fo

ld ch

ange

(e)

0 1 2 3 4 5





PD03DMSO



lowastlowast

lowastlowast

lowastlowast

lowast

(f)





5 2 24

Up in PD03

Up in CHIR

miRNA

(a)

305 57 6

Down in PD03

Down in CHIR

miRNA

(b)

309 64 25PD03CHIR

miRNA

(c)

Pri-miRNA

Mature miRNA

Pre-miRNA

ESC self-renewal

DGCR8 knockoutESCs

ESC differentiation

Mature miRNA

ESC self-renewal

DGCR8 knockoutESCs

ESC differentiation

Drosha

DGCR8

N2B27

PD03CHIR

(d)

0

02

04

06

08

1

12

Relat

ive l

ucife

rase

activ

ity lowast



+

+

minus

+

+

+

minus

minus

minus +

+

minus


psiCHECK2NanogCDS

(e)

0

02

04

06

08

1

12

Relat

ive m

RNA

expr

essio

n lowast


DMSOPD03

+

+

+

+

+

minus

minus minus

minus +

+

minus

(f)

Nanog

Gapdh


(g)





4 Discussion











5 Conclusions






Acknowledgments



References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


mESC media (LIF+)

DMSO (control) PD03

RNA extraction


sequencing

Microarrayanalysis


24h24h

(a)

01

1

10

100

1000

10000

100000

01 10 1000 100000


Expr

essio

n le

vel (

PD03

2590

1)



(b)








09

12

15

18

21

24

DMSO PD03


Relat

ive m

iRN

A fo

ld ch

ange

miR-302-367 cluster

(a)

08

085

09

095

1

105

DMSO PD03

miR-290-295 cluster

Relat

ive m

iRN

A fo

ld ch

ange


(b)

05

07

09

11

13

DMSO PD03

miR-17-92b cluster


Relat

ive m

iRN

A fo

ld ch

ange

(c)

06

065

07

075

08

085

09

095

1

105

DMSO PD03


Relat

ive m

iRN

A fo

ld ch

ange


(d)

08

085

09

095

1

105

DMSO PD03

miR-106b-25 cluster


Relat

ive m

iRN

A fo

ld ch

ange

(e)

0 1 2 3 4 5





PD03DMSO



lowastlowast

lowastlowast

lowastlowast

lowast

(f)





5 2 24

Up in PD03

Up in CHIR

miRNA

(a)

305 57 6

Down in PD03

Down in CHIR

miRNA

(b)

309 64 25PD03CHIR

miRNA

(c)

Pri-miRNA

Mature miRNA

Pre-miRNA

ESC self-renewal

DGCR8 knockoutESCs

ESC differentiation

Mature miRNA

ESC self-renewal

DGCR8 knockoutESCs

ESC differentiation

Drosha

DGCR8

N2B27

PD03CHIR

(d)

0

02

04

06

08

1

12

Relat

ive l

ucife

rase

activ

ity lowast



+

+

minus

+

+

+

minus

minus

minus +

+

minus


psiCHECK2NanogCDS

(e)

0

02

04

06

08

1

12

Relat

ive m

RNA

expr

essio

n lowast


DMSOPD03

+

+

+

+

+

minus

minus minus

minus +

+

minus

(f)

Nanog

Gapdh


(g)





4 Discussion











5 Conclusions






Acknowledgments



References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


09

12

15

18

21

24

DMSO PD03


Relat

ive m

iRN

A fo

ld ch

ange

miR-302-367 cluster

(a)

08

085

09

095

1

105

DMSO PD03

miR-290-295 cluster

Relat

ive m

iRN

A fo

ld ch

ange


(b)

05

07

09

11

13

DMSO PD03

miR-17-92b cluster


Relat

ive m

iRN

A fo

ld ch

ange

(c)

06

065

07

075

08

085

09

095

1

105

DMSO PD03


Relat

ive m

iRN

A fo

ld ch

ange


(d)

08

085

09

095

1

105

DMSO PD03

miR-106b-25 cluster


Relat

ive m

iRN

A fo

ld ch

ange

(e)

0 1 2 3 4 5





PD03DMSO



lowastlowast

lowastlowast

lowastlowast

lowast

(f)





5 2 24

Up in PD03

Up in CHIR

miRNA

(a)

305 57 6

Down in PD03

Down in CHIR

miRNA

(b)

309 64 25PD03CHIR

miRNA

(c)

Pri-miRNA

Mature miRNA

Pre-miRNA

ESC self-renewal

DGCR8 knockoutESCs

ESC differentiation

Mature miRNA

ESC self-renewal

DGCR8 knockoutESCs

ESC differentiation

Drosha

DGCR8

N2B27

PD03CHIR

(d)

0

02

04

06

08

1

12

Relat

ive l

ucife

rase

activ

ity lowast



+

+

minus

+

+

+

minus

minus

minus +

+

minus


psiCHECK2NanogCDS

(e)

0

02

04

06

08

1

12

Relat

ive m

RNA

expr

essio

n lowast


DMSOPD03

+

+

+

+

+

minus

minus minus

minus +

+

minus

(f)

Nanog

Gapdh


(g)





4 Discussion











5 Conclusions






Acknowledgments



References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


5 2 24

Up in PD03

Up in CHIR

miRNA

(a)

305 57 6

Down in PD03

Down in CHIR

miRNA

(b)

309 64 25PD03CHIR

miRNA

(c)

Pri-miRNA

Mature miRNA

Pre-miRNA

ESC self-renewal

DGCR8 knockoutESCs

ESC differentiation

Mature miRNA

ESC self-renewal

DGCR8 knockoutESCs

ESC differentiation

Drosha

DGCR8

N2B27

PD03CHIR

(d)

0

02

04

06

08

1

12

Relat

ive l

ucife

rase

activ

ity lowast



+

+

minus

+

+

+

minus

minus

minus +

+

minus


psiCHECK2NanogCDS

(e)

0

02

04

06

08

1

12

Relat

ive m

RNA

expr

essio

n lowast


DMSOPD03

+

+

+

+

+

minus

minus minus

minus +

+

minus

(f)

Nanog

Gapdh


(g)





4 Discussion











5 Conclusions






Acknowledgments



References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




4 Discussion











5 Conclusions






Acknowledgments



References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




5 Conclusions






Acknowledgments



References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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