Analysis of proteomic profiling of mouse embryonic stem cells derived from fertilized, parthenogenetic and androgenetic blastocysts

doi:10.4236/scd.2011.11001

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Vol.1, No.1, 1-15 (2011)

doi:10.4236/scd.2011.11001

Stem Cell Discovery

Analysis of proteomic profiling of mouse embryonic

stem cells derived from fertilized, parthenogenetic and

androgenetic blastocysts

Xiang-Shun Cui1, Xing-Hui Shen2, Chang-Kwon Lee3, Yo ng-Kook Kang4,

Teruhiko Wakayama5, Nam-Hyung Kim1,*

1Department of Animal Science, Chungbuk National University, Cheongju, Chungbuk, Korea

2Department of Histology and Embryology, Harbin Medical University, Harbin, Heilongjiang Province, China

3Department of Physiology, Konkuk University, Chungju City, Chungbuk, Korea

4Laboratory of Development and Differentiation, Korea Research Institute of Bioscience and Biotechnology, Daejeon, South Korea

5Center for Developmental Biology, RIKEN Kobe, Kobe, Japan; nhkim@chungbuk.ac.kr

Received January 23 2011; revised March 13 2011; accepted March 20 2011.

ABSTRACT

Embryonic stem cells (ESCs) are derived from

the inner cell mass (ICM) of preimplantation

embryos. ESCs exhibit true pluripotency, i.e.,

the ability to differentiate into cells of all three

germ layers in the developing embryo. We used

2-DE MALDI-TOF/TOF to identify differentially

expressed proteins among three types of mouse

embryonic stem cells (ESCs) derived from fer-

tilized, parthenogenetic, and androgenetic (fESC,

pESC and aESC, respectively) blastocysts. We

detected more than 800 proteins on silver-

stained gels of whole protein extracts from each

type of ESC. Of these, 52 differentially expressed

proteins were identified by the MALDI–TOF/TOF

analyzer, including 32 (fESCs vs. pESCs), 28

(fESCs vs. aESCs) and 17 (pESCs vs. aESCs)

prominent proteins with significantly higher or

lower expression relative to the comparison

cells. Among the 32 proteins from fESCs, 12

were significantly increased in expression and

20 were reduced in expression in fESCs com-

pared with pESCs. Similarly, 10 of 28 and 8 of 17

proteins were more highly expressed in fESCs

and pESCs compared with aESCs, respectively.

In contrast, 18 of 28 and 9 of 17 proteins were

reduced in expression in fESCs and pESCs

compared with aESCs, respe ctiv ely . Of the e ight

protein candidates in fESCs, four were in-

creased and four were decreased in expression

relative to both pESCs and aESCs in the 2-DE

analysis. Differential expression of these pro-

teins were confirmed by mRNA expression

analysis using real time RT-PCR. For three pro-

teins, ANXA5, CLIC1 and SRM, Western blot

analysis corroborated the expression patterns

indicated by the 2-DE results. ANXA5 and CLIC1

were increased in expression and SRM was de-

creased in expression in fESCs compared with

both pESCs and aESCs. The differentially ex-

pressed proteins identified in the present study

warrant fu rther inv estigat ion to wards the g oal of

their potential application in ESC therapy.

Keywords: Protein Profiling; Embryonic Stem Cell;

Parthenogenote; Androgenote; Fertilization

1. INTRODUCTION

The field of stem cell biology has attracted increasing

attention in recent years due to the plasticity of stem

cells and their broad potential for use in cell therapy.

Despite this focus, relatively little is known about the

mechanisms underlying the regulation and thereby the

potential for the manipulation of stem cells for clinical

applications. Embryonic stem cells (ESCs) are pluripo-

tent cells derived from the inner cell mass (ICM) of

blastocysts. ES cells can proliferate indefinitely in vitro

and can differentiate into a wide variety of cell types

both in vivo and in vitro [1,2]. Because of their excep-

tional properties, ESCs have enormous potential to be

used for developmental biology studies, drug screening,

tissue engineering and transplantation therapy.

Recently, stem cell research has begun to be applied

in clinical settings. ESCs could be especially helpful

therapeutically in that they could help to overcome im-

mune rejection problems. In particular, parthenogenetic

embryonic stem cells (pESCs) could advance the field of

regenerative medicine by avoiding the immune rejection

X. S. Cui et al. / Stem Cell Discovery 1 (2011) 1-15

problems inherent in transplantation. pESCs are able to

differentiate into cell types from all three germ layers

and are immunologically matched with their oocyte do-

nors [3]. The other uniparental cells, androgenetic ES

Cells (aESCs), can be established from androgenotes,

which cannot develop into viable fetuses. Like pESCs,

aESCs can also be generated with a full complement of

major histocompatibility complex antigens [4]. A previ-

ous report suggested that pESCs were indistinguishable

from ESCs derived from fertilized embryos (fESCs) [5].

However, a detailed understanding of signalling path-

ways and molecular mechanisms involved in biparental

and uniparental pluripotency will be essential before

using them in ESC-based therapies.

Over the past few years, there has been a growing in-

terest in applying proteomics to the study of proteins in

ESCs [6,7]. Although previous studies have generated a

wealth of data, the molecular mechanisms that determine

the characteristics of ESCs remain largely unknown. In

general, little is known about the functional aspects of

ESC-specific proteins. Moreover, in contrast to fESCs,

no detailed comparisons of the proteins expressed in

pESCs and aESCs have been undertaken. In this study,

we used two-dimensional gel electrophoresis (2-DE) and

matrix-assisted laser desorption/ionization time-of-flight

mass spectrometry (MALDI-TOF/TOF MS) to analyze

protein patterns in mouse ESCs. This is the first report to

display the global cellular protein profiles in mono- and

bi-parentally derived ESCs, and in particular to identify

the protein signatures for multipotency in fESCs, pESCs

and aESCs.

