proteomic analysis of neural differentiation of mouse embryonic stem cells

REGULAR ARTICLE

Proteomic analysis of neural differentiation of mouse

embryonic stem cells

Daojing Wang and Ling Gao

Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

Mouse embryonic stem cells (mESCs) can differentiate into different types of cells, and serve as agood model system to study human embryonic stem cells (hESCs). We showed that mESCs dif-ferentiated into two types of neurons with different time courses. To determine the global proteinexpression changes after neural differentiation, we employed a proteomic strategy to analyze thedifferences between the proteomes of ES cells (E14) and neurons. Using 2-DE plus LC/MS/MS,we have generated proteome reference maps of E14 cells and derived dopaminergic neurons.Around 23 proteins with an increase or decrease in expression or phosphorylation after differ-entiation have been identified. We confirmed the downregulation of translationally controlledtumor protein (TCTP) and upregulation of a-tubulin by Western blotting. We also showed thatTCTP was further downregulated in derived motor neurons than in dopaminergic neurons, andits expression level was independent of extracellular Ca21 concentration during neural differ-entiation. Potential roles of TCTP in modulating neural differentiation through binding to Ca21,tubulin and Na,K-ATPase, as well as the functional significance of regulation of other proteinssuch as actin-related protein 3 (Arp3) and Ran GTPase are discussed. This study demonstratesthat proteomic tools are valuable in studying stem cell differentiation and elucidating theunderlying molecular mechanisms.

Received: November 15, 2004Revised: February 25, 2005

Accepted: March 1, 2005

Keywords:

Calcium homeostasis / Mouse embryonic stem cells / Neural differentiation / Transla-tionally controlled tumor protein

4414 Proteomics 2005, 5, 4414–4426

1 Introduction

Stem cells have the ability to self-renew and produce manydifferentiated cell types, and are therefore responsible forgenerating and repairing tissues and organs [1–11]. Al-though the fundamental understanding of the mechanismsfor maintenance of stem cell pluripotency and the pathwaysleading to lineage specification is still lacking, the consensus

is that the property is attributed to the instructive signalsfrom their extracellular microenvironment (or “niche”).Therefore maintaining homeostasis among the “niche”[growth factors, hormones, proteases and extracellularmatrix (ECM) molecules], cell-cell interactions and intracel-lular compartments are critical in determining the specificlineages [8, 12–15]. Mimicking the cellular interactions of themouse embryo by providing appropriate signaling moleculesin culture, such as those for WNT, FGF and TGF-b signalingpathways, has enabled the differentiation of ES cells to bedirected predominantly toward particular lineages [16]. Fur-thermore, soluble factors and the type of ECM both seem tobe critical in directing differentiation of ES cells and the for-mation of tissue-like structures. Studies have shown thatstem cells cultured in 3-D ECM differ significantly from its2-D counterparts in both proliferation and differentiation[17–20]. More recently it was shown that small molecules cancontrol stem cell fate and trigger its dedifferentiation [21–23].These results demonstrated that both chemical and physical

Correspondence: Dr. Daojing Wang, Life Sciences Division, Lawr-ence Berkeley National Laboratory, 1 Cyclotron Road, MS 84-171,Berkeley, CA 94720, USAE-mail: [email protected]: 1-510-486-5730

Abbreviations: m/hESCs, mouse/human embryonic stem cells;Q-TOF, quadrupole/TOF; RA, retinoic acid; TCTP, translationallycontrolled tumor protein; TH, tyrosine hydroxylase; Tuj1, class IIIb-tubulin

2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

DOI 10.1002/pmic.200401304

Proteomics 2005, 5, 4414–4426 Systems Biology 4415

cues are important in stem cell differentiation. In in vivo aswell as in vitro culture systems, free and protein-bound metalions such as Ca21 are distributed throughout the extra-cellular and intracellular environments. Modulation of theirhomeostasis and signaling may influence the environmentalcues and, therefore, specific lineages for stem cell differ-entiation.

Since stem cell differentiation is a highly coordinatedevent and involves myriads of genes, a global survey of tran-scriptome and proteome will generate candidates for detailedstudies. DNA microarray technology using cDNA or oligo-nucleotide arrays allows us to simultaneously compare thedifferential expression of a large number of genes and insome cases the whole genome under a given condition. It hasshown tremendous potential for the analysis of stem cellfunction and differentiation [6, 7, 17, 24–32]. Nevertheless,gene expression at the mRNA level may not correlate wellwith that at the protein level due to mRNA degradation,alternative splicing and protein post-translational modifica-tions. Therefore, direct monitoring of global protein expres-sion and post-translational modifications will identify pro-teins that contribute to stem cell differentiation. Proteomicsquantitatively and qualitatively maps the entire complementof proteins expressed by a particular cell or tissue at a giventime or in response to external stimuli [33, 34]. Rapid tech-nological developments have greatly advanced proteomecharacterization for biomarker discovery [35] and stem cellresearch [36–42].

We have recently demonstrated that proteomic tools arevaluable in studying human adult mesenchymal stem cell(hMSC) differentiation and elucidating the underlying mo-lecular mechanisms [38]. In this study, we adopted a similarstrategy and investigated the proteomic changes during dif-ferentiation of mouse embryonic stem cells (mESCs) intodopaminergic or motor neurons, in the absence or presenceof all-trans retinoic acid (RA), respectively. We used LC/MS/MS to identify differentially expressed proteins in 2-D gels.Preliminary 2-D reference maps of mESCs and dopaminer-gic neurons were generated. Overall 23 protein spots wereconsistently up- or downregulated (over 2-fold) duringneural differentiation. All these regulated proteins wereidentified and among them were three classes of Ca21-related proteins: calreticulin, pyruvate dehydrogenase E1/E2subunits and translationally controlled tumor protein(TCTP). TCTP, a growth-related Ca21- and microtubule-binding protein, was confirmed by Western blotting to bedownregulated in dopaminergic neurons (0.23-fold) and,furthermore, in motor neurons. Our results suggested thepossible involvement of TCTP in neurogenesis throughmodulating tubulin expressions and Ca21 binding. We alsoshowed that TCTP expression in neurons was independentof extracellular Ca21 concentration. These results demon-strated that TCTP and Ca21 homeostasis may play impor-tant roles in mediating ES cell differentiation along neuro-nal lineages and the value of proteomic profiling in eluci-dating underlying mechanisms.

