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Proteomic identification of differentially expressed genes in mouse neural stem cells and neurons differentiated from embryonic stem cells in vitro Kuniko Akama a,b, , Ryosuke Tatsuno b , Masahiro Otsu c , Tomoe Horikoshi a , Takashi Nakayama d , Megumi Nakamura e , Tosifusa Toda e , Nobuo Inoue c a Department of Chemistry, Graduate School of Science, Chiba University, Chiba, Japan b Department of Chemistry, Graduate School of Science and Technology, Chiba University, Chiba, Japan c Laboratory of Regenerative Neurosciences, Graduate School of Human Health Science, Tokyo Metropolitan University, Tokyo, Japan d Department of Biochemistry, Yokohama City University School of Medicine, Yokohama, Japan e Proteomic Collaboration Center, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan Received 1 October 2007; received in revised form 9 January 2008; accepted 4 February 2008 Available online 15 February 2008 Abstract Embryonic stem (ES) cells are pluripotent stem cells and give rise to a variety of differentiated cell types including neurons. To study a molecular basis for differentiation from ES cells to neural cells, we searched for proteins involved in mouse neurogenesis from ES cells to neural stem (NS) cells and neurons by two-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting, using highly homogeneous cells differentiated from ES cells in vitro. We newly identified seven proteins with increased expression and one protein with decreased expression from ES cells to NS cells, and eight proteins with decreased expression from NS cells to neurons. Western blot analysis confirmed that a tumor-specific transplantation antigen, HS90B, decreased, and an extracellular matrix and membrane glycoprotein (such as laminin)-binding protein, galectin 1 (LEG1), increased in NS cells, and LEG1 and a cell adhesion receptor, laminin receptor (RSSA), decreased in neurons. The results of RT-PCR showed that mRNA of LEG1 was also up-regulated in NS cells and down-regulated in neurons, implying an important role of LEG1 in regulating the differentiation. The differentially expressed proteins identified here provide insight into the molecular basis of neurogenesis from ES cells to NS cells and neurons. © 2008 Elsevier B.V. All rights reserved. Keywords: Embryonic stem cell; Differential expression; Neural stem cell; Neuronal differentiation 1. Introduction Neural stem (NS) cells have two essential characteristics, self- renewal and multipotency to differentiate into neural cells. Re- placement of lost neurons by transplantation of NS cells is a promising new approach in the treatment of progressive neu- rodegenerative diseases. Pearce and Svendsen have observed that protein expression of the stem cells from the human fetal brain proliferated by culturing with the mitogens epidermal growth factor and fibroblast growth factor (FGF) differs markedly from stem cells differentiated by removal of the mitogens and addition of serum [1]. Guo and co-workers have identified differentially expressed proteins of neurons derived from human ES cells after retinoic acid induction, which included α-tubulin and vimentin [2]. Maurer and co-workers have developed a proteomic database for NS cells isolated from the adult rat hippocampus to map about 1100 protein spots in a 2-DE proteomic profiling approach, of which 266 were identified [3]. Also, they have developed a database for the expression profiling of differentiation to indicate potential cellular targets mediating the differentiation of neural stem cells [4]. Wang and Gao have generated a proteome reference map of mouse NS cells and dopaminergic neurons differentiated from the ES cells, and identified proteins with altered expression including translationally controlled tumor protein and α-tubulin Available online at www.sciencedirect.com Biochimica et Biophysica Acta 1784 (2008) 773 782 www.elsevier.com/locate/bbapap Corresponding author. Department of Chemistry, Graduate School of Science, Chiba University, Yayoi-Cho 1-33, Inage-Ku, Chiba, Chiba 263- 8522, Japan. Tel.: +81 043 290 2795; fax: +81 043 290 2874. E-mail address: [email protected] (K. Akama). 1570-9639/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2008.02.001

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Page 1: Proteomic identification of differentially expressed genes in mouse neural stem cells and neurons differentiated from embryonic stem cells in vitro

Available online at www.sciencedirect.com

a 1784 (2008) 773–782www.elsevier.com/locate/bbapap

Biochimica et Biophysica Act

Proteomic identification of differentially expressed genes in mouse neuralstem cells and neurons differentiated from embryonic stem cells in vitro

Kuniko Akama a,b,⁎, Ryosuke Tatsuno b, Masahiro Otsu c, Tomoe Horikoshi a, Takashi Nakayama d,Megumi Nakamura e, Tosifusa Toda e, Nobuo Inoue c

a Department of Chemistry, Graduate School of Science, Chiba University, Chiba, Japanb Department of Chemistry, Graduate School of Science and Technology, Chiba University, Chiba, Japan

c Laboratory of Regenerative Neurosciences, Graduate School of Human Health Science, Tokyo Metropolitan University, Tokyo, Japand Department of Biochemistry, Yokohama City University School of Medicine, Yokohama, Japane Proteomic Collaboration Center, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan

Received 1 October 2007; received in revised form 9 January 2008; accepted 4 February 2008Available online 15 February 2008

Abstract

Embryonic stem (ES) cells are pluripotent stem cells and give rise to a variety of differentiated cell types including neurons. To study a molecularbasis for differentiation from ES cells to neural cells, we searched for proteins involved in mouse neurogenesis from ES cells to neural stem (NS) cellsand neurons by two-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting, using highly homogeneous cells differentiated from EScells in vitro. We newly identified seven proteins with increased expression and one protein with decreased expression from ES cells to NS cells, andeight proteins with decreased expression from NS cells to neurons. Western blot analysis confirmed that a tumor-specific transplantation antigen,HS90B, decreased, and an extracellular matrix and membrane glycoprotein (such as laminin)-binding protein, galectin 1 (LEG1), increased in NScells, and LEG1 and a cell adhesion receptor, laminin receptor (RSSA), decreased in neurons. The results of RT-PCR showed that mRNA of LEG1was also up-regulated in NS cells and down-regulated in neurons, implying an important role of LEG1 in regulating the differentiation. Thedifferentially expressed proteins identified here provide insight into the molecular basis of neurogenesis from ES cells to NS cells and neurons.© 2008 Elsevier B.V. All rights reserved.