2. MATERIALS AND METHODS

2.1. Cell Culture, Differentiation in Vitro and

Sample Preparation

Mouse ES cell lines, derived from fertilized (fESC1

and 2, line 1 and 2, respectively), parthenogenetic

(pESC1 and 2), and androgenetic (aESC1 and 2) blasto-

cysts, were kindly provided by Dr. Wakayama (Center

for Developmental Biology, RIKEN Kobe, Japan). Each

type of blastocysts were generated by fertilization, par-

thenogenetically activated by strontium chloride (SrCl2)

following cytochalasin B (CB, Sigma) treatment 6 h and

two sperm heads were injected into enucleated oocytes

for generation of androgenote embryos. Embryos were

cultured in M16 medium for 4 days. ESCs were estab-

lished based on general methods using inner cell mass

(ICM) of single blastocyst. The fESC1, pESC1 and

aESC1 cell lines were used for two-dimensional gel

electrophoresis (2-DE) analysis, and experiments were

repeated three times using passages 6, 7 and 8, respec-

tively. All ESCs were maintained on non-gelatin coated

dishes in DMEM (high glucose; Invitrogen, Carlsbad,

CA, USA) supplemented with 15% fetal calf serum, 0.1

mM b-mercaptoethanol, 1000 U/mL recombinant mouse

LIF (ESGRO; Chemicon International, Temecula, CA,

USA), 1% glutamine (Sigma, St Louis, MO, USA), 0.5%

penicillin/ streptomycin (Sigma), and 1% non-essential

amino acids (Sigma). fESCs, pESCs and aESCs were

differentiated by treatment with 1 mM retinoic acid (RA,

Sigma) for 6 days (fRA, pRA and aRA, respectively).

Protein samples were prepared essentially as de-

scribed in Lee et al. [8] reports. The cultured cells were

harvested with 2-DE lysis buffer containing 8 M Urea, 2

M thiourea, 100 mM DTT, 4% CHAPS and 1 × com-

plete protease inhibitor cocktail (Roche Applied Science,

Germany). The lysates were incubated for 20 min and

centrifuged at 12 000 × g for 10 min at 10℃. The su-

pernatants were diluted with rehydration buffer contain-

ing 7 M Urea, 2 M thiourea, 100 mM DTT, 2% CHAPS

0.5% ampolyte (Bio-Rad) and 0.01 % bromophenol blue,

and then used as the sample to 2-DE analysis.

2.2. 2-DE, in-Gel Digestion and MAL-

DI-TOF/TOF MS

2-DE was performed as described in Lee et al. [8].

Images of silver-stained gels were digitized with a den-

sitometer (VersaDoc Imaging System 1000TM). The gels

were normalized and statistically analyzed with PDQuest

software (Version 7.1.1, Bio-Rad).

In-gel digestion and identification of the altered pro-

tein spots were performed as reported previously. [8]

Briefly, the protein spots were digested with trypsin and

desalted with ZipTip C18 (Millipore). The peptide sam-

ples were mixed with CHCA matrix solution and then

analyzed by MALDI-TOF/TOF (AB4700, Applied Bio-

systems) in the reflector mode.

Spectra were processed and analyzed with the Global

Protein Server Explorer 3.0 software (Applied Biosys-

tems). The internal MASCOR (Matrix Science, UK)

program was used for matching MS and MS/MS data

against database information. The resulting data were

surveyed against mouse databases downloaded from

both NCBI and the Swiss Prot/TrEMBL homepage.

2.3. Genomic DNA Polymerase Chain

Reaction (PCR)

Whole genomic DNA was extracted by phenol/chlo-

roform method. PCR reaction was carried out in 25 μl

volumes, containing distilled water, 2.5 μl of reaction

buffer, 200 μM of dNTPs, 2.0 units of Taq DNA poly-

merase (Promega, USA), 10 mM of each primer for Zinc

finger protein 1, Y linked (Zfy1), Zinc finger protein

X-linked (Zfx) and Ras association domain family

member 1 (Rassf1) genes (Table 1), and about 100 ng of

X. S. Cui et al. / Stem Cell Discovery 1 (2011) 1-15

total DNA. Amplification was performed following con-

ditions: an initial 2-min denaturation at 94˚C was fol-

lowed by 30 cycles of 30 s at 94˚C, 45 s at 55˚C - 60˚C,

and 60 s at 72˚C. The PCR products for 3 genes were

separated on 2.0% agarose gels containing ethidium

bromide.

2.4. Reverse Transcriptase-Polymerase

Chain Reaction (RT-PCR) and Real

Time RT-PCR

Messenger RNA was extracted with the Dynabeads

mRNA Direct Kit (Dynal Asa, Oslo, Norway), standard

cDNA synthesis was achieved by reverse transcription

of RNA using the oligo(dT) 12 - 18 primer and super-

script reverse transcriptase (Invitrogen Co., Grand Island,

NY).

For determination of pluripotency and differentiation,

mRNA expression in the ESCs and differentiated cells

were analyzed by RT-PCR using cDNA as the template,

and following primers: POU domain, class 5, transcrip-

tion factor 1 (Pou5f1, Oct4), Nanog homeobox (Nanog),

Paired box gene 6 (Pax6), Nestin (Nes) and glyceralde-

hyde-3-phosphate dehydrogenase (Gapdh, Table 1).

Table 1. Primers used in PCR.