2 Materials and methods

2.1 Cell culture

Undifferentiated E14 cells obtained from Dr. Yoichi ShinKai(Kyoto University, Japan) were maintained on mitomycin C-inactivated primary embryonic fibroblasts (PEF) in ESC me-dium [Dulbecco’s minimum essential medium supple-mented with 20% FBS, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 0.055 mM b-mercaptoethanol (ME),16 penicillin/streptomycin, and 1000 U/mL leukemia inhi-bitory factor (LIF)]. Before in vitro differentiation, E14 cellsgrown on PEF were treated with trypsin/EDTA for 5 min andresuspended in ESC medium. The mixture was then put on0.1% gelatin-coated dishes for 30 min to allow PEF to attachto the bottom of dishes. E14 cells that remained in thesupernatant were harvested. To induce differentiation [43],E14 cells were seeded on confluent PA6 cell monolayer(stromal cells derived from skull bone marrow, Riken cellbank) at a density of 16105 per 10-cm dish in differentiationmedium (Glasgow minimum essential medium supple-mented with 10% Knockout Serum Replacement, 1 mM

sodium pyruvate, 0.1 mM nonessential amino acids, and0.055 mM b-ME) (day 0). Medium was changed on day 4 andevery other day following that. For all-trans RA-induced dif-ferentiation [10], medium was supplemented with 2 mM RA.The cells were maintained in humidified incubators at 377Cwith 6% CO2. To collect cells on day 10, 10-cm plates werewashed with PBS twice, and then incubated with 7 mL of celldissociation solution at 377C for 15 min. The cells were har-vested and passed through a 100-mm cell strainer to removePA6 cell clumps [44]. The flow through containing neuronswas washed with ice-cold PBS three times, and cell pelletswere either lysed directly or stored at 2807C.

To carry out Ca21-dependent neural differentiation, ap-proximately 4000 single E14 cells were seeded on 60-mmplates with confluent PA6 layer. Each Ca21 concentration wasrun in triplicates. Ca21 was calculated to be 2.3 mM in E14differentiation medium (pH 7.4). To modulate Ca21 con-centration, we added different amounts of stock solution of125 mM EGTA or 250 mM CaCl2 in 10 mM HEPES buffer(pH 7.4), respectively. Free Ca21 was adjusted to 0.6 and1.1 mM with addition of EGTA, and to 3.3 and 4.3 mM withaddition of CaCl2. All other medium components stayed thesame. Media were changed on day 4 and every other day fol-lowing that. After E14 cells were differentiated for 10 days,plates were washed with PBS once, stained with 0.5% crystalviolet (in PBS/formaldehyde, 9:1) for 1 h, and finally washedwith PBS twice. Neuron colonies were then manually count-ed (double-blind) by three individuals and the results fromeach person were averaged.

Cell culture products and other consumable laboratorysupplies were purchased from Fisher Scientific Corp. (Fair-lawn, NJ) and VWR International (Brisbane, CA, USA). Celldissociation solution and all-trans RA were from Sigma (SaintLouis, MO, USA). Fetal bovine serum (FBS) for ES cell cul-

2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de

4416 D. Wang and L. Gao Proteomics 2005, 5, 4414–4426

ture was from HyClone (Logan, UT, USA). LIF was fromChemicon (Temecula, CA, USA). All other culture media andsupplements were from Invitrogen (Carlsbad, CA, USA).

2.2 Cell staining and microscopy

The phase-contrast images of ES cells and neurons werecollected using a Nikon TMS microscope with an6100 magnification and a Hamamatsu Orca100 cooleddigital CCD camera. The images were transferred directlyfrom a frame grabber to the computer storage using C-Im-aging System software (Compix Inc., PA, USA).

For immunostaining, single ES cells collected fromgelatin-coated plates or neurons grown on slide chambers(glass plate) were fixed with 4% paraformaldehyde/PBS for10 min, permeabilized with 0.2% Triton X-100/PBS for15 min. After being blocked with 1% BSA/PBS for 1 h, thecells were double-stained with mouse monoclonal anti-class III b-tubulin (Tuj1) antibody (1:500 dilution in 1% BSA/PBS) and rabbit polyclonal anti-tyrosine hydroxylase (anti-TH; 1:200 dilution in 1% BSA/PBS) for 1 h, and then sec-ondary goat anti-mouse antibody conjugated with Alexa-488(green), and goat anti-rabbit antibody conjugated with Alexa-594 (red), respectively (1:300 dilution each in 1% BSA/PBS,Molecular Probes) for 1 h. The cells were subsequentlystained with 4’,6-diamidino-2-phenylindole (DAPI; 50 ng/mL, Sigma; targeting DNA in the cell nuclei). The imageswere collected by an Olympus BX60 microscope.

2.3 Cell lysis and immunoblotting analysis

Cells were lysed with 2-DE lysis buffer containing 7 M urea,2 M thiourea, 4% CHAPS, 40 mM Tris base and 20 mM DTT.Normally, 1 mL of lysis buffer was used for 16107–26107

cells. The lysates were centrifuged at 15 000 rpm using amicrocentrifuge for 30 min at 207C. The protein concentra-tion of the supernatants was quantified using a modifiedBradford assay (Bio-Rad Laboratories, Hercules, CA, USA).Supernatants were aliquoted and stored at 2807C and readyfor 1-DE and 2-DE.