Keywords: Embryonic stem cell; Differential expression; Neural stem cell; Neuronal differentiation

1. Introduction

Neural stem (NS) cells have two essential characteristics, self-renewal and multipotency to differentiate into neural cells. Re-placement of lost neurons by transplantation of NS cells is apromising new approach in the treatment of progressive neu-rodegenerative diseases. Pearce and Svendsen have observed thatprotein expression of the stem cells from the human fetal brainproliferated by culturing with the mitogens epidermal growth

⁎ Corresponding author. Department of Chemistry, Graduate School ofScience, Chiba University, Yayoi-Cho 1-33, Inage-Ku, Chiba, Chiba 263-8522, Japan. Tel.: +81 043 290 2795; fax: +81 043 290 2874.

E-mail address: [email protected] (K. Akama).

1570-9639/$ - see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.bbapap.2008.02.001

factor and fibroblast growth factor (FGF) differs markedly fromstem cells differentiated by removal of the mitogens and additionof serum [1]. Guo and co-workers have identified differentiallyexpressed proteins of neurons derived from human ES cells afterretinoic acid induction, which includedα-tubulin and vimentin [2].Maurer and co-workers have developed a proteomic database forNS cells isolated from the adult rat hippocampus to map about1100 protein spots in a 2-DE proteomic profiling approach, ofwhich 266 were identified [3]. Also, they have developed adatabase for the expression profiling of differentiation to indicatepotential cellular targets mediating the differentiation of neuralstem cells [4].Wang andGao have generated a proteome referencemap of mouse NS cells and dopaminergic neurons differentiatedfrom the ES cells, and identified proteins with altered expressionincluding translationally controlled tumor protein and α-tubulin

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774 K. Akama et al. / Biochimica et Biophysica Acta 1784 (2008) 773–782

[5]. Hoffrogge and co-workers [6] have reported the results of 2-DE proteome analysis of a proliferating human fetal midbrain stemcell line immortalizedwith the v-myc oncogene tomap 402 proteinspots representing 318 unique proteins, and those differentiatinginto neural cells to map 49 protein spots representing 45 distinctproteins. Recently, Hoffrogge and co-workers [7] have noted thatproteome profiling studies in different systems show limitedoverlap, indicating a lack of common features of NS cells based onthe difference in the origin of the cells, genetic modifications, cellculture conditions, and the total number of passages.

Nakayama and co-workers have established a uniquemethodofproducing NS cells efficiently with high purity from mouse EScells (HK clone, whichwe have established fromC57BL/6mouse)[8] via the formation of neural stem spheres (NSSs) under free-floating conditions in astrocyte-conditioned medium (ACM) [8–10]. During subsequent culture of the NSSs on an adhesivesubstrate with FGF-2, the Nestin-positive NS cells are induced tomigrate onto the substrate, resulting in the efficient production oflarge numbers of NS cells [9,10]. It is shown by RT-PCR analysisthat the NS cells express NS cell-marker genes such as Nestin,Pax6 [9], and Musashi 1 (data not shown), and that expression ofGFAP is not detected in the NS cells. It is revealed byimmunofluorescence analysis that almost all (99.5±0.5%) of theNS cells express Nestin [9]. Furthermore, almost all the NS cellscan be differentiated into functional microtubule-associatedprotein (MAP) 2-positive neurons by changing the mediumfrom Neurobasal B-27 with FGF-2 to ACM [9], or almostexclusively into glial fibrillary acidic protein (GFAP)-positiveastrocytes by withdrawing FGF-2 from the medium [11]. It isshown by RT-PCR analysis that the neurons express neuron-marker genes such as high-molecular-mass neurofilament protein(NF-H) andMAP2, and that expression of GFAP is less than 2.6%[9,11]. It is revealed by immunofluorescence analysis that almostall (98.3±1.2%) of the neurons expresses NF-H, MAP2 andtubulin-beta III, but not GFAP or O4 [9]. However, themechanisms of differentiation from ES cells to NS cells andneurons via NS cells have not been elucidated fully.

In this study, we used a 2-DE-based proteomic approach toidentify differentially regulated proteins in mouse NS cells andneurons derived from ES cells via the formation of NSSs. Wereport signal transduction and stress proteins, and energy-, RNA-,and protein-metabolism proteins that are differentially expressedfromES cells toNS cells and neurons, altered expression ofwhichhas not been reported previously. These provide insight intothe differentiation and induction of ES cells into NS cells andneurons.

2. Materials and methods

2.1. Cell culture

Mouse ES cells (HK clone) [8] weremaintained on a feeder layer ofmitoticallyinactivated mouse embryonic fibroblasts. RT-PCR showed that the mouse ES cellsexpressed undifferentiated ES cell-marker genes such as Oct4, Nanog, and Cripto.Immunostaining indicated that the ES cells expressed Oct4, SSEA-1, Nanog, andalkaline phosphatase (data not shown). The mouse ES cells did not differentiatespontaneously in the presence of LIF, and the expression levels of Nestin, MAP2,and GFAP in ES cells were 2.6%, 0.3%, and 0%, respectively [11]. Colonies of

undifferentiated ES cells were picked up and cultured under free-floating con-ditions in ACM, giving rise to NSSs as described [8–10]. The NSSs were platedonto Matrigel-coated dishes and encouraged to proliferate by culturing inNeurobasal medium (Gibco Invitrogen Corp. Grand Island, NY) supplementedwith 2%B-27 (Gibco Invitrogen Corp.) and 20 ng/ml FGF-2. Followingmigrationof the NS cells from the adhered NSSs to the surrounding areas, the NSSs wereremoved by picking with glass capillaries, and the migrated NS cells wereharvested by treatment with trypsin. The ES cells differentiated to NS cellsdependent on astrocyte-conditioned medium, and did not differentiate sponta-neously. The Nestin-positive NS cells proliferated dependent on FGF-2. Therelative expression levels of Nestin, MAP2 and GFAP in NS cells were 100%,4.6% and 0%, respectively [11]. These cells were collected by centrifugation andsuspended in Neurobasal medium (Gibco Invitrogen Corp.). To induce neuronaldifferentiation, the NS cells were cultured in ACM for 4 days [9,10]. The MAP 2-positive neurons proliferated dependent on ACM, and did not differentiatespontaneously. The relative expression levels of MAP2, GFAP, and O4 in neuronswere 100%, 2.6%, and 0%, respectively [11,9].