Name Acc. No. Sequence Length (bp)PCR

Zfy1 AK076618 F : gtaggaa gaat ctttctcatgctgg

R: tttttgagtgctgatgggtgacgg 217 Genomic DNA

Zfx AL626786 F : atggtacatgtctataatcttagcatt

R: ctaccagggattaaactggttaacat 210 Genomic DNA

Rassf1 NC_000075 F : tgaaacaccttccttcgaaatg

R: cacccttttcaagcttcagagtt 420 Genomic DNA

Oct4 NM_013633 F: tgtggacctcaggttggact

R: cttctgcagggctttcatgt 221 RT

Nanog NM_028016 F: aacgatatggtggctact ct c

R: tcggttcatcatggtacagt 264 RT

Pax6 NM_013627 F: ctgcagacccatgcagatgcaaa

R: aagtcgcatctgagcttcatccg 519 RT

Nes NM_016701 F: aagctgaagctgcatttccttg

R: gtgctaagctgttttctactttt 550 RT

Gapdh NM_008084 F: aaaccrgccaagtat gat ga

R: gtggtccagggtttcttact 273 RT/Real time RT

Anxa5 NM_009673 F: gaagccctcacgactctacg

R: tatcccccaccacatcatct 179 Real time RT

Erp29 NM_026129 F: c ct gaa ga tc atggggaaga

R: ctcctccttctcagcctcct 172 Real time RT

Clic1 NM_033444 F: ctctggctcaagggagtcac

R: atatgtccagtcccgaggtg 232 Real time RT

Po protein X15267 F: tgccacactccatcatcaat

R: cgaagagaccgaatcccata 240 Real time RT

Srm NM_009272 F: gtcctacgggaagtggtgaa

R: gtcctacgggaagtggtgaa 199 Real time RT

Strap NM_011499 F: tcagtcctgatggggaactc

R: tctggaaatccgatctttgg 154 Real time RT

Sod1 AH002084 F: ccagtgcaggacctcatttt

R: ttgtttctcatggaccacca 197 Real time RT

Esrra NM_007953 F: ccaggcttctcctca c t g t c

R: gccccctcttcatctaggac 152 Real time RT

Bcl-xL L35049 F: ttcgggatggagtaaactgg

R:tggatccaaggctctaggtg 157 Real time RT

Birc5 NM_009689 F: gaatcctgcgtttgagtggt

R: aaaacactgggccaaatcag 221 Real time RT

Casp3 BC038825 F: gggcc tgttg aact gaaaaa

R: ccgtcctttgaatttctcca 242 Real time RT

Bax NM_007527 F: tgcagaggatgattgctgac

R: gatcagctcgggcactttag 183 Real time RT

F, forward; R, reverse.

X. S. Cui et al. / Stem Cell Discovery 1 (2011) 1-15

Gapdh amplification was used as a loading control for

the sample. PCR conditions were as follows：95˚C for 30

s, 55˚C - 60˚C for 30 s and 72˚C for 1 min. The PCR

products for 5 genes were separated on 2.0% agarose

gels containing ethidium bromide.

Cell lines from fESC1/2, pESC1/2, and aESC1/2 were

used for real time RT-PCR. Real-time RT-PCR was

performed using the 13 primer sets shown in Table 1

using the DNA Engine OPTICOJ 3 (MJ research, USA).

Relative gene expression was quantified using the

2-ddCt method. Gapdh mRNA, a house keeping gene,

was employed as a control.

2.5. Western Blot Analysis

Western blot analysis was performed as described

previously [9]. Briefly, ESC lysates were separated by

electrophoresis in a CriterionTM Precast Gel (Bio-Rad,

Hercules, CA), followed by transfer to a PVDF mem-

brane (iBlot TM Gel Transfer Stacks, Cat. No.

IB4010-02; Invitrogen). After blocking, the membrane

was incubated with primary anti-ANXA5 (GenWay

Biotech, Inc., CA, USA), anti-CLIC1 (Aviva Systems

Biology, CA, USA), anti-SRM (GeneTex, CA, USA),

and anti-GAPDH (Cell Signaling Technology, Danvers,

MA, USA). Then, the membrane was incubated with

HRP-linked anti-rabbit IgG (Cell Signaling Technology).

Finally, the antibody-binding bands on the membrane

were visualized using Chemiluminescence Luminol Re-

agent (Invitrogen).

2.6. Statistical Analysis

The general linear model (GLM) procedure in the

SAS program [10] was applied to analyze data from all

experiments. Significant differences were determined

using Tukey’s multiple range test [11] and P < 0.05 was

considered statistically significant.

3. RESULTS

3.1. Sex Diagnosis and Characterization of

ESC and Differentiated Cell Lines

ESC lines which were established from Dr. Waka-

yama were well characterized for their pluripotency and

differentiation potential including karyotypping [12,13]

and chimera formation [14]. To determine accuracy of

genotype diagnosis of ESCs, PCR amplification of

Zfy1/Zfx were employed. Genes common to X and Y

chromosomes (ZFY/ZFX, zinc finger protein) can am-

plify by a single primer pair. As shown in Figure 1 (a),

both fESCs and aESCs contain both X and Y chromo-

somes, whereas pESCs only shows X chromosome, the

same to the adult somatic female cells (fm) that were

used as a positive control. Rassf1 was used as a genomic

DNA positive control, expressed all samples except for

negative control, no template group.

(a)

(b)

(c)

(d)

Figure 1. (a), Representative PCR sexing reactions. PCR

products were gene rated from embry onic stem cells (ESCs)

or adult somatic cells (ASCs). Characterization of pluripo-

tency (b) neuroprogenitors (c) and differentiated state 6

days after retinoic acid treatment (d) by RT-PCR. fES:

fertilized embryonic stem cells; aES: androgenetic embry-

onic stem cells; pES: parthenogene tic embryonic stem cells;

fm: adult female cells; m: adult male cells; -: no template.

fRA, aRA and pRA: fESC, aESC and pESC were differen-

tiated by retinoic acid, respectively. Adult somatic cells

(ASCs) from fm (female) and m (male) were used as posi-

tive control in (A); Rassf1 was used as genomic DNA posi-

tive control and no DNA template (-) was used as a negative

control in (a); NIH3T3 cell was used as a negative control

in (b); Gapdh was used as a positive control in (b), (c) and

(d).

X. S. Cui et al. / Stem Cell Discovery 1 (2011) 1-15

Embryonic stem cells and ESC-derived neuroecto-

dermal spheres (NESs) were shown to have characteris-

tics typical of pluripotency and neuroprogenitors. RT-

PCR results showed that ESC marker genes Oct4 and

Nanog were expressed in ESC lines but did not express

in control (NIH3T3) cell line (Figure 1 (b)). An the oth-

er hand, neural stem cell marker genes such as Nes and

Pax6 were expressed at markedly increased levels in the

NESs compared with the ESCs (Figure 1 (c)). In con-

trast, Oct4 and Nanog were not expressed in fRA, pRA

and aRA cells which were treated with retinoic acid for 6

days, and all of them were shown with differentiated

state (Figure 1 (d)).