For immunoblotting analysis, equal amounts of totalproteins (15 mg) were run in SDS-PAGE with 4–20% gra-dient gels (ISC BioExpress, Kaysville, UT, USA) and trans-ferred to a 0.45-mm NC membrane (Bio-Rad Laboratories),which was blocked with 5% nonfat milk, and incubated withthe primary antibody in TBST buffer (25 mM Tris-HCl,pH 7.4, 60 mM NaCl, and 0.075% Tween 20) containing1% BSA. The bound primary antibodies were detected usinga goat anti-mouse or a goat anti-rabbit IgG-horseradish per-oxidase conjugate (Santa Cruz Biotechnologies, Santa Cruz,CA, USA) and the ECL detection system (Amersham Bio-sciences, Piscataway, NJ, USA). The immunoblotting resultswere scanned with an Umax 2100 high-resolution scanner(Umax Technologies, Dallas, TX, USA).

The mouse monoclonal anti-a-tubulin antibody wasfrom Sigma. The mouse monoclonal anti-Tuj1 antibody was

from Covance (Berkeley, CA, USA). The mouse monoclonalantibody against total actin (including all isoforms) and rab-bit polyclonal anti-TH antibody were from Chemicon. Therabbit polyclonal antibody against TCTP was from Medicaland Biological Laboratories International (Nagoya, Japan).

2.4 2-DE and image analysis

The detailed procedures for 2-DE, image analysis and proteinidentification were essentially the same as previously de-scribed [38]. Briefly, the first-dimension IEF was performedusing an Ettan IPGphor unit and the second-dimension(SDS-PAGE) was carried out using an Ettan DALTsix system(Amersham Biosciences). Triplicates of 50 mg of total celllysates were focused on pre-cast 18-cm pH 3–10 NL IPGstrips obtained from Amersham Biosciences. SDS-PAGEwas performed at a constant voltage of 100 V at 107C using 1-mm-thick 10% polyacrylamide gels with a dimension of27.5621-cm2 cast with 30% Duracryl, 0.65% Bis (GenomicSolutions, Ann Arbor, MI, USA). Proteins on gels were visu-alized using silver staining. Stained gels were imaged withan Umax PowerLook 1100 scanner (Umax Technologies)with a defined scan resolution of 250 dpi in a transmissiveand grayscale mode. Protein spots were detected and theirexpression levels were compared using Z3 3.1 software(Compugen, Tel Aviv, Israel). Protein spots that were deter-mined to be differentially expressed (n fold more than 2.0 orless than 0.5; p,0.05, with Student’s t-test) using the auto-matic analyses were verified manually by local pattern com-parison to exclude artifacts.

2.5 In-gel digestion and protein identification

To identify differentially expressed proteins, the spots wereexcised from the gels and digested with trypsin. The trypticpeptides were extracted from gel spots and subjected to LC/MS/MS analysis using a hybrid Quadrupole/OrthogonalTOF mass spectrometer Q-TOF API US (Waters Corp., Mil-ford, MA, USA) interfaced with a capillary LC system(Waters). Around 1–2 mL of sample was injected through anauto-sampler into the LC system at the flow rate of 20 mL/min, and pre-concentrated on a 300 mm65 mm Pep-Map C18 precolumn (Dionex, Sunnyvale, CA, USA). Thepeptides were then eluted onto a 75 mm615 cm Pep-Map C18 analytical column. The column was equilibratedwith solution A (3% ACN, 97% water, 0.1% formic acid) andthe peptide separation was achieved with a solution gradientfrom 3% to 40% solution B (95% ACN, 5% water, 0.1% for-mic acid), over 35 min at a flow rate of 250 nL/min. This flowrate through the column was reduced from 8 mL/min frompumps A and B by flow splitting.

The LC eluent was directed to the electrospray sourcewith a PicoTip emitter (New Objectives, Woburn, MA, USA).The mass spectrometer was operated in positive ion modewith a source temperature of 1007C and a cone voltage of40 V. A voltage of 2 kV was applied to the PicoTip. TOF ana-



lyzer was set in the V-mode. The instrument was calibratedwith a multi-point calibration using selected fragment ionsfrom the CID of Glu-fibrinopeptide B. MS/MS spectra wereobtained in a data-dependent acquisition (DDA) mode inwhich the three multiple-charged (12, 13, 14) peaks withthe highest intensity in each MS scan were chosen for CID.Collision energies were set at 10 V and 30 V, respectively,during the MS and MS/MS scans.

Mass spectra were processed using MassLynx 4.0 softwareand proteins were identified using Protein Global Server 1.0/2.0 software. The protein identities were further confirmed byMascot (http://www.matrixscience.com) using the MS/MSpeak lists exported from MassLynx. The non-redundant data-bases in the molecular weight range of 1000–500 000 Da andpI between 3.0 and 10.0 were used at the website of TheNational Center for Biotechnology Information (NCBI). Pro-tein modifications considered included carbamidomethyla-tion of cysteine, N-terminal acetylation, N-terminal gluta-mine to pyroglutamic acid, oxidation of methionine andphosphorylation of serine, threonine and tyrosine.

