research article survivin improves reprogramming...

Research ArticleSurvivin Improves Reprogramming Efficiency of Human NeuralProgenitors by Single Molecule OCT4

Shixin Zhou1 Yinan Liu1 Ruopeng Feng2 CaiyunWang3 Sibo Jiang4 Xiaoyan Zhang1

Feng Lan5 and Yang Li1

1Department of Cell Biology and Stem Cell Research Center School of Basic Medicine Peking University Beijing China2Baotou Medical College Baotou Inner Mongolia China3Beijing Cellapy Biotechnology Co LTD Beijing China4Department of Pharmaceutics University of Florida 6550 Sanger Road Orlando FL 32827 USA5Beijing Anzhen Hospital Beijing China

Correspondence should be addressed to Feng Lan fenglanccmueducn and Yang Li liyangbjmueducn

Received 5 August 2016 Accepted 1 September 2016

Academic Editor Gary E Lyons

Copyright copy 2016 Shixin Zhou 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

Induced pluripotent stem (iPS) cells have been generated from human somatic cells by ectopic expression of four Yamanaka factorsHere we report that Survivin an apoptosis inhibitor can enhance iPS cells generation from human neural progenitor cells (NPCs)together with one factor OCT4 (1F-OCT4-Survivin) Compared with 1F-OCT4 Survivin accelerates the process of reprogrammingfrom human NPCs The neurocyte-originated induced pluripotent stem (NiPS) cells generated from 1F-OCT4-Survivin resemblehuman embryonic stem (hES) cells inmorphology surfacemarkers global gene expression profiling and epigenetic status Survivinkeeps high expression in both iPS and ES cells During the process of NiPS cell to neural cell differentiation the expression ofSurvivin is rapidly decreased in protein level The mechanism of Survivin promotion of reprogramming efficiency from NPCsmay be associated with stabilization of 120573-catenin in WNT signaling pathway This hypothesis is supported by experiments of RT-PCR chromatin immune-precipitation and Western blot in human ES cells Our results showed overexpression of Survivin couldimprove the efficiency of reprogramming from NPCs to iPS cells by one factor OCT4 through stabilization of the key molecule120573-catenin

1 Introduction

Reprogramming of somatic cells into induced pluripotentstem (iPS) cells holds great promise for the personal regen-erative medicines [1] The injured human cells or tissuescould be replaced by the similar differentiation cells fromhuman iPS cells [2] The method of genomic integrationof transgenes increases the risk of tumor formation andother abnormal issues [3] Nonintegration human iPS cellshave been generated using adenoviral or plasmid transfectionmethods [4 5] Other DNA-free methods include Sendaivirus [6] direct protein delivery and modified mRNA ormicroRNA transformations [7] In this study consideringthe key transcription factor SOX2 is endogenously highlyexpressed in human neural stem cells (NSCs) or progenitors(NPCs) in the process of reprogramming [8 9] we applied an

episomal vector [10] one of no genome integration methodto reprogram human NPCs to iPS cells with only one factorOCT4 (1F-OCT4)

The self-renewal maintenance of human pluripotent stemcells (PSCs) in vitro needs supports of growth factor (egbFGF) and extracellularmatrix (ECM) Better understandingof PSCs self-renewalmakes it possible to develop themediumwith defined chemicals The chemical defined medium is afully defined feeder-free medium formulated for the growthand expansion of human PSCs [11] For ECM element thefeeder-free matrigel which is a gelatinous protein mixturesecreted by mouse Engelbreth-Holm-Swarm (EHS) sarcomacells is widely used in cultivated human PSCs [12] Thedisadvantage of this xeno-proteins originated from mouseEHS may cause antigen response when applying iPS cells

Hindawi Publishing CorporationStem Cells InternationalVolume 2016 Article ID 4729535 11 pageshttpdxdoiorg10115520164729535

2 Stem Cells International

in human regeneration medicine [13] Here we use humanoriginated vitronectin (Xeno-free) instead of matrigel asECM for maintaining iPS pluripotency just for the safetyconcern

There are some reports showing that iPS cells retain anepigenetic memory of their original tissues in mouse andhuman iPS cells [14] Residualmethylation signatures link iPScells to their tissue of origin and even discriminate betweenthe myeloid and lymphoid origins of blood-derived iPS cells[15 16] Incomplete DNA methylation indicates a transcrip-tional memory of somatic cells in human iPS cells especiallyin the early passages [17] All low-passage iPS cells analyzedretain a transcriptional memory of the original cells Sucha memory would be the fingerprint of the iPS cellrsquos somaticorigin [16] iPS cells derived from human pancreatic isletbeta cells demonstrated an increased ability to differentiateinto insulin-producing cells compared with ES cells andisogenic non-beta iPS cells [14] All these evidences indicatethat iPS cells originated from neural progenitors carvedwith epigenetic memory may benefit easier differentiating toneural cells

Survivin is an important member of IAP (inhibitor ofapoptosis) family it functions as an apoptosis inhibitor indifferent types of cell especially in cancer cells Survivinexpression in normal tissue is developmentally regulated andhas been reported to be low in most terminally differentiatedtissues But it has also been showed that Survivin alsoexpressed in ES cell and NSCs (NPCs) OCT4 or SOX2regulates its expression in those cells Survivin expressionis positively related to pluripotency maintenance of ES cellsor iPS cells [18] In our previous research upregulation ofSurvivin could inhibit neural stem cells apoptosis mediatedby SOX2 [19] WNT signaling pathway reported plays animportant role in promoting somatic cell reprogramming themechanism is that 120573-catenin interacts with reprogrammingfactors KLF4 OCT4 and SOX2 to enhance expression ofpluripotency circuitry genes [20] Here we found overexpres-sion of Survivin in NPCs could enhance the efficiency of 1F-OCT4 reprogramming to iPS cells The mechanism of Sur-vivin improving the efficiency of NPCs reprogramming mayrelate to WNT signaling pathway by stabilization of the keymolecule 120573-catenin We found Survivin can directly interactwith120573-catenin in ES cells or iPS cells by immunoprecipitation(IP) analysis Our results indicated that stabilization of 120573-catenin would benefit pluripotency maintenance and couldenhance the efficiency of reprogramming

2 Materials and Methods

21 Cell Isolation and Culture Human embryonic corticaltissues were precisely dissected from naturally miscarriedfetal brains at 6674 and 90 days respectively The authoriza-tion for using human materials and the parentrsquos informedconsent was approved by the Ethics Committee of PekingUniversity Health Science Center and carried out accordingto the guidelines of the World Medical Association Decla-ration of Helsinki (httpwwwwmaneten30publications10policiesb3) Human fetal cortical tissue was treatedwith 025 trypsin for 15 minutes washed with Dulbeccorsquos

minimal essential medium (DMEM Hyclone LaboratoriesLogan UT USA) and dissociated into a single cell suspen-sion The cells were seeded at a density of 100000 cellsmLinto T75 flasks containing 20mL of serum free medium(DMEM F12) supplemented with B27 (2 Life Technolo-gies Carlsbad CA USA) EGF (20 ngmL Peprotech RockyHill NJ USA) FGF2 (20 ngmL Peprotech Rocky HillNJ USA) and heparin (5120583gmL Sigma St Louis MOUSA) NPCs were passaged by a choppingmethod previouslydescribed [21] One day before infection NPCs were digestedinto single cells byAccutase (Sigma St LouisMOUSA) and100000 cells were seeded to poly-ornithine-coated six-wellplates to form a monolayer culture