3.2. Comparative Proteomics in fESCs,

pESCs and aESCs

To identify differentially expressed proteins between

fESCs vs. pESCs, fESCs vs. aESCs, and pESCs vs.

aESCs, proteins were separated using the 2-DE tech-

nique, and experiments were repeated three times for

each ESC type, using different passages. Separated pro-

teins were visualized with silver staining and analyzed

with PDQuest software. Figures 2(a), 3(a) and 4(a )

show typical 2-DE gels of total ESC proteins for the

three groups. In total, more than 800 proteins may be

seen in each gel, with isoelectric pH values of pH 3–10

and molecular weights (MW) of 14 - 180 kDa. The

MALDI–TOF/TOF analyzer identified 52 differentially

expressed proteins among the three types of ESCs, with

gels placed side-by-side for ease of comparison between

groups (Figure 2(b), 3(b) and 4(b)). We identified 32

(fESCs vs. pESCs, Table 2), 28 (fESCs vs. aESCs, Ta-

ble 3), and 17 (pESCs vs. aESCs, Table 4) prominent

proteins in each comparison. Tables 2-4 list these pro-

teins, highlight representative peptide sequences and

sequence coverage, note the theoretical and experimental

isoelectric point (pI) and MW values, and provide acces-

sion numbers from both the Swiss-Prot and NCBI data-

bases.

(a)

(b)

Figure 2. Comparative analysis of protein expression patterns between fESCs and pESC s. (a) Silver-stained 2-DE gel of fESC

and pESC proteins. (b) Enlargement of 2-DE gel images of upregulated or downregulated proteins in fESCs and pESCs. The

statistical data were obtained from 2-DE gels in three independent experiments (P < 0.05). Arrows indicate altered proteins in

fESCs and pESCs. White bars, fESCs; blac k bar s, pESCs.

X. S. Cui et al. / Stem Cell Discovery 1 (2011) 1-15

Table 2. Identification of differentially expressed proteins between fESCs and pESCs.

Protein pI/MW (kDa)

Spot

No.

Fold

Change

(pES/fES)

Protein Name Peptide Sequence SC

(%) Theoretical Experimental

Accession

No.

/Database

High-expression protein spots (relative to fESCs), (12)

1053 –51.53 Unnamed protein product DSNNLCLHFNPR 115.19/11.6 5.20/11.8 52890/NC

P48538/SP

1233 –22.05 Annexin A5 GTVTDFPGFDGR VLTEIIASR

GAGTDDHTLIR 104.83/35.7 4.80/35.4 6753060/NC

P48036/SP

2143 –4.61 Chloride intracellular channel 1 YLSNAYAR EEFASTCPDDEEIE-

LAYEQVAR 125.09/26.9 5.30/30.1

15617203/NC

Q9Z1Q5/SP

3102 –1.63 Prohibitin ILFRPVASQLPR 45.57 /29.8 5.70/29.5 6679299/NC

3134 –6.73

Endoplasmic reticulum protein

ERp29 precursor FDTQYPYGEK ILDQGEDFPASEMAR95.90/28.8 6.10.27.3 19526463/NC

P57759/SP

3233 –17.31 Annexin A3 LTFDEYR NTPAFLAER GAGT-

DEFTLNR 85.33/36.3 5.70/38.8 7304887/NC

O35639/SP

4311 –3.49 Oat protein KTEQGPPSSEYIFER

VLPMNTGVEAGETACK 76.19/48.3 6.20/48.0

14198116/NC

P29758/SP

5231 –2.97

Acidic ribosomal

phosphoprotein P0

GHLENNPALEK AGAIAP-

CEVTVPAQNTGLGPEK 105.91/34.1 6.40/37.5 6671569 /NC

P14869/SP

6124 –1.80 B-cell stimulating factor 3 LGAETLPR 38.70/25.2 7.50/29.0 9910314/NC

Q9QZM3/SP

6229 –3.76 Steroid hormone receptor ERR1 LVLSSLPK 16.84/49.2 7.30//40.5 O08580/SP

7024 –4.64

Trafficking protein particle

complex 5 VLDALVAR 49.69/20.7 8.20/19.3 29165850/NC

7314 –1.77

Branched-chain amino acid

aminotransferase

AGWGPPR LGGNYGPTVAVQR

TWGEFR 77.60/39.7 8.00/41.7 3298579/NC

O35855/SP

Low-expression protein spots (relative to fESCs), (20)

1133 +1.80

Ran-specific GTPase-activating

protein FASENDLPEWK 55.15/23.5 5.30/27.3 P34022/SP

1325 +2.11

Serine/threonine kinase receptor

associated protein FSPDGELYASGSEDGTLR 54.99/38.4 5.20/42.6

6755682/NC

Q9Z1Z2/SP

1332 +1.64 Galactokinase 1 GYALLIDCR LAVLITNSNVR

HSLGSSEYPVR 75.17/42.2 5.20/43.8

16741595/NC

Q9R0N0/SP

2103 +2.48 Ubiquitin thiolesterase PGP9.5 LGVAGQWR NEAIQAAHDSVA-

QEGQCR 115.12/24.7 5.30/25.2 92934/NC

Q9R0P9/SP

2213 +1.80 Spermidine synthase YQDILVFR VLIIGGGDGGVLR

AAFVLPEFTR 105.31/33.9 5.40/35.7 6678131/NC

Q9R0N0/SP

2342 +1.77

SGT1, suppressor of G2 allele

of SKP1

ALEQNPDDAQYYCQR

SLELNPNNCTALLR 85.32/38.1 5.40/43.0

23956176/NC

Q9CX34/SP

3009 +14.57 Stathmin 1 ASGQAFELILSPR 85.76/17.2 5.90/17.5 9789995/NC

P54227/SP

X. S. Cui et al. / Stem Cell Discovery 1 (2011) 1-15

3334 +3.43 Steroid hormone receptor ERR1 LVLSSLPK 16.84/49.2 6.00/41.8 131224731/NC

4009 +2.16 DJ-1 protein VTVAGLAGKDPVQCSR

MMNGSHYSYSESR 156.32/19.9 6.20/22.5

16924002/NC

Q99LX0/SP

4011 +3.36 Cu/Zn-superoxide dismutase KHGGPADEER VISLSGEHSIIGR 146.23/15.9 6.20/16.0 201006 /NC