3 Results

3.1 E14 cells differentiated into two types of neurons

with different time courses

Murine ES cell line E14 is a representative of widely used,multiply passaged, pluripotent ES cell lines [45]. E14 cellsdivided vigorously and grew approximately at the rate of

14–16 h per cell division. To demonstrate that expandedE14 cells had pluripotent differentiation potentials, cells atpassage 10 were tested for neural differentiation in theabsence or presence of RA, following the protocols asdescribed previously [10, 43]. It has been shown that mid-brain dopaminergic neurons could be induced frommESCs by the stromal cell-derived inducing activity(SDIA) that accumulated on the surface of PA6 stromalcells without the use of either RA or embryoid bodies [43].On the other hand, ES cells grown on PA6 cell monolayerand supplemented with RA were shown to differentiateinto spinal progenitor cells, and majority of them even-tually into motor neurons [10]. RA is one of the mostimportant inductive signals in vertebrate ontogeny. Itsembryonic distribution correlates with neural differentia-tion and positional specification in the developmentalcentral nervous system. Dopaminergic neurons (in theabsence of RA, Fig. 1B) and motor neurons (in the pres-ence of RA, Fig. 1C) were produced and showed clearmorphological difference compared to undifferentiatedE14 cells (Fig. 1A). Neuron fibers were observed startingfrom day 5 or 6. For dopaminergic neurons, long processesof neuron fibers surrounded big neuron bodies, whereasfor motor neurons, short processes of neuron fibers wereseen between small neurons, and no big neuron bodieswere formed. Zoom-in view showed that motor neuronshave a cell body on one end, a long axon in the middle anddendrites on the other end, while dopaminergic neuronshave dendrites on both ends, connected by a long axonwith a cell body in the middle.

Figure 1. Morphology and timecourse of neural differentiationof E14 cells on PA6 feeder layerin the absence or presence ofRA. E14 cells were expanded ascontrol (A), or differentiated intoneurons in the absence ofRA (B), or in the presence of 2 mM

RA (C), on PA6 layer for 10 days.Phase-contrast images werecollected at day 10. Images arerepresentatives of three experi-ments. Magnification: (A–C)6100. Western blotting (D)showed different time course ofneuronal marker Tuj1 relative toa-tubulin and actin during thedifferentiation in the absence orpresence of RA. Total proteinloaded per lane was 15 mg andtotal actin was used as the load-ing control.



To further confirm neural differentiation of E14 cells, wecarried out Western blotting (Fig. 1D) and immuno-fluorescence staining (Fig. 2) with anti-Tuj1 antibody, whichrecognizes the neuronal marker Tuj1, and anti-TH antibody,which recognizes dopaminergic neuron maker TH. Day 10neurons differentiated from E14 cells showed positive Tuj1staining in the absence or presence of RA (Fig. 2C, D),whereas undifferentiated E14 cells (Fig. 2B) showed mini-mal (less than 10%) staining, presumably due to sponta-neous neural differentiation. We confirmed that E14 cellsdifferentiated to dopaminergic neurons (TH1) on PA6 layersin the absence of RA (Fig. 2C), whereas to non-dopaminergicneurons (TH2) on PA6 layers in the presence of RA(Fig. 2D). Furthermore, Fig. 1D showed different time cour-ses for Tuj1 expression during neural differentiation into twotypes of neurons. In RA-treated samples (motor neurons) theexpression of Tuj1 started to plateau at around day 6, while

the buildup of Tuj1 continued until day 10 in the absence ofRA (dopaminergic neurons). In contrast, the expression ofa-tubulin stayed relatively constant for RA-treated over 10 days,while in the absence of RA it increased significantly at thelater stage (days 6–10) of differentiation (Fig. 1D). Howeverno significant morphological changes were observed fromdays 6 to 10 in either case.

3.2 Global protein expression was significantly

regulated during neural differentiation

Although a large amount of work has been carried out tocompare the transcriptome of mESCs and neurons usingmicroarray, few studies directly compared the proteome ofES cells and neurons, particularly in the absence of RA. Guoet al. [41] have compared the proteomes of mESCs and RA-induced early-stage neurons. In this study, we profiled the

Figure 2. Immunostaining of Tuj1 and TH in undifferentiated E14 cells and neurons differentiated on PA6 feeder layer in the absence orpresence of RA. (A) Undifferentiated E14 cells stained with secondary antibodies only and indicated as the background. (B) undifferentiatedE14 cells; (C) differentiated neurons in the absence of RA; (D) differentiated neurons in the presence of RA. (B–D) Double-staining withmouse monoclonal anti-Tuj1 and rabbit polyclonal anti-TH, followed by secondary goat anti-mouse antibody conjugated with Alexa-488(green), and goat anti-rabbit antibody conjugated with Alexa-594 (red). DNA was stained with DAPI (blue). Magnification: (A, B) 6400, (C,D) 6100.



proteome changes of E14 cells during differentiation intodopaminergic neurons. The proteins in the cell lysates ofundifferentiated E14 or neurons on day 10 were separated by2-DE, followed by silver staining. Low-density proteomemaps were generated. A representative 2-D reference map(Fig. 3) for dopaminergic neurons shows up- and down-regulated protein spots (labeled with numbers) as comparedto E14. About 1200 protein spots were resolved and identifiedwith high confidence (.95%) from 50 mg of total cell lysates.Overall, 23 protein spots were found to be consistently up- ordownregulated by over 2-fold in triplicate experiments afterE14 differentiation for 10 days. The location of each spot islabeled with a number and an arrow indicating up-(upward)or down-(downward) regulation after differentiation.