2.2. Sample preparation for 2-D electrophoresis

Colonies of ES cells were collected by picking with glass capillaries. NS cellsand neurons were collected using a cell scraper. The cells were washed twice bysuspending in cold PBS followed by centrifugation at 700 ×g for 5 min, and storedas cell pellets at −80 °C. Aliquots of the cell pellets were suspended in lysis buffercontaining 5 M urea, 2 M thiourea, 2% (w/v) 3-[3-cholamidopropyl]dimethylam-monio]-1-propanesulfonate (Dojindo Laboratories, Kumamoto, Japan), 2% (w/v)sulfobetaine 10 (Amresco, Solon, OH), 2% Pharmalyte 3–10 (Amersham Bio-sciences Inc., Piscataway, NJ), without DTT, protease inhibitors (Pierce Chemicals,Rockford, IL) and phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich, St.Louis, MO). The cell suspension was sonicated on ice five times with 1-s burstsevery 15 s at 25-W output, using an ultrasonic vibrator (UR200P; Tomy, Tokyo,Japan). The sonicated cells were centrifuged at 12,000 ×g for 10 min. The su-pernatant was ultracentrifuged at 100,000 ×g for 30 min to remove DNA. Theextracted proteins were reduced and carbamidomethylated with a ReadyPrepReduction-Alkylation Kit (Bio-Rad, Richmond, CA), and desalted with a 2-DClean-Up Kit (GE Healthcare Biosciences, Fairfield, CT) in accordance with themanufacturers' instructions. Alternatively, aliquots of the same cell pellets weresuspended in lysis buffer with 65 mM DTT. The cell suspensions were sonicatedand centrifuged, and the resultant supernatants were ultracentrifuged in the samemanner as described above. The protein content of the supernatant was measuredwith a 2-D Quant Kit (GE Healthcare Biosciences).

2.3. High-resolution 2-D gel electrophoresis

Proteins in the cell extract were separated according to TMIG StandardMethods in Proteomics (http://proteome.tmig.or.jp/2D/2DE_method.html) [12].Briefly, immobilized pH gradient-isoelectric focusing (IPG-IEF) in the first-dimension was carried out on a reswollen Immobiline DryStrip, pH 4–7, 18 cm inlength (Bio-Rad), in a CoolPhoreStar Model 3610 Horizontal IEF apparatus(Anatech, Tokyo, Japan). Aliquots of 50 μg of protein lysate per gel were loadednear the cathode wick on the IPG gel. After electrofocusing with 46,700 Vh, theIPG gel was equilibrated with SDS treatment solution (6 M urea/32 mM DTT/2%SDS/0.0025%w/v Bromophenol Blue (BPB)/25% v/v glycerol/25 mMTris–HCl,pH 6.8) for 30 min. When the proteins without reduction and carbamidomethyla-tion were subjected to electrofocusing, the IPG gel was treated with SDS asdescribed above, and then followed by carbamidomethylation in buffer containingiodoacetamide (4.5% w/v iodoacetoamide/1% w/v SDS/0.0025% w/v BPB/25%v/v glycerol/25 mM Tris–HCl, pH 6.8) for 20 min. An equilibrated gel strip wasplaced on top of a gel slab (7.5% T, 3% C, 20×18 cm). %Tmeans polyacrylamidegel concentration defined as percentage total monomers (i.e. acrylamide plusbisacrylamide, g/100 ml).%C means percentage bisacrylamide crosslinker. SDS-PAGE was run vertically in a Tris–Tricine buffer system using the CoolPhoreStarmodel 3068 electrophoresis apparatus (Anatech). The markers for molecular massand pI were obtained from Bio-Rad and Daiichi-Kagaku (Tokyo, Japan), re-spectively. Proteins on gels were fixed with 40% methanol/10% acetic acid, andsubsequently visualized by staining with SYPRO Ruby (Molecular Probes,Eugene, OR).

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Fig. 1. 2-D protein profiles from mouse ES cells, NS cells, and neurons. Proteinswere separated on the basis of pI (X-axis) and molecular mass (Y-axis), andvisualized by staining with SYPRO Ruby. Each protein was identified byMALDI-TOF-MS, and identified proteins are shown as symbols on the gels.(A) 2-DE pattern from ES cells. The labeled spot indicates identified proteins.(B) 2-DE pattern from NS cells. The up- and down-regulated proteins from EScells to NS cells were indicated by∧ and∨, respectively. The unchanged proteinswere indicated by −. (C) 2-DE pattern from neurons. The down-regulated proteinsfrom NS cells to neurons were indicated by ∨. The unchanged proteins wereindicated by −.

775K. Akama et al. / Biochimica et Biophysica Acta 1784 (2008) 773–782

2.4. Evaluation of protein patterns

2-DE gel images were scanned at an excitation wavelength of 488 nm, andemissionwavelengths over 550 nmwere collected on aMolecular Imager FX (Bio-Rad). Noise reduction, background subtraction, normalization, and quantitativeprofiling of proteins in 2-DE gels were carried out using PDQuest software version8.0 (Bio-Rad). For spot quantification, spot volumes were calculatedwith the built-in feature involving the application of a fixed multiple of Gaussian radius of thespot as a background intensity function. Subsequently, relative spot intensities,defined as percentage of spot volume to the sumof total spot volumes on the parentgel, were extracted from a spreadsheet generated by the software and used forstatistical analysis. All of the spots were roughlymatched by an automatic programin PDQuest software, which was followed by a more detailed manual matchingprocess to correct inappropriatematching pairs. Triplicate experimentswere carriedout using three separate samples. Student's t-test was used to determine thesignificance of stage-to-stage differences. As Student's t-test provides valid resultsonly when the variances of each sample are equal, a preliminary Fisher equality ofvariance test was applied. When variances were not equal between the two sets ofdata, a version of the Student's t-test comparison of twomeans including theWelchcorrection was used. Novel spots that appeared at a later stage were included in thecomparison as increasing spots.