22 Episomal Vectors to Cells by Electroporation The episo-mal vectors (EBNA-1) are a well-described system for pro-ducing transgene-free virus-free iPS cells [10] The episomalvector containing pCXLE-OCT4 (1F-OCT4) was introducedinto human NPC by electroporation (Amaxa Nucleofec-tor 2B LabWrench Canada) on day 0 The transfectedcells were plated onto vitronectin-coated culture dishes Weused Reproeasy chemical defined medium (Cellapy BeijingChina) to cultivate 1F-OCT transfected cells from day 1to day 14 Mediums were changed to PSCeasy chemicaldefined medium (Cellapy Beijing China) from day 15 onvitronectin-coated culture vessels using 05mMEDTA as thepassaging reagent according to the manufacturerrsquos instruc-tions Survivin knockdown shRNAs (CCAGTGTTTCTTCT-GCTTCAA) were cloned into the adenovirus shuttle vectorpDC316-EGFP with U6 promoter and full-length SurvivincDNA was cloned into the pDC316-EGFP vector Recom-binant adenovirus was generated using the AdMax system(MicrobixBiosystems) Adenovirus was purified using theViraBindTM Adenovirus Purification Kit (Cell Biolabs) TheNPCs were infected at aMOI (multiplicity of infection) of 60

23 Immunofluorescence and Immunochemistry Cells weregrown on cover slides and fixed with 4 paraformaldehydeThe following antibodies to SOX2 NANOG Nestin SSEA-4TRA-1-60 and TRA-1-81 were all purchased from Chemicon(Temecula CA USA) and anti-OCT4 was from Santa CruzBiotechnology (Santa Cruz CA USA) Rabbit polyclonalantibody of Survivin was from Cell Signaling TechnologyInc (China) and120573-catenin antibodywas from Sigma-Aldrich(St Louis MO USA) The second antibodies (Alexa FluorSeries) were from Invitrogen (Grand Island NY USA) Thecell AP activity was analyzed by an alkaline phosphatase bluemembrane substrate solution kit (Sigma-Aldrich St LouisMO USA) according to the manufacturerrsquos guidelines

24 Gene Expression Profiling and Methylation AnalysisThe global gene expression profiling was performed byAffymetrix Human U133 plus 20 microarrays (AffymetrixSanta Clara CA USA) For transcriptome profiling 5 120583g oftotal RNA was used as input for labeled cRNA synthesisfollowing the manufacturerrsquos instructions (IVT 12 hours)Quality-checked cRNA samples were hybridized as biologicalfor 16 hours onto Human U133 plus 20 31015840 expressionmicroarrays All the steps including washing staining and

Stem Cells International 3

scanning signals followed suggestions by the manufacturerThemicroarray data were analyzed by Affymetrix ExpressionConsole Software 13 (Affymetrix SantaClara CAUSA)Theanalysis of MvA plot was used to compare two microarrays(cell types) identity The method also used Pearsonrsquos cor-relation (R2) value to indicate the similar The hierarchicalclustering analysis was carried out using Cluster 30 ([22] asupdated by Michiel de Hoon) and Treeview software usingmean-centered Pearson correlation and complete linkageDNA methylation analysis on OCT4 and NANOG wasdescribed as Freberg et al [23]

25 Luciferase Activity Assays The Survivin promoters wereamplified by PCR from human genome DNA digestedwith XhoI and HindIII and then ligated into the reportervector pGL3-Basic (Promega USA) The primers of Survivinpromoter for PCR were forward 51015840AGCTCGGCGGGG-TGGGAGGGGTGGGGAG31015840 and reverse 51015840AGCTTAGTA-GAGGCGGGGCGGCGCG31015840 HEK 293 cells were seeded ona 24-well plate and transiently transfected with the Survivinpromoter reporter vector and SOX2 or OCT4 expressionplasmid using Lipofectamine 2000 (Invitrogen USA) pRL-CMV (Promega USA) expressing Renilla luciferase wastransfected as an internal control After 48 h the transfectedHEK293 cells were lysed in passive lysis buffer and luciferaseactivity was measured with the dual-luciferase reporter assaysystem (Promega USA) using a Centro LB960 96-wellluminometer (Berthold Technologies Germany)

26 Chromatin Immunoprecipitation (ChIP) andWestern BlotAnalysis To generate chromatin about 1 times 107 infectedES cells or iPS cells were collected and fixed with 1polyformaldehyde The formaldehyde was inactivated by theaddition of 125mm glycine Cellular lysates were prepared byincubating the cells in lysis buffer (50mM Tris-HCl pH 80150mMNaCl 05 NP40) for 20min at 4∘CThe chromatincontaining genomic DNA in lysis buffer was fragmentedby sonication to the ranges of 150ndash500 bp The fragmentedchromatin is then precleared with protein A and G sepharosebeads and immunoprecipitated with antibodies of interestand sepharose beadsThematerial is thenwashed eluted andpurified with the Qiagen PCR purification kit The samplesare then analyzed by qPCR ChIP assays were performed asdescribed previously [8] For PCR 1120583L of DNA extractionwas used and amplified for 21ndash25 cycles The primers ofproximal promoter of Survivin were forward 51015840GACGCC-CTGCTTTGCGAAGG31015840 and reverse 51015840CTTCTGGAG-TCAGGGGCCAGGG31015840 Primers of NANOG promoter wereforward 51015840GCTGGTTTCAAACTCCTGACTTC31015840 and re-verse 51015840CAACAGAACCTGAAGACAAAC31015840 Primers of 120573-actin promoter were forward 51015840AGAAAATCTGGCACC-ACACC31015840 and reverse 51015840CGAGCCATAAAAGGCAACTTT-CGGA31015840

The RT-qPCR reactions were set up in triplicate with theSYBR Premix assay (Takara Japan) Reactions were run on aStratagene 3000P real-time PCR machine (Stratagene USA)with 40 cycles of 30 s at 95∘C 30 s at 58∘C and 30 s at 72∘C[24]

For Western blotting cells were lysed with ice-cold lysisbuffer and a protease inhibitor cocktail [19] Equal amounts ofextracted proteins (50ndash80120583g) were resolved with SDSPAGE(10 gels) electrophoresis then the gel was transferred onto 02 120583m PVDF membranes After blocking in 5 skimmedmilk for 1 h the membranes were incubated in the primaryantibodies at 4∘C overnight The antibodies used were rabbitpolyclonal anti-SOX2 anti-Survivin and mouse monoclonalanti-120573-actin The membranes were washed with PBST buffer3 times After incubation in labeled fluorescent secondaryantibodies (Rockland USA) for 1 h at room temperatureimages on membrane were developed with the OdysseyWestern blotting system (LI-COR Bio USA)

27 Reverse Transcription PCR (RT-PCR) RNAwas extractedusing the RNeasy Plus Mini Kit (Qiagen Germany) inaccordance with the manufacturerrsquos instructions For reversetranscription-quantitative PCR (RT-qPCR) SYBR Premixassay (Takara Japan) was used to perform real-time PCRwithMX3000PThe following thermal profile was used for allPCR experiments 95∘C for 5min 40 cycles at 95∘C for 30 sannealing temperature 58ndash60∘C for 30 s and it is terminatedby a final extension at 72∘C for 10min