All 23 spots were identified successfully with high con-fidence using in-gel trypsin digestion followed by MS/MS asdescribed in Sect. 2. For all proteins identified, two searchengines (ProteinLynx and MASCOT) gave the same proteinhits with high confident scores and at least two peptidessequenced with good MS/MS spectra (Table 1). The pre-dicted molecular weights and pIs of unmodified proteinsusing the Z3 program agreed well with their theoreticalvalues (610%). The identity of the proteins along with theirfold changes and major functions are listed in Table 1.Interestingly, majority (74%) of the proteins are upregulated,

and they are involved in either protein synthesis (e.g., calre-ticulin and ERp29) or actin remodeling (e.g., actin-relatedprotein 3 and heat shock 27-kDa protein). Multiple isoformsof a-tubulin (spots 2358 and 2516) were significantly upre-gulated with an average of 3-fold, consistent with the West-ern blotting data (Figs. 1D, 4). Tubulin, a heterodimer whichconsists of a- and b-tubulin, is the major building block ofmicrotubules. For downregulated proteins in neurons mostof them are metabolic enzymes such as aldose reductase(spot 1050), uridine phosphorylase 1 (spot 1074) and gluta-thione S-transferase (spot 1202), indicating active metabo-lism in ES cells during differentiation (hence a higherabundance than in neurons). Ran GTPase (spot 1080), aGTP-binding protein involved in nucleocytoplasmic trans-port, chromatin condensation and control of cell cycle, is alsodownregulated (0.50-fold). Among the regulated proteinswere three classes of Ca21-related proteins: calreticulin(spot 1368), pyruvate dehydrogenase E1 and E2 subunits(spot 1308 and 1578), and TCTP (spot 1064). TCTP wasdownregulated (0.23 fold), while calreticulin and pyruvatedehydrogenase E1/E2 were upregulated (5.3-, 2.6- and 3.8-fold, respectively) after differentiation. TCTP is a growth-related Ca21- and microtubule-binding protein. The pyruvatedehydrogenase complex contains multiple copies of threeenzymatic components: pyruvate dehydrogenase (E1), dihy-

Figure 3. A 2-D reference mapfor dopaminergic neuronsshowing up- and down-regulated protein spots in com-parison to undifferentiatedE14 cells. Cells were lysed and50 mg of total protein lysateswere subjected to 2-DE, fol-lowed by silver staining andimage analysis. Results werequantified from three sets of2-DE. A representative 2-DE gelafter silver staining for neuronsis shown here. IEF (pH 3–10 non-linear gradient) is in the hor-izontal direction. PAGE(10% gel) is in the vertical direc-tion. Upward arrows indicateupregulation, and downwardarrows indicate down-regulation. The spots of interestwere excised from the gels anddigested with trypsin. Theresulting peptides were used forLC/MS/MS analysis. The identi-fied proteins are listed inTable 1.



Table 1. Differentially expressed proteins identified after E14 cells differentiated to dopaminergic neurons

Spotno.

n-fold

p value(t-test)

Protein name No. ofpeptidesequenced

Sequencecoverage

Swiss-Protaccessionno.

Molecularmass(kDa)

pI Major functionsa)

1458 6.3 0.0060 Isocitrate dehydrogenase[NAD], subunit alpha

7 22% P50213 40.0 6.5 Catalyzes conversion ofIsocitrate to oxoglutarate

1368 5.4 0.032 Calreticulin 12 31% P14211 48.0 4.3 Molecular calcium bindingchaperone

2176 4.8 0.010 Endoplasmic reticulumprotein ERp29

2 9% P57759 28.8 5.9 Plays an important role in theprocessing of secretoryproteins within the ER

1698 4.1 0.0083 Peroxiredoxin 4 3 13% O08807 31.1 6.7 Probably involved in redoxregulation of the cell

1578 3.8 0.032 Dihydrolipoamide acetyl-transferase component ofpyruvate dehydrogenasecomplex

4 8% P10515 65.8 5.8 E2 component of the pyruvatedehydrogenase complexthat catalyzes the overallconversion of pyruvate toacetyl-CoA and CO2

1640 3.8 0.0083 Ubiquinol-cytochrome-C reductase complexcore protein I

12 25% Q9CZ13 52.8 5.8 Part of the mitochondrialrespiratory chain and mayalso mediate formation ofthe complex betweencytochromes c and c1

1828 3.8 0.049 Triosephosphate isomerase 9 59% P17751 26.6 7.1 Catalyzes conversion of d-gly-ceraldehyde 3-phosphate toglycerone phosphate

1600 3.6 0.0098 Actin-related protein 3 10 33% Q99JY9 47.4 5.6 Part of a complex implicated inthe control of actinpolymerization in cells

2358b) 3.6 0.024 Tubulin alpha-2 chain 3 10% P05213 50.2 5.0 Major constituent ofmicrotubules

Tubulin alpha-3/alpha-7 chain

P05214 50.0 5.0 Major constituent ofmicrotubules

1668b) 3.1 0.050 60-kDa heat shock protein 5 11% P63038 61.0 5.9 Implicated in mitochondrialprotein import andmacromolecular assembly

Hydroxymethylglutaryl-CoAsynthase

2 4% P17425 57.4 5.6 Condenses acetyl-CoA withacetoacetyl-CoA to formHMG-CoA

FK506-binding protein 4 2 5% P30416 51.4 5.6 May have a rotamase activityand play a role in the intra-cellular trafficking of hetero-oligomeric forms of steroidhormone receptors

1218 3.0 0.012 Protein disulfide-isomerase A3

15 34% P27773 56.6 6.0 Catalyzes the rearrangementof both intrachain and inter-chain disulfide bonds inproteins to form the nativestructures

2516b) 2.8 0.00038 Tubulin alpha-6 chain 10 34% P05216 50.0 5.0 Major constituent ofmicrotubules

Tubulin alpha-3/alpha-7 chain

P05214 50.0 5.0 Major constituent ofmicrotubules



Table 1. Continued

Spotno.

n-fold

p value(t-test)


Sequencecoverage


Molecularmass(kDa)


1922 2.8 0.0009 Electron transferflavoprotein alpha-subunit

9 41% Q99LC5 35.0 8.6 Transfers the electrons to themain mitochondrial re-spiratory chain via ETF-ubi-quinone oxidoreductase(ETF dehydrogenase)