P08228/SP

4128 +1.73 Heat shock protein 27 LFDQAFGVPR AV-

TQSAEITIPVTFEAR 136.12/22.8 6.30/24.6 204665/NC

P14602/SP

4222 +8.40 Estrogen-related receptor alpha LV LVLSSLPK 15.77/45.0 6.30/33.2

6679693/NC

O08580/SP

5106 +2.89

Acyl-protein thioesterase 1 (Lyso-

phospholipase I) ASFSQGPINSANR 56.14/24.6 6.40/23.3 P97823/SP

5212 +3.50 Uridine phosphorylase 1 FVCVGGSSSR EYPNICAGTDR

MLYHAR CSNITIIR 116.12/34.0 6.50/33.3 6678515/NC

P52624/SP

6011 +1.60

Similar to basic transcription factor

3 VQASLAANTFTITGHAETK 347.85/19.4 7.60/18.5 51762066/NC

6106 +3.87 GTP-binding nuclear protein Ran FNVWDTAGQEK NVPNWHR VCE-

NIPIVLCGNK SIVFHR 177.01/24.4 7.00/24.5 P62827/SP

6128 +1.82

Glutathione S-transferase,

alpha 4

EKEESYDLILSR FLQPGSQR

KPPPDGPYVEVVR 146.77/25.5 7.10/23.3 6754082/NC

P24472/SP

6223 +2.67

L-lactate dehydrogenase

chain M

VIGSGCNLDSAR SLNPELGTDAD-

KEQWK SADTLWGIQK 117.62/36.4 7.50/37.6 65923/NC

P06151/SP

8011 +3.16

Proteasome (prosome, macropain)

subunit, beta type 5

RGPGLYYVDSEGNR GPGLY-

YVDSEGNR RAIYQATYR AIYQATYR

DAYSGGAVNLYHVR

146.52/28.5 8.50/21.3 6755204/NC

O55234/SP

8021 +413.80 Hypothetical protein VGPMLSPR 49.69/21.0 8.50/19.6

51767674/NC

Q7TQH0/SP

pES/fES: pESCs/fESCs.

Table 3. Identification of differentially expressed proteins between fESCs and aESC.

Protein pI/MW (kDa)

Spot

No.

Fold

Change

(aES/fES)

Protein Name Peptide Sequence SC

(%) Theoretical Experimental

Accession No.

/Database

High-expression protein spots (relative to fESCs), (10)

1053 –37.68 Unnamed protein DSNNLCLHFNPR 115.19/11.6 5.20/11.8 52890/NC

P48538/SP

1233 –3.23 Annexin A5 GTVTDFPGFDGR VLTEIIASR

GAGTDDHTLIR 104.83/35.7 4.80/35.4 6753060/NC

P48036/SP

2143 –3.51

Chloride intracellular

channel 1

YLSNAYAR

EEFASTCPDDEEIELAYEQVAR 125.09/26.9 5.30/30.1

15617203/NC

Q9Z1Q5/SP

3134 –13.01

Endoplasmic reticulum

protein ERp29 precursor

FDTQYPYGEK

ILDQGEDFPASEMAR 9 5.90/28.8 6.10.27.3 19526463/NC

P57759/SP

3233 –5.48 Annexin A3 LTFDEYR NTPAFLAER

GAGTDEFTLNR 8 5.33/36.3 5.70/38.8 7304887/NC

O35639/SP

3307 –1.61 Calponin 3, acidic RFDEGK CASQAGMTAYGTR GAS-

QAGMLAPGTR GMSVYGLGR 125.46/36.4 5.80/41.6

55391513/NC

Q9DAW9/SP

X. S. Cui et al. / Stem Cell Discovery 1 (2011) 1-15

3312 –1.50 Unnamed protein APIQWEER CSDFTEEICR 4 6.27/39.6 5.90/41.4 26339056/NC

4311 –2.16 Oat protein KTEQGPPSSEYIFER

VLPMNTGVEAGETACK 7 6.19/48.3 6.20/48.0 14198116/NC

P29758/SP

5231 –5.55

Acidic ribosomal

phosphoprotein P0

GHLENNPALEK AGAIAP-

CEVTVPAQNTGLGPEK 105.91/34.1 6.40/37.5 6671569/NC

P14869/SP

5309 –1.94

Heterogeneous nuclear

ribonucleoprotein A/B EVYQQQQYGSGGR 4 7.68/30.8 6.50/43.6 Q99020/SP

Low-expression protein spots (relative to fESCs)， (18)

1109 +2.97 14-3-3 protein gamma LAEQAER NVTELNEPLSNEER

MKGDYYR 114.80/28.3 4.80/28.1 3065929/NC

P68510/SP

1325 +1.89

Serine/threonine kinase

receptor-associated protein FSPDGELYASGSEDGTLR 5 4.99/38.4 5.20/42.6

6755682/NC

Q9Z1Z2/SP

1331 +2.05

B-cell stimulating factor-3

precursor (BSF-3) (Novel

neurotrophin-1) (NNT-1)

LGAETLPR 3 8.70/25.2 5.20/42.0 Q9QZM3/SP

2213 +1.76 Spermidine synthase YQDILVFR VLIIGGGDGGVLR

AAFVLPEFTR 105.31/33.9 5.40/35.7 6678131/NC

Q9R0N0/SP

2221 +1.83

Similar to hypothetical

protein MGC36907 NKYEDEINKR 3 5.42/36.2 5.50/36.3 34868312/NC

2317 +2.17

Ubiquitin-like 1 activating

enzyme E1A VDQICHR 2 5.24/38.5 5.40/43.7 Q9R1T2/SP

4009 +2.15 DJ-1 protein VTVAGLAGKDPVQCSR

MMNGSHYSYSESR 156.32/19.9 6.20/22.5

16924002/NC

Q99LX0/SP

4011 +2.47

Cu/Zn-superoxide

dismutase KHGGPADEER VISLSGEHSIIGR 146.23/15.9 6.20/16.0 201006/NC

P08228/SP

4222 +5.09

Estrogen-related

receptor alpha LV LVLSSLPK 1 5.77/45.0 6.30/33.2 6679693/NC

O08580/SP

4305 +1.76 eIF3-p44 CPYKDTLGPMQK

CPYKDTLGPMQK 3 6.08/35.3 6.10/47.8 4097873/NC

Q9Z1D1/SP

5212 +3.03

Uridine

phosphorylase 1

FVCVGGSSSR EYPNICAGTDR

MLYHAR CSNITIIR 116.12/34.0 6.50/33.3 6678515/NC

P52624/SP

6011 +1.56

Similar to basic

transcription factor 3 VQASLAANTFTITGHAETK 347.85/19.4 7.60/18.5 51762066/NC

6106 +1.95

GTP-binding nuclear

protein Ran

FNVWDTAGQEK NVPNWHR VCE-

NIPIVLCGNK SIVFHR 177.01/24.4 7.00/24.5 P62827/SP

6128 +1.88

Glutathione S-transferase,

alpha 4

EKEESYDLILSR FLQPGSQR

KPPPDGPYVEVVR 146.77/25.5 7.10/23.3 6754082/NC

P24472/SP

7011 +3.06

Thymidylate kinase;