The primers used for RT-PCR were SOX2 forward51015840GCCGAGTGGAAACTTTTGTCG31015840 and reverse 51015840GGC-AGCGTGTACTTATCCTTCT10158403 and Survivin forward51015840AGGACCACCGCATCTCTACAT31015840 and reverse 51015840AAG-TCTGGCTCGTTCTCAGTG31015840

28 ES (iPS) Cell Differentiation with Retinoic Acid (RA) ESor iPS cells were detached and dissociated into single cellswith 05mM EDTA and then plated onto a bacteriologicaldish in 10mLof a-MEM(Gibco) supplementedwith 10FBSsodiumbicarbonate (3mM) and 01mM2-ME (EBmedium)at a density of 5 times 104 cellsmL On day 2 Retinoic Acid(Sigma-Aldrich St Louis USA) of 500 nM was added to theculture medium and maintained for 96 h [25] The cells ofeach 24 h were collected for further analysis

3 Results

31 The Reprogramming Process of Human NPCs with 1F-OCT4 First we identified markers of human NPCs withimmunofluorescent staining The cultivated human NPCswere stained with antibodies of SOX2 and Nestin the keytranscription factor and surface marker by immunofluores-cent analysis It was observed that SOX2 and Nestin wereendogenously expressed in most human NPCs (Figure 1)Then we transfected 1F-OCT4 to human NPCs by electro-poration Three days after the transfection the cells wereplated at about 1 times 105 cells per well in a 35mm dishprecoated with vitronectin As seen under phase contrastmicroscopy the cell colony did not show up until 15 daysafter transfection (Figure 1(b)) In the following days thetypical colonies appeared flat and compact like human EScells in morphology (Figure 2(b)) We called these coloniesNiPS cells Typically 100ndash200 1F-OCT4 colonies could begenerated from 1 times 105 NPCs (reprogramming efficiency 01ndash02)


SOX2DAPI SOX2NestinNestinDAPI

(a)Day 3 Day 7 Day 13

Day 15 Day 19 Day 21Day 15 Day 19 Day 21

(b)

Figure 1 SOX2 and Nestin were endogenously expressed in human NSC or NPCs (a) SOX2 (green) and Nestin (red) were endogenouslyexpressed in human NSC or NPCs Nuclei were stained with DAPI (blue) (b) Reprogramming process of NPCs under phase contrastmicroscopy Scale bars 50 120583m

32 The Identification of Pluripotent Markers of NiPS CellsThese NiPS cells could be passaged by EDTA and exhib-ited positive features by alkaline phosphatase (AP) staining(Figures 2(a) 2(b) and 2(c)) Immunofluorescence stainingconfirmed human NiPS cells from 1F-OCT4 transfectionwidely expressed human pluripotent cell markers includingalkaline phosphatase OCT4 SOX2 NANOG SSEA4 TRA-1-60 and TRA-1-81 (Figure 2(d))These results demonstrated

that human iPS cells could be generated from human NPCsby a factor OCT4 alone

33 Gene Expression Profiling DNA Methylation and Dif-ferentiation In Vivo The global gene expression profiling ofNPCs NiPS and hES cells was performed by AffymetrixHuman U133 plus 20 microarrays The comparison of twomicroarrays was performed by Affymetrix Expression Con-sole Software 13 We use MvA plots to compare expression


(a) (b) (c)

OCT4DAPI SOX2DAPI NANOGDAPI

SSEA4DAPI TRA-1-60DAPI TRA-1-80DAPI

(d)

Figure 2 Morphology of NiPS colonies by 1F-OCT4 Low (a) and high (b) magnification of NiPS colonies 1F-OCT4 human NiPS colonieswere stained for alkaline phosphatase (c) immunofluorescent analysis of pluripotency and surface markers (d) The pluripotent markers(OCT4 SOX2 and NANOG) and surface markers (SSEA4 TRA-1-60 and TRA-1-81) were stained with antibodies in human 1F-OCT4 NiPScells Nuclei are stained with DAPI (blue) Scale bars 100120583m

profiling of NiPS and NPC and NiPS and hES cells Pearsonrsquoscorrelation (R2) of NiPS andNPC andNiPS and hES cells was087 and 097 respectively The result indicated expressionsof NiPS cells are more similar to hES cells than those ofNPCs (Figure 3(a)) The hierarchical clustering analysis onhuman NPCs ESCs and NiPS cells showed the similar result

(see Supplementary Figure S1 in Supplementary Materialavailable online at httpdxdoiorg10115520164729535)To testify epigenetic status in reprogrammed cells we usedbisulphite sequencing analysis to determine the degree ofDNA methylation of the OCT4 and NANOG promoters(Figure 3(b)) Similar to human ES cells H1 both promoter


NPC

N-iP

S

N-iP

SES

C

(a)

NPC1 2 3 4 5 6

1 2 3 4 5 6 7 8 9 10

41 74 86 110

172

197

210

234

238

290

1 2 3 4 5 6 7 8 9 10

41 74 86 110

172

197

210

234

238

290

1 2 3 4 5 6 7 8 9 10

41 74 86 110

172

197

210

234

238

290

46 55 137

147

205

332

1 2 3 4 5 6

46 55 137

147

205

332

1 2 3 4 5 6

46 55 137

147

205

332

OCT4

NANOG

NiPS hES

(b)

(c)

Figure 3 Analyses of DNAmethylation and gene expression profiling in NiPS NPC and hES cells (a) Analysis of gene expression profilingthe MvA plot is a comparison plot comparing two microarraysThe 119910-axis is displayed on a log2 scale with green threshold lines for two-foldchanges The lower parts of MvA plot represent signals of NiPS cells while the upper parts represent signals of NPC or hES cells The colorcoding of the plot indicates the density of probes represented by that data point (b) bisulphite sequencing analysis of OCT4 and NANOGpromoter regions in human NSCs 1F-OCT4 human NiPS clones and human ES cells (c) Hematoxylin and Eosin (HE) stain of teratomasgenerated from 1F-OCT4 derived iPS cells injected subcutaneously into immunocompromised SCID mice Structures derivative of all threegerm layers could be identified (i) Neural tissue (ectoderm left) (ii) cartilage (mesoderm middle) and (iii) gut-like epithelium (endodermright)

regions were demethylated in 1F-OCT4 human NiPS cellsrelative to the cultivated human NSCs or NPCs Our resultsshowed a single transcription factor OCT4 could reprogramhuman NSCs into iPS cells that are very similar to human EScells at the promoters of two genes

The pluripotency of both NiPS cells was also testedin vivo Eight weeks after injection of 1 times 106 NiPS cellsinto the testis of SCID mice tumor mass was isolated andanalyzed by immunostaining As shown in Figure 3(c) the

tumor tissues contained neural tissues (ectoderm left panel)cartilage (mesoderm middle panel) and gut-like epithelium(endoderm right panel) These results indicated the featureof teratoma for NiPS cells and revealed the pluripotency ofthese cells

34 Survivin Expression Related to Efficiency of Reprogram-ming Survivin is an important factor that related to ES cellpluripotency maintenance we checked Survivin expression


The n

umbe

r of A

P po

sitiv

e col

onie

s

0

50

100

150

200

250

300

350

Mock SurvivinControl(a)

The n

umbe

r of A

P po

sitiv

e col

onie

s

0

50

100

150

200

Survivin RNAiControl Nonsilence(b)

Mock SurvivinControl

Surv

ivin

relat

ive m

RNA

leve

l

0

1

2

3

4

(c)Survivin RNAiControl Nonsilence

Surv

ivin

relat

ive m

RNA

leve

l

0

05

1

15

(d)