1308 2.6 0.010 Pyruvate dehydrogenase E1component beta subunit

6 19% P49432 38.9 5.9 E1 component of the pyruvatedehydrogenase complexthat catalyzes the overallconversion of pyruvate toacetyl-CoA and CO2

1168 2.6 0.013 Heat shock 27 kDa protein 7 34% P14602 23.0 6.1 Involved in stress resistanceand actin organization

1662 2.2 0.030 Protein disulfide-iso-merase A3 (probablyphosphorylated)

18 42% P27773 56.6 6.0 Catalyzes the rearrangementof both intrachain and inter-chain disulfide bonds inproteins to form the nativestructures

1080 0.5 0.021 Ran GTPase 8 44% P62827 24.4 7.0 GTP-binding and involved innucleocytoplasmic trans-port and chromatin conden-sation and control of cellcycle

1050 0.4 0.032 Aldose reductase 15 47% P45376 35.6 6.8 Catalyzes the NADPH-depend-ent reduction of a wide va-riety of carbonyl-containingcompounds to their corre-sponding alcohols with abroad range of catalyticefficiencies

1132b) 0.3 0.031 Inosine-5’-monophosphatedehydrogenase 2

7 19% P24547 55.8 6.8 Rate limiting enzyme in the denovo synthesis of guaninenucleotides and therefore isinvolved in the regulation ofcell growth. It may also havea role in the development ofmalignancy and the growthprogression of some tumors

ATP synthase alpha chain 3 7% Q03265 59.8 9.2 Produces ATP from ADP in thepresence of a protongradient across themembrane. The alpha chainis a regulatory subunit

1074 0.3 0.0039 Uridine phosphorylase 1 6 30% P52624 34.1 6.1 Catalyzes the reversible phos-phorylytic cleavage ofuridine and deoxyuridine touracil and ribose- or deoxy-ribose-1-phosphate

1202 0.3 0.0073 GlutathioneS-transferase 5.7

4 18% P24472 25.6 6.8 Conjugates reduced glutathi-one to a wide number ofexogenous and endogenoushydrophobic electrophiles



Table 1. Continued

Spotno.

n-fold

p value(t-test)


Sequencecoverage


Molecularmass(kDa)


1064 0.2 0.039 Translationally controlledtumor protein

9 37% P63028 19.5 4.8 Calcium binding, microtubulesstabilization and regulationof apoptosis

3143 Unique 0.050 Actin, fetal skeletal/adultcardiac muscle – mouse(fragment)

2 8% Q61275 39.2 5.8 Ubiquitously expressed andmay involved in cell motility

a) Adapted from http://us.expasy.org/sprot/-b) For spots 2358, 1668, 2516, and 1132, more than one protein was identified from the digested peptides, suggesting that these proteins

co-migrated in the gels.

drolipoamide acetyltransferase (E2) and lipoamide dehy-drogenase (E3). It catalyzes the overall conversion of pyru-vate to acetyl-CoA and CO2. The complex is located in themitochondrial matrix and responds to Ca21 by increasing therate of ATP synthesis [46]. Calreticulin is both an ER Ca21

storage protein and a chaperone involved in protein synthe-sis and folding [47].

3.3 RA coordinated the decrease of TCTP and

increase of Æ-tubulin in neurons

To verify the regulation in protein expression obtained byproteomic profiling, and to examine the interdependence ofa-tubulin and TCTP expression (see discussion), we investi-gated these two proteins in total lysates from E14, dopami-nergic (no RA) and motor neurons (1RA) using Westernblotting. Total actin was probed as the loading control, andTuj1 was probed as the neuron marker. As shown in Fig. 4,Tuj1 expression in undifferentiated E14 cells was barelydetected, indicating minimal spontaneous neural differ-entiation under our cell culture conditions. TCTP was

Figure 4. TCTP expression in undifferentiated E14 cells and neu-rons. The conditions for Western blotting were the same as inFig. 1. Total protein loaded per lane was 15 mg and total actin wasused as the loading control.

downregulated by 4–5-fold while a-tubulin was upregulatedby 2–3-fold in dopaminergic neurons (no RA) compared toE14, consistent with the results from 2-DE (Table 1, Fig. 1D).Interestingly, the addition of RA further reduced TCTPexpression, while alleviating the increase of a-tubulinexpression in the resulted motor neurons (1RA). Theseresults confirmed the regulation of TCTP and a-tubulin atprotein level during neural differentiation, and suggestedthat RA may coordinate the increase of a-tubulin and Tuj1expression and the decrease of TCTP expression. Our resultsimplicated the cross-talk between Ca21 and RA signalingpathways, which has been previously demonstrated in leu-kemia HL60 cells [48].

3.4 Neural differentiation was dependent on

extracellular Ca21 concentration, while TCTP

expression was independent

We observed that TCTP expression was decreased, while a-tubulin expression was increased, after E14 cells differ-entiated into neurons. It was shown that Ca21 and tubulinbind TCTP at the same domain [49]. To directly determinewhether neural differentiation, neuron survival, and expres-sion of TCTP and a-tubulin were dependent on Ca21 con-centration in the media, we differentiated E14 cells undervarious Ca21 concentrations. We used EGTA to reduce Ca21

concentration because the relatively low affinity of EDTA forCa21 compared to Mg21 and Mn21 makes EGTA a betterchoice to study Ca21 without influencing other Mg21 orMn21 enzyme activities. As shown in Fig. 5A, the number ofneurons generated was greatly impacted by Ca21 concentra-tion in the culture media, with the apparent optimal value ataround 2 mM. No survival was observed below 0.6 mM Ca21

resulting from addition of 1.7 mM EGTA. However, neuronsgenerated under different Ca21 conditions (1.1, 2.3, 3.3 and4.3 mM, respectively) did not show any obvious morphologi-cal changes (Fig. 5B).