2.5. Mass spectrometry

In-gel digestion was performed according to the TMIG Standard Methods inProteomics (http://proteome.tmig.or.jp/2D/2DE_method.html) [12]. Briefly, theSYPRO Ruby-stained protein spots were cut out of the gel with a spot cutter (Bio-Rad), and washed twice with 50%methanol/50mM ammonium bicarbonate, threetimes with 50% acetonitrile/50 mM ammonium bicarbonate, and washed with100% acetonitrile. The supernatant was removed, and the gel fragments were driedat room temperature for 20 min. The gels were then incubated in trypsin solutioncontaining 5 μg/ml trypsin (V511A; Promega, Madison, WI) in 30% acetonitrile/50mM ammonium bicarbonate at 30 °C overnight. The supernatant was subjectedto mass spectrometry (MS). Samples (1μl) weremixedwith 1 μl of matrix solutioncontaining 10 mg/ml CHCA (Sigma-Aldrich) in 50% acetonitrile/40% methanol /0.1% trifluoroacetic acid, and 2-μl samples were applied to the target plate. MSanalysis was performed on a MALDI-TOF mass spectrometer (AXIMA-CFR;Shimadzu, Kyoto, Japan) in reflectron mode and with the measurement range of500–3500m/z. The background noise was removed by subtraction of mass signalsobtained from a control gel. Protein spots were identified by matching all thepeptide masses against the Swiss-Prot and NCBInrMus musculis databases usingMascot Search (http://www.matrixscience.com/search_form_select.html) and MSFit (http://prospector.ucsf.edu/ucsfhtml4.0/msfit.htm). In general, mass toleranceof ±0.2 Da, one missed trypsin cleavage, oxidation of Met, and fixed modificationof carbamidomethyl cysteine were selected as matching parameters in the searchprogram.

2.6. Western blotting

For 1-DWestern blotting analysis, an equal volume of a 4% SDS solution with125 mM Tris–HCl, 30% glycerol, 4% 2-mercaptoethanol, and 0.02% Bromophe-nol Blue, pH 6.8, was added to the cell lysate, and themixturewas boiled for 3min.An equal amount of protein for each sample (1 or 3 μg per lane) was separated bySDS-PAGE in a Tris–Tricine buffer system on a 7.5% gel [12]. Electrical transferonto a PVDFmembrane (Millipore, Bedford, MA)was carried out with a semi-dryelectroblotting apparatus at ca. 2 mA/cm2 for 1 h at room temperature using abuffer containing 25 mM Tris, 192 mM glycine, 0.08% SDS, and 20% methanol.The membrane was treated with 3% ECL blocking agent (GE HealthcareBiosciences) in 0.05%Tween, 10mMTris–HCl, 150mMNaCl, 1 mMCaCl2, and1 mMMgCl2, pH 7.4 (T-TBS), for 1 h for blocking nonspecific binding, and thenincubated with primary antibody overnight at 4 °C. The following primaryantibodies were used: anti-HB90B (anti-Hsp84 rabbit polyclonal antibody, PA3-012, 1:1000; Affinity BioReagents, Golden, CO), which recognizes Hsp84 but notHsp86; anti-RSSA (anti-laminin-R rabbit polyclonal antibody, sc-20979, 1:1000;Santa Cruz Biotechnology, Santa Cruz, CA); and anti-LEG1 (anti-galectin-1 goatpolyclonal antibody, sc-19277, 1:1000; Santa Cruz Biotechnology). Alternatively,anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH mouse monoclo-

nal antibody, MAB374, 1:2000; Chemicon International, Temecula, CA) was usedas the primary antibody to confirm equal protein loading in each lane.

Subsequently, the membranes were washed three times in T-TBS, and boundantibodies were detected using appropriate horseradish peroxidase-conjugatedsecondary antibodies (bovine anti-goat IgG and donkey anti-rabbit IgG, sc-2350and sc-2313, respectively, 1:2000; Santa Cruz Biotechnology), followed by anECLWestern blotting Detection System (GE Healthcare Biosciences).

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2.7. Real-time RT-PCR analysis

Poly(A)+ mRNA from undifferentiated ES colonies, NS cells, and neurons wasprepared using a QuickPrep Micro mRNA Purification Kit (GE HealthcareBiosciences). Each mRNA was reverse transcribed into cDNA using randomhexamer primers, in accordance with the manufacturer's instructions (AppliedBiosystems, Foster City, CA). Quantitative real-time PCR was performed on anABI PRISM 7300 (Applied Biosystems) using SYBRGreen PCRMaster Mix andthe primer pairs shown in Supplementary Table 1, designed using Primer Expresssoftware (Applied Biosystems), in accordance with the manufacturer's instruc-tions. Expression of each target gene was normalized relative to that of GAPDHmRNA, with relative quantitative evaluation of the initial template copy numberdetermined relative to a standard curve of GAPDH cDNA (Applied Biosystems).All samples were analyzed in five replicates.

3. Results

3.1. Differential expression of proteins during neuraldifferentiation

To identify proteins that may be differentially regulated inES cells (HK clone) [8], NS cells and neurons, we used highlyhomogeneous mouse NS cells and neurons differentiated fromES cells by the NSS method [8–10]. Preliminary experimentsusing a broader pH range of pH 3–10 showed that about2/3 and 1/3 of all spots were in pH 4–7 and in pH 7–10,respectively. However, the resolution of proteins on the gel wasnot fully satisfactory. Then, we used IPG strip gel of pH 4–7 asthe first step, which had higher resolution in pH 4–7 than thestrip gel of pH 3–10. Protein preparation for 2-DE was carriedout by two methods. First, the reduction and carbamidomethyla-tion of extracted proteins was performed before IEF. Alterna-tively, they were reduced and carbamidomethylated after IEF.