Figure 4 AP positive numbers in Survivin overexpression or Survivin knockdown in the reprogramming of NPCs (a) AP positive numbersof 1F-OCT4 mock (vector control) and Survivin overexpression on 35 cm diameter well from the left to right (b) AP positive numbersof 1F-OCT4 nonsilence and Survivin-RNAi on the same size dishes (from the left to right) A representative experiment is shown in theleft panels Counting AP+ colonies in the same experiment mean values + SD are shown in the right panels (c) The relative expression ofSurvivin in mRNA level

manner during ES cell differentiation and reprogrammingWe evaluated the effect of 1F-OCT4 infected NPCs withhigh level or low level of Survivin in the process of NPCreprogramming At days 12ndash15 after infection most alkalinephosphatase-positive (AP+) colonies appeared in the group

of 35 cm diameter well with Survivin overexpression com-pared to groups of the control or mock vectorThe number ofcolonies in the group of Survivin overexpressionwas two-foldcompared to the group of 1F-OCT4 control and mock vector(119899 = 3 119901 lt 005) (Figure 4(a)) On the contrary low Survivin


70

60

50

40

30

20

10

000

SOX2 (ng)OCT4 (ng)

0200 0

200200200

Rela

tive l

ucife

rase

activ

ity

(a)009

008

007

006

005

004

003

002

001

000NANOGpromoter

IgGSOX2 Ab

Birc5promoter

ACT8promoter

Inpu

t (

)

lowastlowast

(b)

NANOGpromoter

IgGOCT4 Ab

Birc5promoter

ACT8promoter

009

008

007

006

005

004

003

002

001

000

Inpu

t (

)

lowast

lowast

(c)

Figure 5 SOX2 and OCT4 synergistically regulate expression of Survivin in ES cells ChIP-qPCRs were conducted using SOX2 and OCT4antibodies and primers specific for promoter of NANOG Survivin (Birc5) and the 120573-actin genes DNA lysates of cells were as input NANOGwas the positive control while 120573-actin was the negative control

expression (about 70 reducing) by RNAi leads to a halfAP+ colonies formation compared to the 1F-OCT4 controlor nonsilence (119899 = 3 119901 lt 005) (Figure 4(b)) The mRNArelative level of Survivin overexpression group was about 35-fold compared with control and mock groups (Figure 4(c))When Survivin was inhibited by RNAi the mRNA relativelevel was decreased 3 times compared with the control andnonsilence groups (Figure 4(d))These studies suggested thatsilencing of Survivin reduces iPS cells generation and that theexpression of the self-renewal regulator Survivin is absolutelyessential for cellular reprogramming

35 SOX2 and OCT4 Synergistically Regulate Expressionof Survivin We constructed Survivin promoter sequencesand then measured Survivin transcriptional activity in

HEK 293 cells upon adding OCT4 and SOX2 plasmids byluciferase assay The results showed Survivin promoter driv-ing luciferase expression was positively regulated by OCT4and SOX2 These two transcription factors had synergisticeffects in the regulation of Survivin in vitro (Figure 5(a))To find out how did Survivin participates in ES or iPS cellpluripotencymaintenance and reprogramming we did chro-matin immunoprecipitation (ChIP) in ES cells ChIP-qPCRanalysis was conducted using SOX2 and OCT4 antibodiesand primers specific for promoter of NANOG Survivin(Birc5) and the 120573-actin genes The results showed a higherlevel of enrichment on NANOG promoter as positive controland Survivin (Birc5) promoters in ES cells However thepromoter of120573-actin (the negative control) had no enrichment(Figure 5(b)) ChIP analysis revealed the occupancy of SOX2


Input Normal IgG Survivin IP

120573-catenin

Survivin

HSP90

(a)

120573-catenin

120573-actin

Survivin

Nonsilence Survivin knockdown

(b)

Figure 6 ChIP (chromatin immunoprecipitation) analysis of Survivin in ES (iPS) cells (a) ChIP of 120573-catenin and Survivin promoter HSP90(hot shock protein 90) was the positive control (b) Survivin-RNAi (knockdown) made 120573-catenin reducing expression by the analysis ofWestern blot

120573-actin

Survivin

OCT4

SOX2

0h 24h 36h 48h 72hRA treat(a)

00

02

04

06

08

10

12Su

rviv

in re

lativ

e mRN

A le

vel

RA treat 0h 48h 72h 96h24h(b)

Figure 7 The expression of Survivin was decreased with ES cell differentiation (a) Expression of Survivin was downregulated with ES celldifferentiation with Retinoic Acid (RA) The cells of 0 h 24 h 36 h 48 h and 72 h were collected for Western blot analysis The proteins ofSOX2 OCT4 and Survivin were analyzed in RA-treated differentiation and 120573-actin as housekeeping control (b) The change of SurvivinmRNA levels in the process of RA-treated differentiation for ES cells The expressions of mRNA decreased gradually and sharply withprolonged RA-treatment time

and OCT4 in the promoters of SurvivinThis result indicatedSOX2 and OCT4 could regulate Survivin by binding to itspromoter synergistically

36 Survivin Controls Pluripotency by Regulating 120573-CateninProtein Stability It has been shown that activation of thecanonicalWNTpathway is sufficient tomaintain self-renewalof both hES and mES cells [26] To find out the role ofSurvivin in reprogramming we did immunoprecipitationwith Survivin in human ES cell Coimmunoprecipitation(Co-IP) analysis showed 120573-catenin could be pulled downby Survivin (Figure 6(a)) This proved that Survivin coulddirectly interact with 120573-catenin which is also related to EScell pluripotency and reprogramming If Survivin expressiondecreased by shRNA the expression of 120573-catenin was also

downregulated (Figure 6(b)) These results indicate knock-down of Survivin leads to reduction of 120573-catenin

37 Decreased Survivin Expression in the Process of Neu-ral Differentiation of Human ES Cells In serum-containingcultures Retinoic Acid (RA) is essential to induce efficientneural differentiation from ESCs [27] In the process of ESCdifferentiation Survivinwas sharply downregulated after 24 htreatment of RA byWestern blot analysisThen it maintainedmedium expression in the differentiation to neural cells(Figure 7(a)) The pluripotent genes such as OCT4 andSOX2 were dramatically decreasing in neural differentiation(Figure 7(a)) However Survivin was gradually decreasingin mRNA level with treatment time prolonged with RA byRT-qPCR (Figure 7(b)) Both RT-qPCR and Western blot


analysis showed Survivin expressions were decreased in theprocess of ES cells differentiation to neural cells This resultindicated Survivin was another factor relative to maintainingpluripotency of human pluripotent stem cells

4 Discussion

The ability of reprogramming of somatic cells provides greatpotential for developing personalized treatments for diseasesand for drug screening [3 6] Here we presented that humaniPS cells are reprogrammed by a single factor OCT4 fromneural progenitor or stem cells with episomal vector HumaniPS cells reprogrammed by OCT4 cultivated with this systemappeared to be similar to human ES cells in morphologysurface markers and the capacity of differentiation to threeembryonic germ layers [10] The formulated and feeder-free mediums were advantageous for NSC reprogrammingand experiment repeats Episomal vectors could be sub-sequently removed from cells by culturing them (gradualloss of cellular episomal vectors from proliferating cells)Generation of patient-derived iPS cells serves as a greatsource for neurodegeneration therapies [28] Reproeasy andPSCeasy medium which were designed and developed byCellapy Biotechnology Corporation are mediums of com-plete chemically defined formulation designed for feeder-free reprogramming maintenance and expansion of bothhuman ES cells and iPS cells This kind of cultivated systemwith defined ingredients mediums feeder-free and Xeno-free ECM to maintain human iPS cells has higher safety forclinical applications