Figure 5. Ca21-dependent neural differentiation. Number of neuron colonies were averaged through triplicateexperiments and plotted with different Ca21 concentration in the differentiation media (A). Error bar indicates SD.Phase-contrast images (B) of neurons collected at day 10 were representatives of three experiments. Expression ofTCTP, Tuj1 and a-tubulin was shown by Western blotting (C). Total protein loaded per lane was 15 mg and total actinwas used as the loading control.

We also compared the expression of TCTP, Tuj1 and a-tubulin in day 10 neurons under three Ca21 concentrations(2.3, 4.3 and 1.1 mM). Figure 5C showed that extracellularCa21 concentration did not significantly regulate the expres-sion levels of these proteins in neurons. This is presumablydue to complex mechanisms to maintain the intracellularCa21 homeostasis within the concentration range we applied(see discussion).

4 Discussion

A major challenge in stem cell research is to elucidate themechanisms underlying its differentiation along specificlineage. We have chosen mESC differentiation along neuro-nal lineage as a model system because mESCs and hESCs

share a myriad of conserved paths that regulate self-renewaland differentiation [29, 30, 50], and elucidation of neurogen-esis will provide insights into embryo developments. In thepost-genomic era, there is an increasing need to decipher thetemporal and spatial functions and interactions of proteinsunder different physiological conditions. Transcriptomic andproteomic profiling provides a systematic and quantitativeapproach to identify unique protein markers and elucidatesignaling mechanisms during ES cell differentiation. Al-though still in its development stage, proteomic profiling ispoised to play an essential role in this endeavor. If we canidentify and validate unique protein markers and elucidateinter-connections between different cellular signaling path-ways through the proteome-wide screening, it will facilitatethe characterization of stem cell differentiation. The prote-omic profiling may elucidate connections between broad



cellular pathways that were neither apparent nor predictablethrough traditional biochemical analysis in the past. How-ever, there has been very few detailed proteomic studies onthe neural differentiation of ES cells particularly into dopa-minergic neurons derived in the absence of RA.

In this study, we have compared the global proteinexpression in mESCs and differentiated dopaminergic neu-rons. Using a proteomic approach, we have generated 2-Dreference maps for both mESCs and derived dopaminergicneurons. Based on this map, higher resolution proteomemaps of MSCs with pre-fractionated cell lysates and zoom-inpI range 2-D gels can be generated in the future. We havealso identified all the 23 spots differentially regulated duringneural differentiation and confirmed the findings for select-ed proteins using Western blotting. For example, we haveshown that a-tubulin, which is generally used as a loadingcontrol in Western blotting, was significantly upregulated inneurons. We have further demonstrated that a-tubulin wasregulated differently for different types of neurons such asdopaminergic and motor neurons, which differ both inmorphology and functionalities [4, 10, 43]. Interestingly, aprevious proteomic study also showed regulation of a-3/a-7 tubulin during early-stage differentiation of mESCs intoneural cells induced by RA [41]. This is consistent with ourresults showing that not only neuronal Tuj1 but also total a-tubulin is significantly regulated during neurogenesis. Wehave identified multiple functionally significant proteins thatare regulated during neural differentiation. For example,actin-related protein 3, part of a seven-subunit 220-kDaArp2/3 complex implicated in controlling of actin polymeri-zation in cells [51], is upregulated. We have recently demon-strated that TGF-b coordinates the increase of a-actin and thedecrease of gelsolin to promote human MSC differentiation[38]. Both Arp 2/3 complex and gelsolin actively participatein actin dynamics. A recent quantitative proteomic study onphosphotyrosine-dependent signaling networks in HeLacells showed that Arp2/3 and gelsolin have the same activa-tion profiles upon EGFR stimulation [52]. The inter-dependence of Arp 2/3 complex and gelsolin expressionsand dynamics, as well as their participation in phosphotyr-osine-dependent signaling in controlling actin remodelingduring stem cell differentiation awaits further investigation.Another interesting protein is Ran GTPase that was down-regulated in neurons. One of the major effects of growthfactor stimulation and cellular differentiation is the rearrang-ement of the actin cytoskeleton through small GTPases[53]. Ran GTPase is a GTP-binding protein involved innucleocytoplasmic transport. It is required not only for theimport of proteins into the nucleus but also for RNA export.Furthermore, it is involved in chromatin condensation andcontrol of cell cycle [54, 55]. Its higher abundance in ES cells(hence downregulation in neurons) implies its active parti-cipation in proliferation and differentiation. Persistentexpression of Ran GTPase was shown in proliferating neuraltissue, neural crest derived dorsal root ganglions and sensorypits during early development of the mouse embryo [56]. We

should point out that these changes may be ubiquitous incell differentiation. Future detailed functional studies ofthese regulated proteins in the context of stem cell differ-entiation may help elucidate cell-type-specific mechanisms.