Table 1Differentially expressed proteins of mouse NS cells and neurons differentiated from

Symbol Protein name Relative spot intensity (Mean±S

ES cells NS cells Neuro

ACTB Actin, cytoplasmic 1/2 0.79±0.12 1.70±0.22 1.46±KCRB Creatine kinase B 0.93±0.07 7.58±0.95 5.62±HNRPK Heterogeneous ribonucleoprotein K 0.53±0.15 0.79±0.08 0.33±IF5A1 Eukaryotic translation initiation

factor 5A-12.31±0.99 3.07±0.31 0.86±

CALR Calreticulin 0.42±0.45 2.02±0.34 0.33±PDIA1 Protein disulfide isomerase precursor 0.58±0.13 1.40±0.36 0.71±GDIR Rho GDP dissociation inhibitor 1 0.77±0.20 1.86±0.17 0.88±CALU Calumenin precursor NA f 0.42±0.20 0.27±HS90B HSP90-β (Hsp84) 1.60±0.40 0.29±0.50 0.65±PHB Prohibitin 0.17±0.16 0.67±0.12 0.51±RSSA 40S ribosomal protein SA

(laminin receptor 37 kDa)1.59±0.14 1.66±0.22 0.76±

THIO Thioredoxin 1 0.35±0.61 6.49±1.52 2.42±LEG1 Galectin 1 0.15±0.14 4.42±0.60 2.62±FABPB Fatty acid binding-protein, brain NA f 8.81±0.89 9.90±a Up (▲) or down (▼)-regulation of proteins toward the older developmental stagb ES cells vs. NS cells. For protein spots that showed statistical significance, the Pc NS cells vs.neurons. For protein spots that showed statistical significance, the Pd Swiss-Prot accession numbers are given for proteins.e Two missed trypsin cleavages.f NA: not detectable on the gel.

Proteins from ES cells, NS cells, and neurons were separated by2-DE, and the protein patterns from the cells at three devel-opmental stages were compared. Approximately 500 proteinspots were detected by SYPRO Ruby staining. Quantitativecomparison was performed using PDQuest software to assessthe relative abundance of altered proteins on 2-D gel maps of thecells.

The protein specimens prepared by reduction and carbami-domethylation before IEF yielded more spots of differentiallyexpressed proteins than those after IEF, suggesting that protein-degrading activity was almost completely inhibited by reductionand carbamidomethylation of the proteins before IEF. We ob-tained reproducible 2-DE profiles and relative spot intensitiesfrom all samples in experiments performed in triplicate. Fig. 1shows typical gel maps of proteins from mouse ES cells, NScells, and neurons. To investigate the changes in the proteinpatterns between the three stages, we compared the patternsbetween ES cells and NS cells, and between NS cells andneurons. Table 1 shows the relative intensities of the cor-responding spots in two stages. We found 11 protein spots thatwere up-regulated and one that was down-regulated in NS cellsas compared with ES cells.We found nine protein spots that weredown-regulated in neurons as compared with NS cells. Theseproteins were selected for subsequent analysis by MS. Peptidemass fingerprinting (PMF) of the selected spots followed by adatabase search revealed the identities of these proteins (Table 1and Fig. 1). Consequently, we found ACTB, KCRB, HNRPK,CALR, PDIA1, GDIR, CALU, THIO, LEG1 and FABPB withincreased expression and HS90B with decreased expressionfrom ES cells to NS cells, and KCRB, HNRPK, IF5A1, CALR,PDIA1, GDIR, RSSA, THIO and LEG1 with decreased

ES cells by the NSS method

D) Quantitative changes a Accessionnumber d

Mowsescore

Matchedpeptides

Sequencecoverage (%)

ns NS cells b Neurons c

0.35 ▲ (0.05) − Q6ZWM3 116 9 310.40 ▲ (0.01) ▼ (0.05) Q04447 145 9 270.15 ▲ (0.05) ▼ (0.01) P61979 72 6 200.10 − ▼ (0.01) P63242 75 5 42

0.24 ▲ (0.01) ▼ (0.01) P14211 105 e 8 140.18 ▲ (0.05) ▼(0.05) P09103 88 8 190.05 ▲ (0.01) ▼ (0.01) Q99PT1 124 7 420.13 ▲ (0.05) − O35887 115 7 250.24 ▼(0.05) − P11499 111 10 150.05 ▲ (0.05) − P67778 148 10 430.20 − ▼ (0.01) P14206 126 8 36

1.15 ▲ (0.01) ▼ (0.05) P10639 99 6 650.08 ▲ (0.01) ▼ (0.05) P16045 86 5 483.47 ▲ (0.01) − P51880 133 8 73

e. (−): no change.-values are shown in parentheses (t-test).-values are shown in parentheses (t-test).

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Table 2Annotations of proteins with altered expression in differentiation from ES cells to NS cells and neurons by the NSS method

Symbol Chr Class Function Neurogenesis Ref.