TMK YAFSGVAFTGAK GEFGLER 8 9.16/25.5 8.00/22.8 1836042/NC

P97930/SP

7023 +1.63

Component C5 of

proteasome LSEGFSIHTR DVFISAAER 8 8.29/24.6 8.20/23.1 1165123/NC

O09061/SP

8011 +2.74

Proteasome (prosome,

macropain) subunit,

beta type 5

RGPGLYYVDSEGNR GPGLY-

YVDSEGNR RAIYQATYR AIY-

QATYR DAYSGGAVNLYHVR

146.52/28.5 8.50/21.3 6755204/NC

O55234/SP

8021 +562.88 Hypothetical protein VGPMLSPR 4 9.69/21.0 8.50/19.6

51767674/NC

Q7TQH0/SP

aES/fES: aESCs/fESCs.

X. S. Cui et al. / Stem Cell Discovery 1 (2011) 1-15

Table 4. Summary of differentially expressed proteins betwee n pESCs and aE SCs.

Protein pI/MW (kDa)

Spot

No.

Fold

Change

(aES/pES)

Protein Name Peptide Sequence SC

(%) Theoretical Experimental

Accession No.

/Database

High-expression protein spots (relative to pESCs), (8)

3009 –2.13 Stathmin 1 ASGQAFELILSPR 8 5.76/17.2 5.90/17.5 9789995/NC

P54227/SP

3112 –2.00 Heat shock protein beta 1 LFDQAFGVPR

AVTQSAEITIPVTFEAR 126.45/22.8 5.90/25.2 7305173/NC

P14602/SP

3334 –2.27 Steroid hormone receptor

ERR1 LVLSSLPK 1 6.84/49.2 6.00/41.8 131224731/NC

4222 –1.65 Estrogen related

receptor alpha LVLSSLPK 1 5.77/45.0 6.30/33.2 6679693/NC

O08580/SP

5231 –2.38 Acidic ribosomal

phosphoprotein P0

GHLENNPALEK

AGAIAPCEVTVPAQNTGLGPEK 105.91/34.1 6.40/37.5 6671569/NC

P14869/SP

6106 –1.98 GTP-binding nuclear

protein Ran

FNVWDTAGQEK NVPNWHR

VCENIPIVLCGNK SIVFHR 177.01/24.4 7.00/24.5 P62827/SP

6223 –1.90 L-lactate dehydrogenase

chain M

VIGSGCNLDSAR

SLNPELGTDADKEQWK SADTLWGIQK 117.62/36.4 7.50/37.6 65923/NC

P06151/SP

8021 –1.90 Hypothetical protein VGPMLSPR 4 9.69/21.0 8.50/19.6 51767674/NC

Q7TQH0/SP

Low-expression protein spots (relative to pESCs), (9)

1233 +4.88 Annexin A5 GTVTDFPGFDGR VLTEIIASR

GAGTDDHTLIR 104.83/35.7 4.80/35.4 6753060/NC

P48036/SP

4311 +1.62 Oat protein KTEQGPPSSEYIFER

VLPMNTGVEAGETACK 7 6.19/48.3 6.20/48.0 14198116/NC

P29758/SP

6124 +2.00

B-cell stimulating

factor 3 LGAETLPR 3 8.70/25.2 7.50/29.0 9910314/NC

Q9QZM3/SP

6126 +1.52 Unnamed protein FNVWDTAGQEKNVPNWHR

VCENIPIVLCGNK SIVFHR 177.01/24.3 7.60/23.7 12846283/NC

P62827/SP

7011 +1.59

Thymidylate kinase;

TMK YAFSGVAFTGAK GEFGLER 8 9.16/25.5 8.00/22.8 1836042/NC

P97930/SP

7024 +1.87

Trafficking protein

particle complex 5 VLDALVAR 4 9.69/20.7 8.20/19.3 29165850/NC

7314 +1.59

Branched-chain amino

acid aminotransferase

AGWGPPR LGGNYGPTVAVQR

TWGEFR 7 7.60/39.7 8.00/41.7 3298579/NC

O35855/SP

8107 +1.63 Unnamed protein FDPENPQTLR 5 9.36/18.2 8.50/23.6 12845642/NC

8212 +1.65

Heterogeneous nuclear

ribonucleoprotein A2/B1

EKEQFRK NYYEQWGK

GGNFGFGDSR GGNFGFGDSR

NMGGPYGGGNYGPGGSGGSGGYGGR

198.67/35.9 8.70/35.6 3329498/NC

O88569/SP

aES/pES: aESCs/pESCs.

X. S. Cui et al. / Stem Cell Discovery 1 (2011) 1-15

3.3. Identification of Proteins in fESCs,

pESCs and aESCs

Table 2 lists the majority of the protein spots dis-

playing different protein expression in fESCs and pESCs.

There are 32 spots with significant differences in

post-intensities between fESCs and pESCs (Figure 2

(b)). Among these 32, 12 were expressed at significantly

higher levels and 20 were expressed at significantly

lower levels in fESCs than in pESCs. The identities of

the 32 proteins were determined by comparing the re-

corded masses of fingerprint peptides with the theoreti-

cal peptide masses derived from known mouse peptides

in the protein database.

Similarly, Table 3 and 4 show proteins that were dif-

ferentially displayed between fESCs vs. aESCs and

pESCs vs. aESCs, respectively. Individual protein spots

for these groups are shown in Figure 3(b) and Figure

4(b). Ten out of the 28 proteins differentially expressed

in fESCs vs. aESCs were decreased in fESCs as com-

pared with pESCs, and 8 out of the 17 proteins differen-

tially expressed in pESCs vs. aESCs were decreased in

pESCs as compared with aESCs. In contrast, 18 and 9

proteins were increased in expression for fESCs vs.

aESCs and pESCs vs. aESCs, respectively.