One of the most interesting findings from this proteomicstudy is that TCTP and tubulin are significantly regulatedduring neural differentiation of ES cells, and their regulationdepends on the type of neurons generated (i.e., 1/2RA).TCTP, also known as IgE-dependent histamine-releasingfactor, is highly conserved and abundantly expressed in alleukaryotic organisms. It is a growth-related protein and itsexpression is highly regulated at both transcriptional andtranslational level and by a wide range of extracellular signals[49]. Although TCTP is described as a cytoplasmic protein, itsnuclear localization [57] and extracellular secretion [58] hasalso been reported. The 3-D structure of TCTP reveals a do-main similar to the Mss4/Dss4 family, which bind to theGDP/GTP-free form of Rab proteins [59]. TCTP has beenshown to be involved in calcium binding and microtubulesstabilization, and implicated in regulation of apoptosis, cellgrowth and cell cycle [49]. The Ca21-binding and tubulin-binding domains of TCTP were mapped to the same a-heli-cal region. Most recently, the C terminus of TCTP (aminoacids 102–172) was shown to bind to Na,K-ATPase, and thusinhibit Na,K-ATPase activity in HeLa cells [60]. Na,K-ATPaseis the ion pump that controls signal transductions in neu-rons. A previous study has established different expression,activity and distribution patterns of Na,K-ATPase isoforms(a, b, g) during in vitro neuronal differentiation of mESCs[61]. The upregulation of tubulin and downregulation ofTCTP may work coordinately to promote microtubuledynamics and intracellular Ca21 transport. This is consistentwith increased Na,K-ATPase activity in neurons. In addition,mitotic polo-like kinases (Plk) phosphorylate TCTP on twoserine residues in vitro and in vivo. The phosphorylationdecreases the microtubule-stabilizing activity of TCTP andincreases microtubule dynamics occurring after metaphase[62]. Another recent study has identified TCTP as a target fortumor reversion and showed that drugs to decrease TCTPexpression could kill tumor cells [63]. Since microtubuledevelopment is important in tumorigenesis as well as neu-rogenesis, we postulate that changes in expression andphosphorylation of TCTP play important roles during ES celldifferentiation to neuron. We have not yet identified thephosphorylated forms of TCTP, presumably due to its lowabundance and/or the modification being transient. Weshowed that TCTP was downregulated after neural differ-entiation, and was further downregulated in motor neuronscompared to dopaminergic ones. As mentioned before, RAcan drive ES cells to differentiate into motor-type neurons onPA6 feeder layer [10]. It has been established that there is across-talk between RA receptor and calcium-dependent sig-naling in leukemia cells HL60 and NB4, in which the all-trans RA-induced cell differentiation is either preceded bydownregulation of autonomous generation of inositol lipid-derived Ca21 [64], or enhanced by inhibition of calcium



accumulation into the ER [48]. Since TCTP binds Ca21, it ispossible that the regulation of TCTP expression and RA sig-naling are interconnected. Different TCTP expression levelscould be correlated to the levels of Na,K-ATPase activities indifferent types of neurons. Therefore, TCTP may playimportant roles in neural differentiation through modulat-ing its binding to Ca21, tubulin and Na,K-ATPase. In agree-ment with this hypothesis, a recent genome-wide screen forphenotypes of C. elegans using RNAi established that theknockdown of TCTP results in a slow-growth phenotype [65].Works to investigate the functional consequences in neuraldifferentiation after siRNA knockdown of TCTP and to dis-sect the interconnection between the Ca21 and RA signalingin ES cells are ongoing.

We also observed the regulation of other classes of Ca21

binding proteins such as calreticulin and pyruvate dehy-drogenase complex during neural differentiation. Thisimplies that Ca21 plays an important role in neural differ-entiation, although our Ca21-dependent experiments did notshow apparent regulation of TCTP expression in neurons inresponse to the concentration of extracellular Ca21. Intracel-lular Ca21 homeostasis is maintained through a complexinterplay between Ca21 influx and efflux through ion chan-nels, Ca21 buffering and internal Ca21 storage with Ca21

binding proteins. Under physiological conditions, these pro-cesses enable multiple Ca21-regulated signaling cascades tooccur independently within the same cell. Ca21 homeostasisgoverns a multitude of cellular processes, including cellgrowth, differentiation, synaptic activity, neurotransmitterrelease, and neuronal degeneration. Calcium regulates geneexpression through modulation of transcription factors suchas CREB, CBP, NFATc1-4 and DREAM [66]. It also mediatespost-translational modifications including phosphorylation-dephosphorylation through modulation of protein kinases,phosphatases, or calcium-sensitive adenylate cyclases [67].For example, it was shown that an activity-dependent tran-scription of pituitary adenylate cyclases activating polypep-tide (PACAP) is induced by the Ca21 signals in neurons [68].Calcium regulates intercellular communication through gapjunctions and triggers the terminal differentiation programsof cells. Dysregulation of Ca21 homeostasis may cause over-activating Ca21-dependent proteases, lipases, kinases, phos-phatases, and endonucleases. This can lead to apoptosis andcell death, and eventually aging, cancer, neurodegenerationand other brain disorders [66, 69–74]. Especially in the ER,disruption of the Ca21 homeostasis triggers ER stressresponses, which result in neurodegeneration and death ofneuronal cells. It has been demonstrated that dyshomeos-tasis of Ca21 is a common underlying factor in brain pathol-ogies including Alzheimer’s and other neurodegenerativediseases [75–77]. In our system, Ca21 presumably mediatesneural differentiation through modulating the communica-tion between PA6 feeder layer and E14 cells, as well as be-tween E14 cells through gap junctions [72]. We have observedsignificant reduction of neuron numbers under stressedCa21 concentrations (Fig. 5A). However, details of Ca21

homeostasis and signaling, its influence on neuron survival,and regulation of particular Ca21-related proteins duringES cell differentiation remain to be determined.

Finally, the findings from this study may be applicable toneural differentiation of hESCs. Future work to expand cur-rent proteomic and biochemical studies to hESs and to carryout in vivo studies will have significant impacts on stem cellbiology and engineering.

We thank Dr. Yoichi ShinKai (Kyoto University) for undif-ferentiated E14 cells, and Drs. David J. Chen, Priscilla K. Cooperand Terumi Kohwi-Shigematsu for their support. This work wassupported by the Office of Science (BER) Low Dose RadiationResearch Program, U.S. Department of Energy, at the Universityof California/Lawrence Berkeley National Laboratory undercontract no. DE-AC03-76SF00098.

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