ACTB 11E2 D ATP-binding structural constituent of cytoskeleton Increase in neuronal differentiation fromhuman NS cell line

[6]

KCRB 12F1 EM Catalysis of transfer of phosphate between ATPand various phosphogens

Increase in neuronal differentiation fromhuman NS cell line

[6]

FABPB 10B4 LM Fatty acid-binding activity involved in transportor propagation process extending from the cell

Maintenance of neuroepithelial cells of rat cortex [21–24]Functional links to glutamate receptor

PHB 15A1 E Inhibition of DNA synthesis to regulate proliferation Increase in neural differentiation from mouse ES cells [22]HNRPK 13B1 RM Pre-mRNA-binding proteins in the nuclear

metabolism of hnRNAsDecrease in neuronal differentiation from humanNS cell line, Control of mouse neuronal differentiation

[6,21]

IF5A1 13B1 PM Promotion of formation of the firstpeptide bond in protein biosynthesis

Present in human NS cell line [6]

PDIA1 11D-E PM Catalysis of rearrangement of disulfide bonds in proteins Present in human NS cell line [6]CALR 8C3 PM Calcium ion binding Increase in mouse dopaminergic neurons [5,6]

Lectin for refolding of glycoprotein Present in human NS cell lineGDIR 11E2 S Regulation of GDP/GTP exchange reaction of Rho proteins Present in human NS cell line [6]CALU 6A3.3 S Calcium ion binding, calcium sensors and calcium

signal modulatorsmRNA expression in mouse whole head onembryonic day 16.5

[16]

THIO 4B3 C Catalysis of redox reactions Increase in mouse brain at 1 week and decreaseat 8 weeks after birth

[18]

HS90B 10C1 C Molecular chaperone with ATPase activity inprocessing and transport of secreted proteins

Present in human NS cell line [6]

RSSA 9 71.0cM PM Laminin-binding activity to initiate a change in cell activity Present in NS cell line [6]LEG1 15E S Galactoside binding and signal transducer activity Promotion of proliferation of NS cells in adult brain [20,42–45]

44.9cM Involved in neurite outgrowth and degradationof neuronal processes

Molecular functions, chromosomal locations (Chr), and possible roles in neurogenesis are shown for the proteins identified in this study. Molecular functions wereobtained from the Gene Ontology Consortium (http://www.geneontology.org/). Listed proteins were classified into eight groups: D, cytoskeleton; C, heat shock/stressproteins; S, signal transduction; PM, protein metabolism; LM, lipid metabolism; EM, energy metabolism; RM, RNA metabolism; E, Cell cycle.

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expression from NS cells to neurons. Table 2 summarizes theannotations with possible functions of the differentially reg-ulated proteins in differentiation from ES cells to neural cells.We classified the molecular functions of the proteins intofive groups: seven were involved in metabolism, one in thecytoskeleton, three in signal transduction, two in stress re-sponses, and one in the cell cycle. Of the metabolism-relatedproteins, four (HNRPK, IF5A1, PDIA1, and CALR) are knownto play roles in post-transcriptional regulation, protein biosynth-esis, protein folding, and glycoprotein folding, respectively.Together with the results shown in Table 1, these suggest veryactive protein synthesis and folding in NS cells. A cytoskeletonprotein, ACTB, was up-regulated in NS cells as compared to EScells. Cytoskeletal networks including actin serve multiple rolesin neurons [13]. Of the signal transduction-related proteins,GDIR and CALU were up-regulated in NS cells as compared toES cells. GDIR is known to be involved in regulation of the actincytoskeleton [14]. CALU has been implicated in calcium signalmodulation, and its mRNA has been reported to be expressed inwhole head on embryonic day 16.5 [15,16]. These suggest aprobable link between active signal transduction and changes inthe cytoskeleton during the differentiation from ES cells to NScells.

Of the heat shock/stress proteins, HS90B was down-regulatedin NS cells and neurons. HS90B, also known as tumor-specifictransplantation antigen Hsp84, inhibits tumor growth in animalsreceiving antigen injection prior to tumor challenge [17]. On theother hand, THIO was up-regulated in NS cells and down-

regulated in neurons, suggesting active oxidoreduction in NScells. THIO has been reported to show altered expression in thedeveloping mouse brain after birth [18].

RSSA (40S ribosomal protein SA), also identified as lamininreceptor or 37 kDa oncofetal protein, appears to be a multi-functional protein involved in initiation of changes in cellularactivity and the translational machinery. The laminin receptor, amember of the integrin family of cell adhesion receptors, wasdown-regulated in neurons.

LEG1 was up-regulated in NS cells, and down-regulated inneurons. LEG1 has been reported to be expressed in a subset ofmouse subventricular zone astrocytes, including the adult NScells, and to promote proliferation of adult NS cells [19].

3.2. Comparison of protein and mRNA expression levels

To investigate the mRNA expression of the identified proteins,we performed real-time RT-PCR analyses for KCRB, FABP,PHB, CALR, CALU, THIO, HSP90B, RSSA, and LEG1[Supplementary Table 2]. Fig. 2 shows a comparison of proteinand mRNA expression. The mRNA expression levels of CALU,PHB, and LEG1 changed in a similar manner to the respectiveprotein expression levels (Fig. 2D, G, and H). In the case of THIO(Fig. 2C), its mRNA expression level was up-regulated from EScells to NS cells in a similar manner to the protein expressionlevel, but unchanged from NS cells to neurons, in contrast to theobserved decrease in its protein expression. In the case of KCRB,FABPB, HSP90B, and RSSA (Fig. 2A, B, E, and F), the

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Fig. 2. Protein and RNA expression of eight genes identified. RNA expression determined by real-time RT-PCR together with protein expression determined by 2-DEare plotted against each differentiation stage (ES cells, NS cells, and neurons). Gray bars refer to protein expression and white bars refer to RNA expression. Thevertical axis on the left shows the normalized volume of each protein spot on 2-DE, and that on the right shows the relative amounts of RNA normalized with respect tosignals from ubiquitously expressed GAPDH mRNA. ES, ES cells; NSC, NS cells. ⁎ and ⁎⁎ indicate Pb0.05 and Pb0.01, respectively, when compared between thecorresponding expression of two stages.

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expression patterns of their mRNAs differed from those of theproteins.