Our results demonstrated that Survivin was one of thefactors to participate in ES or iPS cell pluripotency mainte-nance We also found overexpression of Survivin in NPCscould enhance the efficiency of 1F-OCT4 reprogramming toiPS cells Themechanism of Survivin to maintain pluripotentstate of ESCs was that it could interact with 120573-catenin inWNT signal pathway WNT signals and 120573-catenin werealso reported relative to ESCrsquos pluripotency and iPS cellsrsquoreprogramming [20 29] The WNT3a-conditioned mediapromoted reprogramming of mouse embryonic fibroblasts(MEF) [30] The evidence proposed by other groups alsosupported our observation nuclear 120573-catenin protectedcells from apoptosis during the reprogramming processBased upon these results they proposed that WNT120573-cateninCBP signaling leaded to improvement of repro-gramming efficiency [31] So stabilization of 120573-catenin bySurvivin was helpful for maintaining pluripotent state ofES or iPS cells Other researches showed the inhibition ofubiquitin-proteasome pathway blocked ESC differentiationand enhanced cellular reprogramming through stabilizationof c-Myc [32] So the other possibility for that study was thatinhibition of ubiquitin-proteasome systemmight stabilize the120573-catenin protein Here we showed Survivin interacted with120573-catenin in ES cell and the mechanism might be due tothe fact that Survivin-120573-catenin interaction could prevent 120573-catenin from degradation

Differentiation capacity of iPS cells to neural cells hadno influence in the formulated-ingredient-medium The keypoint of iPS cells inmedical application depended onwhether

there are well-established differentiation systems to generateneural cell types [15] The iPS cells exhibited diverse prop-erties in different cell cultural system The cultural systemof defined ingredients mediums feeder-free and Xeno-freeECM for iPS cells could reduce batch differences in theprocess of reprogrammingThe studies of cell-of-origin indi-cated that the parental cell could influence the differentiationcapacity of the resultant iPS cells [33] There were somereports that showed iPS cells derived from nonhematopoieticcells (neural progenitors and fibroblasts) retained resid-ual methylation [34ndash36] Some evidences showed iPS cellsretained a residual transcriptional memory of the somaticcells and provided data in support of inefficient promoterDNAmethylation as the underlyingmechanism [14 17] Sucha memory would be the fingerprint of the iPS cellrsquos somaticorigin These original memories may help iPS cells fromhuman NSC in more easy and efficient differentiation toneural cells compared with human fibroblast-originated iPScells

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Shixin Zhou Yinan Liu and Ruopeng Feng contributed tothis work equally

Acknowledgments

This work was funded by National Natural Science Foun-dation of China (31301212 31571517 81422003 81570215 and21233003)

References

[1] J B Kim B GreberM J Arazo-Bravo et al ldquoDirect reprogram-ming of human neural stem cells byOCT4rdquoNature vol 461 no7264 pp 649ndash653 2009

[2] B-YHu J PWeick J Yu et al ldquoNeural differentiation of humaninduced pluripotent stem cells follows developmental principlesbut with variable potencyrdquo Proceedings of the National Academyof Sciences of the United States of America vol 107 no 9 pp4335ndash4340 2010

[3] K Okita T Ichisaka and S Yamanaka ldquoGeneration ofgermline-competent induced pluripotent stem cellsrdquo Naturevol 448 no 7151 pp 313ndash317 2007

[4] A Nanbo A Sugden and B Sugden ldquoThe coupling of synthesisand partitioning of EBVrsquos plasmid replicon is revealed in livecellsrdquoThe EMBO Journal vol 26 no 19 pp 4252ndash4262 2007

[5] B Thyagarajan K Scheying H Xue et al ldquoA single EBV-basedvector for stable episomal maintenance and expression of GFPin human embryonic stem cellsrdquo Regenerative Medicine vol 4no 2 pp 239ndash250 2009

[6] M Auger ldquoRapid prescreening in gynecologic cytology a moreefficient quality assurance methodrdquo Cancer Cytopathology vol119 no 6 pp 357ndash360 2011

[7] L Warren P D Manos T Ahfeldt et al ldquoHighly efficientreprogramming to pluripotency and directed differentiation of


human cells with syntheticmodifiedmRNArdquoCell StemCell vol7 no 5 pp 618ndash630 2010

[8] S Zhu R Ambasudhan W Sun et al ldquoSmall moleculesenable OCT4-mediated direct reprogramming into expandablehuman neural stem cellsrdquo Cell Research vol 24 no 1 pp 126ndash129 2014

[9] J B Kim V Sebastiano G Wu et al ldquoOct4-induced pluripo-tency in adult neural stem cellsrdquo Cell vol 136 no 3 pp 411ndash4192009

[10] K Okita Y Matsumura Y Sato et al ldquoA more efficient methodto generate integration-free human iPS cellsrdquo Nature Methodsvol 8 no 5 pp 409ndash412 2011

[11] G Chen D R Gulbranson Z Hou et al ldquoChemically definedconditions for human iPSC derivation and culturerdquo NatureMethods vol 8 no 5 pp 424ndash429 2011

[12] C S Hughes L M Postovit and G A Lajoie ldquoMatrigel acomplex protein mixture required for optimal growth of cellculturerdquo Proteomics vol 10 no 9 pp 1886ndash1890 2010

[13] S R Braam L Zeinstra S Litjens et al ldquoRecombinant vit-ronectin is a functionally defined substrate that supports humanembryonic stem cell self-renewal via 120572V1205735 integrinrdquo StemCellsvol 26 no 9 pp 2257ndash2265 2008

[14] O Bar-Nur H A Russ S Efrat and N Benvenisty ldquoEpigeneticmemory and preferential lineage-specific differentiation ininduced pluripotent stem cells derived from human pancreaticislet beta cellsrdquo Cell Stem Cell vol 9 no 1 pp 17ndash23 2011

[15] D A Robinton and G Q Daley ldquoThe promise of inducedpluripotent stem cells in research and therapyrdquoNature vol 481no 7381 pp 295ndash305 2012

[16] K Kim A Doi B Wen et al ldquoEpigenetic memory in inducedpluripotent stem cellsrdquo Nature vol 467 no 7313 pp 285ndash2902010

[17] Y Ohi H Qin C Hong et al ldquoIncomplete DNA methylationunderlies a transcriptional memory of somatic cells in humaniPS cellsrdquo Nature Cell Biology vol 13 no 5 pp 541ndash549 2011

[18] A N Mull A Klar and C S Navara ldquoDifferential localizationand high expression of SURVIVIN splice variants in humanembryonic stem cells but not in differentiated cells implicate arole for SURVIVIN in pluripotencyrdquo Stem Cell Research vol 12no 2 pp 539ndash549 2014

[19] R Feng S Zhou Y Liu et al ldquoSox2 protects neural stemcells from apoptosis via up-regulating survivin expressionrdquoBiochemical Journal vol 450 no 3 pp 459ndash468 2013