(a)

(b)

Figure 3. Comparative analysis of protein expression patterns betwee n fESCs and aESCs. (a) Silver-stained 2-DE gel of fESC

and aESC proteins. (b) Enlargement of 2-DE gel images of upregulated or downregulated proteins in fESCs and aESCs. The

statistical data were obtained from 2-DE gels in three independent experiments (P < 0.05). Arrows indicate altered proteins in

fESCs and aESCs. White bars, fESCs; black bars, aESCs.

Openly accessible at

X. S. Cui et al. / Stem Cell Discovery 1 (2011) 1-15

1111

(a)

(b)

Figure 4. Comparative analysis of protein expression patterns between pESCs and aESCs. (a) Silver-stained 2-DE gel of

pESC and aESC proteins. (b) Enlargement of 2-DE gel images of upregulated or downregulated proteins in pESCs and

aESCs. The statistical data were obtained from 2-DE gels in three independent experiments (P < 0.05). Arrows indicate al-

tered proteins in pESCs and aESCs. White bars, pESCs; black bars, aESCs.

X. S. Cui et al. / Stem Cell Discovery 1 (2011) 1-15

3.4. mRNA and Protein Expression

To investigate the changes in gene expression at the

mRNA level, some proteins identified by MS were veri-

fied by real time RT-PCR to detect mRNA transcription

(Figure 5 (a)-(c)). First, transcription levels were deter-

mined for eight protein candidates displaying differential

expression in 2-DE. Four of these exhibited increased

expression (Figure 5 (a)) and the other four exhibited

reduced expression (Figure 5 (b)) in fESCs compared

with both pESCs and aESCs. Additionally, apoptosis

related four genes that were not identified by MS were

also analyzed: Bcl-xL, Baculoviral IAP repeat-containing

5 (Birc5, Survivin ), Caspase 3 (Casp3) and BCL2-asso-

ciated X protein (Bax, Figure 5 (c)). As seen in Figure 5,

analyses of Annexin A5 (Anxa5), Chloride intracellular

channel 1 (Clic1) and Spermidine synthase (Srm) mRNA

(Figure 5 (a) & (b)) showed the same expression pat-

terns as the 2-DE results, i.e., the mRNA expression of

Anxa5 and Clic1 was higher in fESCs than in pESCs and

aESCs, and, SRM was low expressed in the fESCs

compared with pESCS and aESCs. In addition, we con-

firmed ANXA5, CLIC1 and SRM protein expression by

Western blot analysis, and these expression patterns

were in agreement with the mRNA analyses (Figure 5

(b)). Endoplasmic reticulum protein ERp29 precursor

(Erp29) and Cu/Zn-superoxide dismutase (Sod1) were

increased and reduced in expression, respectively, in

fESCs compared with pESCs, though no differences

were observed between fESCs and aESCs. For three

other candidates, Acidic ribosomal phosphoprotein P0

(Po protein), Serine/threonine kinase receptor associated

protein (Strap) and Estrogen-related receptor alpha (Es-

rra), no differences were observed among the three

types of ESCs (Figure 5 (a) & (b)). For the apoptosis

related genes, the anti-apoptotic gene Bcl-xL was highly

expressed in the fESCs compared with the pESCs and

aESCs; in contrast, the pro-apoptotic gene Bax was

higher in the pESCs and aESCs than in the fESCs (Fig-

ure 5 (c)).

(a)

(b)

(c)

4. DISCUSSION (d)

Figure 5. Relative mRNA expression levels of differentially

expressed protein candidates with high (A) or low (B) ex-

pression levels in fESCs compared with both pESCs and

aESCs in 2-DE analysis. Apoptosis-related genes (C) were

analyzed by real time RT-PCR. Gapdh mRNA expression

was used as an internal standard and its mRNA level in

fESCs was designated as one-fold (baseline). Black bars,

fESCs; white bars, pESCs; grey bars, aESCs. Statistically

significant differences are indicated: *, P < 0.05; **, P <

0.01. Values are means ±SEM for two independent cell

lines. (D), Protein expre ssion in fESCs (fES), pESCs (pES),

and aESCs (aES). GAPDH protein expression was used as

the control.

Embryonic stem cells derived from fertilized, andro-

genetic and parthenogenetic blastocyst (fESCs, aESCs

and pESCS, respectively) were established and well-

characterized by Dr. Wakayama. All ESC lines estab-

lished in Dr. Wakayama’s laboratory were shown posi-

tive for ESC-specific markers, and negative for differen-

tiation markers [14-16]. We further performed for pluri-

potency/differentiation and sex analysis using RT- PCR

or genomic DNA PCR. fESCs, aESCs and pESCs which

were used to microarray were shown positive for

ESC-specific marker genes such as Oct4 and Nanog, and

Openly accessible at

X. S. Cui et al. / Stem Cell Discovery 1 (2011) 1-15

1313

negative for differentiated cell markers, Pax6 and Nes.

Sex diagnosis results showed that both fESCs and aESCs

contained both X and Y chromosomes.

Several transcription factors are essential for ES cell

pluripotency. Octamer-binding protein 3 or 4 (Oct3/4,

Oct4), a POU family member, is one such factor. Oct4 is

downregulated in response to differentiation, and its

upregulation induces differentiation. Therefore, a critical

amount of Oct4 is required to sustain stem-cell

self-renewal [17]. Also, Nanog, a homeodomain protein,

was found to be capable of maintaining ES cell

self-renewal, independent of the LIF/STAT3 pathway

[18,19]. In the present study, Oct4 and Nanog were ex-

pressed in the fESCs, pESCs, and aESCs. The data par-

tially suggested that pluripotency of pESCs and aESCs

did not differ from that of fESCs. This supports evidence

from a previous study [5], in which chimeras produced

from pESCs generated by diploid blastocysts developed

well postnatally, with no growth retardation, and the age

of the chimeras did not affect the proportions of tissues

contributed by pESCs. Furthermore, pESCs have the

capacity to differentiate into all tissue types in the body;

Surprisingly, even in organisms as complex as mice,

pESCs can support full-term development, resulting in a

pESC-derived newborn [5]. aESCs also exhibit a sur-

prising ability to differentiate. Dinger et al. [20] ob-

served widespread contributions from aESCs in fetal

chimeric mice and reported that their neural differentia-

tion potential, in terms of self-renewal properties of

neural stem cells, did not differ from that of normal bi-

parental fESCs [20]. In addition, aESCs are able to dif-

ferentiate into various cell types of all three embryonic

germ layers [21]. Together, homozygous ESCs, or at

least pESCs, are indistinguishable from fESCs with re-

spect to tissue/organ contribution. Reliable derivation of

pluripotent pESCs or aESCs is a critical step towards the

feasibility of female or male patient-specific ES cell

therapy in regenerative medicine.