The results of proteomic and RT-PCR analyses showed thatLEG1 was up-regulated in NS cells and down-regulated inneurons, implying an important role of LEG 1 in regulating thedifferentiation. In the case of HSP90B and RSSA (Fig. 2E and F),the expression patterns of their mRNAs conflicted with those ofthe proteins. In addition, their functions were interesting inregulating the differentiation, because HS90B and RSSA havea role in tumor suppression [17] and a cell adhesion receptoractivity (http://www.geneontology.org/), respectively. Then, toconfirm the differential expression of HS90B, RSSA (laminin

receptor), and LEG1proteins, we performed 1-DWestern blottinganalyses of HS90B, RSSA (laminin receptor), and LEG1 fromEScells, NS cells, and neurons using the same specimens as used for2-DE. As shown in Fig. 3, the level of expression of HS90B(Hsp84) was higher in ES cells than in NS cells and neurons. Incontrast, expression of LEG1 was up-regulated in NS cells anddown-regulated in neurons,while that of RSSA (laminin receptor)was down-regulated in neurons. These results were consistentwith the data obtained in the proteomic study using 2-DE,indicating that the approach used in this study was reliable.

Consequently, the comparison of protein and mRNA expres-sion levels suggested that the up-regulation of KCRB and FABPB

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Fig. 3. 1-D Western blotting analysis with anti-HS90B, anti-RSSA, and anti-LEG1 antibodies. Lanes 1–3: lysate from ES cells, NS cells, and neurons,respectively. For Western blotting analysis with anti-HS90B (anti-Hsp84) andanti-RSSA (anti-laminin receptor), aliquots of 1 μg of protein were subjected toSDS-PAGE, and for that with anti-LEG1 and anti-GAPDH, aliquots of 3 μg ofprotein were subjected to SDS-PAGE.

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proteins in NS cells, down-regulation of KCRB and THIO pro-teins in neurons, and the comparable expression of FABPBprotein in NS cells and neurons, occur at the post-transcriptionallevel. In addition, the down-regulation of HS90B protein in NScells and neurons, and that of RSSA (laminin receptor) protein inneurons, also occurs at the post-transcriptional level.

4. Discussion

In this study, we examined differentially regulated proteins inmouse NS cells and neurons derived from ES cells. We newlyidentified seven proteins with increased expression and oneprotein with decreased expression from ES cells to NS cells, andeight proteins with decreased expression from NS cells toneurons, using highly homogeneous cells differentiated from EScells in vitro. The data suggested that protein synthesis andfolding, oxidoreduction, signal transduction, and changes in thecytoskeletonwere up-regulated fromES cells to NS cells, and thatprotein synthesis and folding, oxidoreduction, and signaltransduction except for calcium signal modulation by CALUwere down-regulated from NS cells to neurons (Tables 1 and 2).Interestingly, at the transcriptional level, CALU also increasedfrom ES cells to NS cells, and unchanged from NS cells toneurons (Fig. 2). At least for the proteins that we examined, thedata obtained in this study were thought to be consistent withcharacteristics of NS cells and neurons.

Among the proteins identified, some have been reported tobe differentially regulated in mammalian neurogenesis from EScells to NS cells and neurons. For example, Hoffrogge and co-workers have reported that the expression of ACTB, KCRB,and HNRPK was altered in neural cells differentiated from thehuman NS cell line ReNcell VM [6]. HNRPK has beensuggested to control neuronal differentiation [20]. FABPB hasbeen reported to be expressed in neuroepithelial cells, includingNS cells and differentiating neurons downstream of thetranscription factor Pax6, and to be essential for theirmaintenance during early embryonic development of the ratcortex [21]. FABPB is expressed in mouse radial glia, whichserve as neuroprogenitors in the adult brain [22]. FABPB isstrongly expressed in radial glia and immature astrocytes in pre-and perinatal brain, but its expression is remarkably attenuatedin the astrocytes of adult brain [23]. FabpB, which is also calledFabp7, has functional links to the glutamate receptor, N-methyl-D-aspartic acid receptor [24]. These observations sup-

port the reliability of the data obtained by this proteomicanalysis procedure.

On the other hand, Guo and co-workers have reported thatα-3/α-7 tubulin and vimentin are down-regulated from mouse EScells to neurons [2]. Wang and Gao have mentioned thattranslationally controlled tumor protein was down-regulated,but tubulinα-6 and actin-related protein 3 are up-regulated frommouse ES cells to neurons [5]. Hoffrogge and co-workers havealso reported that proliferating cell nuclear antigen andperoxiredoxin 4 are down-regulated from proliferating humanneuronal stem cells (ReNcell VM) to differentiating neuronalstem cells [6]. These observations were different from ourobservations that the expression levels of these proteins wereunchanged. In addition, Maurer and co-workers have reportedthat ACTB and Hsp84 are up-regulated from adult rathippocampus NS cells to neurons [4]. However, ACTB wasunchanged and Hsp84 was down-regulated, from the NS cells toneurons in our observations. These may have been due to thedifferences in the origin of the cell line, cell homogeneity, cultureconditions used, and developmental stages of differentiation fromES cells to neurons. It has been reported that NS cells change theircompetency over time during development [25–28].

Among the proteins identified, HS90B, LEG1 and RSSA(laminin receptor) were particularly notable proteins thought tobe involved in modulation of the differentiation from ES cells toNS cells and neurons.

Mouse and human HSP90 exist in two forms, Hsp84 andHsp86, encoded by related but separate genes [29,30]. We foundthe down-regulation of HS90B (Hsp84, a tumor-specific antigento inhibit tumor growth) from ES cells to NS cells as describedabove (Table 1, Figs. 2 and 3).

On the other hand, PHB also has a potential role in devel-opment, senescence, and tumor suppression, which can directlyinteract with p53 [31,32]. In contrast, both protein and mRNAexpression of PHB were up-regulated from ES cells to NS cells(Table 1 and Fig. 2). The up-regulation of PHB protein duringmouse neural differentiation from ES cells agrees with recentreport of Battersby et al. [33].

We found the down-regulation of RSSA (laminin receptor)protein from NS cells to neurons (Table 1, Figs. 2 and 3). Thelaminin receptor carries a stage-specific embryonic antigen-4epitope defined by the monoclonal antibody Raft.2 [34]. The37-kDa precursor of the 67-kDa laminin receptor is apolypeptide the mRNA expression of which is consistentlyup-regulated in aggressive carcinomas [35]. Laminin receptoris found at 9-fold higher levels of mRNA expression in coloncarcinoma than in adjacent normal colonic epithelium [36], andup-regulated in lung cancer cell line SB03 as compared withlung normal cell line IMR90 [37]. Whether this up-regulationof laminin receptor in tumor cells occurs at the proteinexpression level remains unclear.