[20] P Zhang W-H Chang B Fong et al ldquoRegulation of inducedpluripotent stem (iPS) cell induction by Wnt120573-catenin signal-ingrdquo The Journal of Biological Chemistry vol 289 no 13 pp9221ndash9232 2014

[21] C N Svendsen M G ter Borg R J E Armstrong et al ldquoA newmethod for the rapid and long term growth of human neuralprecursor cellsrdquo Journal of Neuroscience Methods vol 85 no 2pp 141ndash152 1998

[22] M B Eisen P T Spellman P O Brown and D Botstein ldquoClus-ter analysis and display of genome-wide expression patternsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 95 no 25 pp 14863ndash14868 1998

[23] C T Freberg J A Dahl S Timoskainen and P CollasldquoEpigenetic reprogramming of OCT4 and NANOG regulatoryregions by embryonal carcinoma cell extractrdquoMolecular Biologyof the Cell vol 18 no 5 pp 1543ndash1553 2007

[24] Q Hu L Zhang J Wen et al ldquoThe EGF receptor-sox2-EGFreceptor feedback loop positively regulates the self-renewal ofneural precursor cellsrdquo Stem Cells vol 28 pp 279ndash286 2010

[25] M Bibel J Richter E Lacroix and Y-A Barde ldquoGenerationof a defined and uniform population of CNS progenitors andneurons from mouse embryonic stem cellsrdquo Nature Protocolsvol 2 no 5 pp 1034ndash1043 2007

[26] N Sato L Meijer L Skaltsounis P Greengard and A HBrivanlou ldquoMaintenance of pluripotency in human and mouseembryonic stem cells through activation of Wnt signaling bya pharmacological GSK-3-specific inhibitorrdquo Nature Medicinevol 10 no 1 pp 55ndash63 2004

[27] T Glaser and O Brustle ldquoRetinoic acid induction of ES-cell-derived neurons the radial glia connectionrdquo Trends inNeurosciences vol 28 no 8 pp 397ndash400 2005

[28] H Okano M Nakamura K Yoshida et al ldquoSteps toward safecell therapy using induced pluripotent stem cellsrdquo CirculationResearch vol 112 no 3 pp 523ndash533 2013

[29] D ten Berge D Kurek T Blauwkamp et al ldquoEmbryonic stemcells require Wnt proteins to prevent differentiation to epiblaststem cellsrdquoNature Cell Biology vol 13 no 9 pp 1070ndash1075 2011

[30] A Marson R Foreman B Chevalier et al ldquoWnt signalingpromotes reprogramming of somatic cells to pluripotencyrdquo CellStem Cell vol 3 no 2 pp 132ndash135 2008

[31] F Lluis E Pedone S Pepe and M P Cosma ldquoThe Wnt120573-catenin signaling pathway tips the balance between apoptosisand reprograming of cell fusion hybridsrdquo STEMCELLS vol 28no 11 pp 1940ndash1949 2010

[32] S M Buckley B Aranda-Orgilles A Strikoudis et al ldquoReg-ulation of pluripotency and cellular reprogramming by theubiquitin-proteasome systemrdquo Cell Stem Cell vol 11 no 6 pp783ndash798 2012

[33] Q Hu A M Friedrich L V Johnson and D O CleggldquoMemory in induced pluripotent stem cells reprogrammedhuman retinal-pigmented epithelial cells show tendency forspontaneous redifferentiationrdquo STEMCELLS vol 28 no 11 pp1981ndash1991 2010

[34] P-L LuuH R Scholer andM J Arauzo-Bravo ldquoDisclosing thecrosstalk among DNA methylation transcription factors andhistone marks in human pluripotent cells through discovery ofDNAmethylation motifsrdquo Genome Research vol 23 no 12 pp2013ndash2029 2013

[35] K Kim R Zhao A Doi et al ldquoDonor cell type can influencethe epigenome and differentiation potential of human inducedpluripotent stem cellsrdquo Nature Biotechnology vol 29 pp 1117ndash1119 2011

[36] G Hargus M Ehrlich M J Arauzo-Bravo et al ldquoOrigin-dependent neural cell identities in differentiated human iPSCsin vitro and after transplantation into the mouse brainrdquo CellReports vol 8 no 6 pp 1697ndash1703 2014

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


in human regeneration medicine [13] Here we use humanoriginated vitronectin (Xeno-free) instead of matrigel asECM for maintaining iPS pluripotency just for the safetyconcern

There are some reports showing that iPS cells retain anepigenetic memory of their original tissues in mouse andhuman iPS cells [14] Residualmethylation signatures link iPScells to their tissue of origin and even discriminate betweenthe myeloid and lymphoid origins of blood-derived iPS cells[15 16] Incomplete DNA methylation indicates a transcrip-tional memory of somatic cells in human iPS cells especiallyin the early passages [17] All low-passage iPS cells analyzedretain a transcriptional memory of the original cells Sucha memory would be the fingerprint of the iPS cellrsquos somaticorigin [16] iPS cells derived from human pancreatic isletbeta cells demonstrated an increased ability to differentiateinto insulin-producing cells compared with ES cells andisogenic non-beta iPS cells [14] All these evidences indicatethat iPS cells originated from neural progenitors carvedwith epigenetic memory may benefit easier differentiating toneural cells

Survivin is an important member of IAP (inhibitor ofapoptosis) family it functions as an apoptosis inhibitor indifferent types of cell especially in cancer cells Survivinexpression in normal tissue is developmentally regulated andhas been reported to be low in most terminally differentiatedtissues But it has also been showed that Survivin alsoexpressed in ES cell and NSCs (NPCs) OCT4 or SOX2regulates its expression in those cells Survivin expressionis positively related to pluripotency maintenance of ES cellsor iPS cells [18] In our previous research upregulation ofSurvivin could inhibit neural stem cells apoptosis mediatedby SOX2 [19] WNT signaling pathway reported plays animportant role in promoting somatic cell reprogramming themechanism is that 120573-catenin interacts with reprogrammingfactors KLF4 OCT4 and SOX2 to enhance expression ofpluripotency circuitry genes [20] Here we found overexpres-sion of Survivin in NPCs could enhance the efficiency of 1F-OCT4 reprogramming to iPS cells The mechanism of Sur-vivin improving the efficiency of NPCs reprogramming mayrelate to WNT signaling pathway by stabilization of the keymolecule 120573-catenin We found Survivin can directly interactwith120573-catenin in ES cells or iPS cells by immunoprecipitation(IP) analysis Our results indicated that stabilization of 120573-catenin would benefit pluripotency maintenance and couldenhance the efficiency of reprogramming

2 Materials and Methods

21 Cell Isolation and Culture Human embryonic corticaltissues were precisely dissected from naturally miscarriedfetal brains at 6674 and 90 days respectively The authoriza-tion for using human materials and the parentrsquos informedconsent was approved by the Ethics Committee of PekingUniversity Health Science Center and carried out accordingto the guidelines of the World Medical Association Decla-ration of Helsinki (httpwwwwmaneten30publications10policiesb3) Human fetal cortical tissue was treatedwith 025 trypsin for 15 minutes washed with Dulbeccorsquos

minimal essential medium (DMEM Hyclone LaboratoriesLogan UT USA) and dissociated into a single cell suspen-sion The cells were seeded at a density of 100000 cellsmLinto T75 flasks containing 20mL of serum free medium(DMEM F12) supplemented with B27 (2 Life Technolo-gies Carlsbad CA USA) EGF (20 ngmL Peprotech RockyHill NJ USA) FGF2 (20 ngmL Peprotech Rocky HillNJ USA) and heparin (5120583gmL Sigma St Louis MOUSA) NPCs were passaged by a choppingmethod previouslydescribed [21] One day before infection NPCs were digestedinto single cells byAccutase (Sigma St LouisMOUSA) and100000 cells were seeded to poly-ornithine-coated six-wellplates to form a monolayer culture