One of the proteins highly expressed in fESCs was

identified as ANNEXIN A5 (ANXA5, spot no. 1233), a

35 kD plasma protein. The membrane-binding capacity

of ANXA5 has multiple functions, including the modu-

lation of signalling events, a function as a Ca-channel,

involvement in calcification processes, and a function as

a receptor for viruses [22]. ANXA5 can also interfere

with calcium and phospholipid signalling pathways [23].

Of note, ANXA5 mRNA and protein have been shown

to be expressed in the zebrafish oocyte [24]. It is possi-

ble that the expression level of Anex5 is higher in fESCs

than in pESCs and aESCs, and that this may be related to

fertilization and calcium oscillation upon sperm penetra-

tion into the oocyte. As seen in a previous report [25],

after sperm capacitation, Anex5 binding sites were found

mainly in the post-acrosomal region of the sperm head

plasma membrane. After induction of the acrosome re-

action, the Anex5 binding sites were found almost only

in the acrosomal region and with higher numbers of

binding sites in the equatorial area.

Spot no. 2143 was also highly expressed in fESCs

compared with pESCs and aESCs. This protein spot was

identified as CLIC1. CLIC1, also known as NCC27, is a

member of the Clic family of chloride channels, which

can function as chloride channels in vitro [26,27]. These

proteins have significant structural homology with glu-

tathione-S-transferase [28]. In somatic cells, the expres-

sion of CLIC1 is localized mainly in the nuclear and

vesiculo-cytoplasmic membranes. Furthermore, vesiculo-

cytoplasmic CLIC1 colocalizes with mitochondria, and

CLIC1 may play a role in the regulation of osteoblastic

differentiation from mesenchymal progenitors [29].

CLIC1 protein is expressed in Xenopus oocytes in com-

bination with the cystic fibrosis transmembrane conduc-

tance regulator (CFTR) [30]. To date, no study has re-

ported any function associated with CLIC1 in ESCs or

embryos. However, one study did report that CLIC1

might play important roles in gallbladder carcinoma me-

tastasis, including in cell motility and invasion [31]. It

would be considering that CLIC1 may be associated

with lower efficiency in derivation of pESC line than

fESC line [5]. During ICM outgrowth, MAPK signalling

is noticeably reduced in parthenogenetic blastocysts

compared with fertilized blastocysts. Though no similar

study has been done for aESCs, as uniparental ESCs,

aESCs may undergo intracellular processes more similar

to those of pESCs than to those of fESCs. CLIC1, highly

expressed in fESCs compared with pESCs and aESCs, is

required further observation of its function during pre-

implantation embryogenesis and ESC generation in both

uniparental and biparental chromosomes.

In the present study, the expression level of sper-

midine synthase (SRM，spot no. 2213) was higher in

pESCs and aESCs than in fESCs. Spermidine synthase,

an aminopropyltransferase, catalyzes the biosynthesis of

polyamine spermidine from putrescine. Spermidine syn-

thase and spermine synthase are important contributors

to ion channel regulation, transcription, translation, and

enzyme activities. Previously, we identified several

genes that were differentially expressed in blasto-

cyst-stage porcine parthenotes. One of these, SRM, was

highly expressed only in the blastocysts of porcine par-

thenotes [31]. Another previous study showed upregula-

tion of SRM mRNA following the addition of exogenous

polyamines [32]. Furthermore, this upregulation was

influenced by apoptosis and apoptotic-related gene ex-

pression. Higher expression of SRM in pESCs and

aESCs could be related to the relatively low overall

X. S. Cui et al. / Stem Cell Discovery 1 (2011) 1-15

quality and high levels of apoptosis in pESCs and aESCs

compared with fESCs. In the present study, significantly

higher expression level of Casp3 and lower expression

level of Bcl-xL in pESCs and aESCs vs. fESCs provided

strong evidence in support of this idea, although no dif-

ferentially expressed protein spot was identified by MS.

Previous reports have indicated that polyamines were

essential for normal cell growth [33] and required for

apoptosis with Caspase activation [34]. Spermidine

synthase gene is also essential for survival of Arabidop-

sis [35]. In plant cells, spermidine acts as a signalling

regulator in stress signalling pathways, leading to a

build-up of stress tolerance mechanisms under stress

conditions [36]. Apoptosis occurs during the normal

development of mammalian embryos because it helps to

remove unnecessary cells, an important developmental

process [37]. In pESCs and aESCs, SRM may act as a

Caspase activator for cells which are needed to undergo

apoptosis.

This study describes analyses of the expressed pro-

teins in fESCs as compared with pESCs and aESCs. This

information contributes to our understanding of the in-

tracellular processes in uniparental- or biparental-de-

rived homozygous or heterozygous ESCs, and should

serve to provide insight into the functional capabilities of

these distinct cell types. Although we believe that the

proteins identified in this study are important for ESC

therapy in the clinical setting, we should also point out

that the actual function of these proteins in these types of

stem cells, especially during differentiation, is at present

unknown. Studying each protein individually, using gain

of function, loss of function, and dominant-negative

mutants, may reveal how and when these molecules

contribute to the self-renewal and differentiation of

ESCs.

In conclusion, two protein candidates, ANXA5 and

CLIC1, were more highly expressed in fESCs compared

with pESCs and aESCs. In contrast, SRM was more

highly expressed in pESCs and aESCs than in fESCs.

Further study of these protein candidates is needed to

identify and clarify their functions, including functions

related to the avoidance of immune rejection problems

during ESC therapy, the maintenance of pluripotency,

and the properties of ESC differentiation.

5. ACKNOWLEDGEMENTS

This work is supported by grant No. 20100301-061-224-001-50-00

from the Biogreen 21 program, RDA, Republic of Korea.

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