In the case of LEG1, both protein and mRNA expression wereup-regulated from ES cells to NS cells, and down-regulated fromNS cells to neurons, as described above (Table 1, Figs. 2 and 3).LEG1 binds to basement membrane laminin and fetally derivedfibronectin, which carry polylactosamine glycans and tetra-antennary complex-type glycans, respectively [38], suggesting a

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probable link between LEG1 and RSSA (laminin receptor). LEG1binds to the membrane receptor, CD45, and inhibits its proteinphosphatase activity, and therefore dephosphorylation of Lyntyrosine kinase to induce cell cycle arrest andmodulate the viabilityof Burkitt's lymphoma B cells [39]. LEG1 interacts with the α5β1fibronectin receptor to restrict epithelial carcinoma cell growth viainhibition of the Ras-MEK-ERK pathway and consecutivetranscriptional induction of p21 and p27, which are cyclin-dependent kinase inhibitors that maintain G0 and inhibit G1traverse [40]. Transcriptional activation of p21 by vitamin D3 leadsto the induction of differentiation of the myelomonocytic cell lineU937 [41]. LEG1 has also been suggested to be involved inneuronal path-finding, neurite outgrowth, and fasciculation [42,43].In LEG1nullmice, a subpopulation of primary sensory neurons failto project their axons to the correct target sites in the caudalolfactory bulb [43]. In addition, rat transient forebrain ischemiacauses selective induction in the brain of ΔFosB, a truncated formof FosB that inhibits transcriptional activity of Fos/Jun (compo-nents of transcription activator protein-1), followed by induction ofnestin (a marker for neuronal precursors) or LEG1. ΔFosB andLEG1 are involved in enhancing the proliferation and survival ofneuronal precursor cells after transient forebrain ischemia [44].These observations suggest that LEG1 plays an important role as asignal transducer that restricts tumor cell growth and regulates thedifferentiation from ES cells to NS cells and neurons. Recently,Plachta and co-workers have reported that LEG1 causes thedegeneration of neuronal processes using engineered mouse EScells, which express neurotrophin receptor p75NTR when ES cell-derived progenitors start elongating neural processes to lead to anincrease in the level of LEG1 [45]. They have demonstrated thatLEG1 causes the processes of wild type neurons to degenerate,followed by the cell bodies in vitro. The up-regulation of p75NTR

by the application of a glutamate receptor agonist leads to anincrease in the amount of LEG1 and to the degeneration of neuronsin vivo. These data also support our results that LEG 1 is down-regulated from NSC to neurons, and regulates the differentiation.

These results clearly showed that the proteomic approach usedhere is useful for gaining insight into the differentiation andinduction from ES cells to NS cells via NSS, and from NS cells toneurons. In addition, this study supported the suggestion that theNSS method for the differentiation and induction from ES cells toNS cells and neurons in vitro is useful to provide an experimentalmodel of neurogenesis from ES cells to neural cells. A furtherapplication of proteomics to the detailed stages of the differentia-tion should provide a more complete picture of early neurogenesis,and enable us to elucidate the differences in characteristics of fetaland adult NS cells and neurons in the future. Studies to describe thepathways that are activated or suppressed during differentiationfrom ES cells to NS cells and neurons will help to elucidate thefunctional roles of newly identified proteinswith altered expressionin these three cell stages. The studies of biological functions of theproteins, of which differentiation behavior was elucidated in thisstudy, are in progress. Recently, Watanabe and co-workers havereported that Fabp7/FabpB is a candidate gene for prepulseinhibition, of which deficits are biological marker for schizo-phrenia, by the use of quantitative trait loci analysis on mice [46].They have demonstrated that Fabp7/FabpB-deficient mice show

decreased prepulse inhibition and attenuated neurogenesis, and thathuman Fabp7/FabpB shows altered expression in schizophrenicbrains and genetic association with schizophrenia [46].

5. Conclusion

A proteomic approach to search for genes involved in neuro-genesis frommouse ES cells to NS cells and neurons, using highlyhomogeneous cells differentiated from ES cells in vitro, showedseven proteins with increased expression and one protein withdecreased expression from ES cells to NS cells, and eight proteinswith decreased expression from NS cells to neurons, alteredexpression of which has not been reported previously. Particularly,HS90Bwas down-regulated andLEG1 up-regulated fromES cellsto NS cells. LEG1 and RSSA (laminin receptor) were down-regulated from NS cells to neurons. At the transcriptional level,LEG1 was also up-regulated in NS cells and down-regulated inneurons, suggesting an important role of LEG1 in regulating thedifferentiation from ES cells to NS cells and neurons. Thedifferentially expressed proteins identified here provide insightinto themolecular basis for understanding cellular properties ofNScells and neurons, and will be useful for further characterization ofneurogenesis from ES cells to NS cells and neurons.

Acknowledgements

This work was supported in part by Grant-in-Aid for ScientificResearch of Japan (17500256) and Selective Research Fund ofTokyo Metropolitan University (to N. I.). The authors areindebted to Professor Takashi Obinata, Dr. Hiroshi Abe, and Dr.Naruki Sato of Chiba University, Japan, for ultracentrifugaltreatment in the sample preparation for 2-D electrophoresis.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.bbapap.2008.02.001.

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Glossary

ES: embryonic stemNS: neural stem2-DE: two-dimensional gel electrophoresisNSS: neural stem sphereACM: astrocyte-conditioned mediumFGF: fibroblast growth factor

MAP: microtubule-associated proteinGFAP: glial fibrillary acidic proteinIPG: immobilized pH gradientIEF: isoelectric focusingGAPDH: glyceraldehyde-3-phosphate dehydrogenaseT-TBS: TBS containing 0.05% TweenMS: mass spectrometryPMF: peptide mass fingerprinting