22 Episomal Vectors to Cells by Electroporation The episo-mal vectors (EBNA-1) are a well-described system for pro-ducing transgene-free virus-free iPS cells [10] The episomalvector containing pCXLE-OCT4 (1F-OCT4) was introducedinto human NPC by electroporation (Amaxa Nucleofec-tor 2B LabWrench Canada) on day 0 The transfectedcells were plated onto vitronectin-coated culture dishes Weused Reproeasy chemical defined medium (Cellapy BeijingChina) to cultivate 1F-OCT transfected cells from day 1to day 14 Mediums were changed to PSCeasy chemicaldefined medium (Cellapy Beijing China) from day 15 onvitronectin-coated culture vessels using 05mMEDTA as thepassaging reagent according to the manufacturerrsquos instruc-tions Survivin knockdown shRNAs (CCAGTGTTTCTTCT-GCTTCAA) were cloned into the adenovirus shuttle vectorpDC316-EGFP with U6 promoter and full-length SurvivincDNA was cloned into the pDC316-EGFP vector Recom-binant adenovirus was generated using the AdMax system(MicrobixBiosystems) Adenovirus was purified using theViraBindTM Adenovirus Purification Kit (Cell Biolabs) TheNPCs were infected at aMOI (multiplicity of infection) of 60

23 Immunofluorescence and Immunochemistry Cells weregrown on cover slides and fixed with 4 paraformaldehydeThe following antibodies to SOX2 NANOG Nestin SSEA-4TRA-1-60 and TRA-1-81 were all purchased from Chemicon(Temecula CA USA) and anti-OCT4 was from Santa CruzBiotechnology (Santa Cruz CA USA) Rabbit polyclonalantibody of Survivin was from Cell Signaling TechnologyInc (China) and120573-catenin antibodywas from Sigma-Aldrich(St Louis MO USA) The second antibodies (Alexa FluorSeries) were from Invitrogen (Grand Island NY USA) Thecell AP activity was analyzed by an alkaline phosphatase bluemembrane substrate solution kit (Sigma-Aldrich St LouisMO USA) according to the manufacturerrsquos guidelines

24 Gene Expression Profiling and Methylation AnalysisThe global gene expression profiling was performed byAffymetrix Human U133 plus 20 microarrays (AffymetrixSanta Clara CA USA) For transcriptome profiling 5 120583g oftotal RNA was used as input for labeled cRNA synthesisfollowing the manufacturerrsquos instructions (IVT 12 hours)Quality-checked cRNA samples were hybridized as biologicalfor 16 hours onto Human U133 plus 20 31015840 expressionmicroarrays All the steps including washing staining and










3 Results






(b)






(a) (b) (c)



(d)





NPC

N-iP

S

N-iP

SES

C

(a)

NPC1 2 3 4 5 6

1 2 3 4 5 6 7 8 9 10

41 74 86 110

172

197

210

234

238

290

1 2 3 4 5 6 7 8 9 10

41 74 86 110

172

197

210

234

238

290

1 2 3 4 5 6 7 8 9 10

41 74 86 110

172

197

210

234

238

290

46 55 137

147

205

332

1 2 3 4 5 6

46 55 137

147

205

332

1 2 3 4 5 6

46 55 137

147

205

332

OCT4

NANOG

NiPS hES

(b)

(c)







The n

umbe

r of A

P po

sitiv

e col

onie

s

0

50

100

150

200

250

300

350


The n

umbe

r of A

P po

sitiv

e col

onie

s

0

50

100

150

200



Surv

ivin

relat

ive m

RNA

leve

l

0

1

2

3

4


Surv

ivin

relat

ive m

RNA

leve

l

0

05

1

15

(d)





70

60

50

40

30

20

10

000

SOX2 (ng)OCT4 (ng)

0200 0

200200200

Rela

tive l

ucife

rase

activ

ity

(a)009

008

007

006

005

004

003

002

001

000NANOGpromoter

IgGSOX2 Ab

Birc5promoter

ACT8promoter

Inpu

t (

)

lowastlowast

(b)

NANOGpromoter

IgGOCT4 Ab

Birc5promoter

ACT8promoter

009

008

007

006

005

004

003

002

001

000

Inpu

t (

)

lowast

lowast

(c)







120573-catenin

Survivin

HSP90

(a)

120573-catenin

120573-actin

Survivin


(b)


120573-actin

Survivin

OCT4

SOX2


00

02

04

06

08

10

12Su

rviv

in re

lativ

e mRN

A le

vel









4 Discussion





Competing Interests




Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










3 Results






(b)






(a) (b) (c)



(d)





NPC

N-iP

S

N-iP

SES

C

(a)

NPC1 2 3 4 5 6

1 2 3 4 5 6 7 8 9 10

41 74 86 110

172

197

210

234

238

290

1 2 3 4 5 6 7 8 9 10

41 74 86 110

172

197

210

234

238

290

1 2 3 4 5 6 7 8 9 10

41 74 86 110

172

197

210

234

238

290

46 55 137

147

205

332

1 2 3 4 5 6

46 55 137

147

205

332

1 2 3 4 5 6

46 55 137

147

205

332

OCT4

NANOG

NiPS hES

(b)

(c)







The n

umbe

r of A

P po

sitiv

e col

onie

s

0

50

100

150

200

250

300

350


The n

umbe

r of A

P po

sitiv

e col

onie

s

0

50

100

150

200



Surv

ivin

relat

ive m

RNA

leve

l

0

1

2

3

4


Surv

ivin

relat

ive m

RNA

leve

l

0

05

1

15

(d)





70

60

50

40

30

20

10

000

SOX2 (ng)OCT4 (ng)

0200 0

200200200

Rela

tive l

ucife

rase

activ

ity

(a)009

008

007

006

005

004

003

002

001

000NANOGpromoter

IgGSOX2 Ab

Birc5promoter

ACT8promoter

Inpu

t (

)

lowastlowast

(b)

NANOGpromoter

IgGOCT4 Ab

Birc5promoter

ACT8promoter

009

008

007

006

005

004

003

002

001

000

Inpu

t (

)

lowast

lowast

(c)







120573-catenin

Survivin

HSP90

(a)

120573-catenin

120573-actin

Survivin


(b)


120573-actin

Survivin

OCT4

SOX2


00

02

04

06

08

10

12Su

rviv

in re

lativ

e mRN

A le

vel









4 Discussion





Competing Interests




Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





(b)






(a) (b) (c)



(d)





NPC

N-iP

S

N-iP

SES

C

(a)

NPC1 2 3 4 5 6

1 2 3 4 5 6 7 8 9 10

41 74 86 110

172

197

210

234

238

290

1 2 3 4 5 6 7 8 9 10

41 74 86 110

172

197

210

234

238

290

1 2 3 4 5 6 7 8 9 10

41 74 86 110

172

197

210

234

238

290

46 55 137

147

205

332

1 2 3 4 5 6

46 55 137

147

205

332

1 2 3 4 5 6

46 55 137

147

205

332

OCT4

NANOG

NiPS hES

(b)

(c)







The n

umbe

r of A

P po

sitiv

e col

onie

s

0

50

100

150

200

250

300

350


The n

umbe

r of A

P po

sitiv

e col

onie

s

0

50

100

150

200



Surv

ivin

relat

ive m

RNA

leve

l

0

1

2

3

4


Surv

ivin

relat

ive m

RNA

leve

l

0

05

1

15

(d)





70

60

50

40

30

20

10

000

SOX2 (ng)OCT4 (ng)

0200 0

200200200

Rela

tive l

ucife

rase

activ

ity

(a)009

008

007

006

005

004

003

002

001

000NANOGpromoter

IgGSOX2 Ab

Birc5promoter

ACT8promoter

Inpu

t (

)

lowastlowast

(b)

NANOGpromoter

IgGOCT4 Ab

Birc5promoter

ACT8promoter

009

008

007

006

005

004

003

002

001

000

Inpu

t (

)

lowast

lowast

(c)







120573-catenin

Survivin

HSP90

(a)

120573-catenin

120573-actin

Survivin


(b)


120573-actin

Survivin

OCT4

SOX2


00

02

04

06

08

10

12Su

rviv

in re

lativ

e mRN

A le

vel









4 Discussion





Competing Interests




Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b) (c)



(d)





NPC

N-iP

S

N-iP

SES

C

(a)

NPC1 2 3 4 5 6

1 2 3 4 5 6 7 8 9 10

41 74 86 110

172

197

210

234

238

290

1 2 3 4 5 6 7 8 9 10

41 74 86 110

172

197

210

234

238

290

1 2 3 4 5 6 7 8 9 10

41 74 86 110

172

197

210

234

238

290

46 55 137

147

205

332

1 2 3 4 5 6

46 55 137

147

205

332

1 2 3 4 5 6

46 55 137

147

205

332

OCT4

NANOG

NiPS hES

(b)

(c)







The n

umbe

r of A

P po

sitiv

e col

onie

s

0

50

100

150

200

250

300

350


The n

umbe

r of A

P po

sitiv

e col

onie

s

0

50

100

150

200



Surv

ivin

relat

ive m

RNA

leve

l

0

1

2

3

4


Surv

ivin

relat

ive m

RNA

leve

l

0

05

1

15

(d)





70

60

50

40

30

20

10

000

SOX2 (ng)OCT4 (ng)

0200 0

200200200

Rela

tive l

ucife

rase

activ

ity

(a)009

008

007

006

005

004

003

002

001

000NANOGpromoter

IgGSOX2 Ab

Birc5promoter

ACT8promoter

Inpu

t (

)

lowastlowast

(b)

NANOGpromoter

IgGOCT4 Ab

Birc5promoter

ACT8promoter

009

008

007

006

005

004

003

002

001

000

Inpu

t (

)

lowast

lowast

(c)







120573-catenin

Survivin

HSP90

(a)

120573-catenin

120573-actin

Survivin


(b)


120573-actin

Survivin

OCT4

SOX2


00

02

04

06

08

10

12Su

rviv

in re

lativ

e mRN

A le

vel









4 Discussion





Competing Interests




Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


NPC

N-iP

S

N-iP

SES

C

(a)

NPC1 2 3 4 5 6

1 2 3 4 5 6 7 8 9 10

41 74 86 110

172

197

210

234

238

290

1 2 3 4 5 6 7 8 9 10

41 74 86 110

172

197

210

234

238

290

1 2 3 4 5 6 7 8 9 10

41 74 86 110

172

197

210

234

238

290

46 55 137

147

205

332

1 2 3 4 5 6

46 55 137

147

205

332

1 2 3 4 5 6

46 55 137

147

205

332

OCT4

NANOG

NiPS hES

(b)

(c)







The n

umbe

r of A

P po

sitiv

e col

onie

s

0

50

100

150

200

250

300

350


The n

umbe

r of A

P po

sitiv

e col

onie

s

0

50

100

150

200



Surv

ivin

relat

ive m

RNA

leve

l

0

1

2

3

4


Surv

ivin

relat

ive m

RNA

leve

l

0

05

1

15

(d)





70

60

50

40

30

20

10

000

SOX2 (ng)OCT4 (ng)

0200 0

200200200

Rela

tive l

ucife

rase

activ

ity

(a)009

008

007

006

005

004

003

002

001

000NANOGpromoter

IgGSOX2 Ab

Birc5promoter

ACT8promoter

Inpu

t (

)

lowastlowast

(b)

NANOGpromoter

IgGOCT4 Ab

Birc5promoter

ACT8promoter

009

008

007

006

005

004

003

002

001

000

Inpu

t (

)

lowast

lowast

(c)







120573-catenin

Survivin

HSP90

(a)

120573-catenin

120573-actin

Survivin


(b)


120573-actin

Survivin

OCT4

SOX2


00

02

04

06

08

10

12Su

rviv

in re

lativ

e mRN

A le

vel









4 Discussion





Competing Interests




Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


The n

umbe

r of A

P po

sitiv

e col

onie

s

0

50

100

150

200

250

300

350


The n

umbe

r of A

P po

sitiv

e col

onie

s

0

50

100

150

200



Surv

ivin

relat

ive m

RNA

leve

l

0

1

2

3

4


Surv

ivin

relat

ive m

RNA

leve

l

0

05

1

15

(d)





70

60

50

40

30

20

10

000

SOX2 (ng)OCT4 (ng)

0200 0

200200200

Rela

tive l

ucife

rase

activ

ity

(a)009

008

007

006

005

004

003

002

001

000NANOGpromoter

IgGSOX2 Ab

Birc5promoter

ACT8promoter

Inpu

t (

)

lowastlowast

(b)

NANOGpromoter

IgGOCT4 Ab

Birc5promoter

ACT8promoter

009

008

007

006

005

004

003

002

001

000

Inpu

t (

)

lowast

lowast

(c)







120573-catenin

Survivin

HSP90

(a)

120573-catenin

120573-actin

Survivin


(b)


120573-actin

Survivin

OCT4

SOX2


00

02

04

06

08

10

12Su

rviv

in re

lativ

e mRN

A le

vel









4 Discussion





Competing Interests




Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


70

60

50

40

30

20

10

000

SOX2 (ng)OCT4 (ng)

0200 0

200200200

Rela

tive l

ucife

rase

activ

ity

(a)009

008

007

006

005

004

003

002

001

000NANOGpromoter

IgGSOX2 Ab

Birc5promoter

ACT8promoter

Inpu

t (

)

lowastlowast

(b)

NANOGpromoter

IgGOCT4 Ab

Birc5promoter

ACT8promoter

009

008

007

006

005

004

003

002

001

000

Inpu

t (

)

lowast

lowast

(c)







120573-catenin

Survivin

HSP90

(a)

120573-catenin

120573-actin

Survivin


(b)


120573-actin

Survivin

OCT4

SOX2


00

02

04

06

08

10

12Su

rviv

in re

lativ

e mRN

A le

vel









4 Discussion





Competing Interests




Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



120573-catenin

Survivin

HSP90

(a)

120573-catenin

120573-actin

Survivin


(b)


120573-actin

Survivin

OCT4

SOX2


00

02

04

06

08

10

12Su

rviv

in re

lativ

e mRN

A le

vel









4 Discussion





Competing Interests




Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



4 Discussion





Competing Interests




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

research article survivin improves reprogramming...

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