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First published online August 11, 2005
Stem Cells Vol. 23 No. 10 November 2005, pp. 1502 -1513
doi:10.1634/stemcells.2004-0307; www.StemCells.com
© 2005 AlphaMed Press

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Heat Shock 70-kDa Protein 8 Isoform 1 Is Expressed on the Surface of Human Embryonic Stem Cells and Downregulated upon Differentiation

Yeon Sung Sona,d, Jae Hyun Parka, Young Kook Kanga, Jin-Sung Parka, Hong Seo Choia, Ji Young Lima, Jeoung Eun Leec, Jung Bok Leeg, Myoung Seok Koe, Yong-Sam Kimb, Jeong-Heon Kob, Hyun Soo Yoonc, Kwang-Woong Leed, Rho Hyun Seongd, Shin Yong Moonf, Chun Jeih Ryua, Hyo Jeong Honga

a Laboratory of Antibody Engineering and
b Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejon, Korea;
c Department of Anatomy & Cell Biology, Hanyang University, Seoul, Korea;
d School of Biological Sciences, Seoul National University, Seoul, Korea.
e R&D Center, Aprogen, Inc., Daejon, Korea;
f College of Medicine, Seoul National University, Seoul, Korea;
g MizMedi Medical Research Center, Seoul, Korea

Key Words. Human embryonic stem cells • Heat shock protein 70 • Cell-surface marker

Correspondence: Chun Jeih Ryu, Ph.D., Laboratory of Antibody Engineering, Korea Research Institute of Bioscience and Biotechnology, 52, Oun-Dong, Yusong-Gu, Daejon 305-333, Republic of Korea. Telephone: 82-42-860-4249; Fax: 82-42-860-4597; e-mail: cjryu{at}kribb.re.kr; and Hyo Jeong Hong, Ph.D., Laboratory of Antibody Engineering, Korea Research Institute of Bioscience and Biotechnology, 52, Oun-Dong, Yusong-Gu, Daejon 305-333, Republic of Korea. Telephone: 82-42-860-4122; Fax: 82-42-860-4597; e-mail: hjhong{at}kribb.re.kr


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cell-surface markers used routinely to define the undifferentiated state and pluripotency of human embryonic stem cells (hESCs) are those used in mouse embryonic stem cells (mESCs) because of a lack of markers directly originated from hESC itself. To identify more hESC-specific cell-surface markers, we generated a panel of monoclonal antibodies (MAbs) by immunizing the irradiated cell clumps of hESC line Miz-hES1, and selected 26 MAbs that were able to bind to Miz-hES1 cells but not to mESCs, mouse embryonic fibroblast cells, and STO cells. Most antibodies did not bind to human neural progenitor cells derived from the Miz-hES1 cells, either. Of these, MAb 20-202S (IgG1, {kappa}) immunoprecipitated a cell-surface protein of 72-kDa from the lysate of biotin-labeled Miz-hES1 cells, which was identified to be heat shock 70-kDa protein 8 isoform 1 (HSPA8) by quadrupole time-of-flight tandem mass spectrometry. Immunocytochemical analyses proved that the HSPA8 protein was also present on the surface of hESC lines Miz-hES4, Miz-hES6, and HSF6. Two-color flow cytometric analysis of Miz-hES1 and HSF6 showed the coexpression of the HSPA8 protein with other hESC markers such as stage-specific embryonic antigen 3 (SSEA3), SSEA4, TRA-1-60, and TRA-1-81. Flow cytometric and Western blot analyses using various cells showed that MAb 20-202S specifically bound to the HSPA8 protein on the surface of Miz-hES1, contrary to other anti-HSP70 antibodies examined. Furthermore, the surface expression of the HSPA8 protein on Miz-hES1 was markedly downregulated upon differentiation. These data indicate that a novel MAb 20-202S recognizes the HSPA8 protein on the surface of hESCs and suggest that the HSPA8 protein is a putative cell-surface marker for undifferentiated hESCs.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human embryonic stem cells (hESCs) derived from the inner cell mass of preimplantation embryos have been shown to give rise to stable pluripotent cell lines that appear to proliferate infinitely under specific culture conditions [1–3]. They are able to differentiate into a wide range of cell types in vitro and form teratomas containing derivatives of all three embryonic germ layers in immune-deficient mice. They also share some features in common with mouse embryonic stem cells (mESCs), for example, high level expression of alkaline phosphatase and stem cell transcription factor, Oct-4. Nevertheless, hESCs show marked differences from their mouse counterparts. In addition to morphological differences, hESCs and mESCs differ in growth conditions and cytokine requirements to maintain self-renewal and pluripotency in culture. Actually, the number of human stemness genes shared by mESCs appeared by recent microarray analysis to be quite low [4, 5].

The cell-surface markers used routinely to identify undifferentiated hESCs were initially characterized as markers for mESCs, mouse embryonic carcinomas (ECs), or human EC cells. Stage-specific embryonic antigen 1 (SSEA1), SSEA3, and SSEA4 are widely used as cell-surface markers to define both human and mouse ESCs [6, 7]. SSEA1 is expressed on undifferentiated mESCs or differentiated hESCs, whereas SSEA3 and SSEA4 are expressed on undifferentiated hESCs but not on undifferentiated mESCs [8]. Two human EC cell antigens, TRA-1-60 and TRA-1-81, are also used to mark undifferentiated hESCs [9]. However, the epitopes of the surface antigens are carried by carbohydrates, and the exact functions of the carbohydrate antigens in ESCs are not known [10, 11]. In addition, the definitive phenotype of undifferentiated hESCs has not yet been identified.

The systematic identification and characterization of cell-surface molecules of hESCs can be of practical use in identification and analysis of specific cell population and in purification of cells for cell transplantation therapy. Besides, as cell-surface molecules play important roles in regulating the development and differentiation of hESCs, determination of the molecules involved in maintaining the undifferentiated and pluripotent state of hESCs and elucidation of the mechanism by which information is transferred from the surface of a cell into metabolic reactions are major goals of stem cell researches. To approach these issues, the cell-surface molecules, as well as the genes that control their synthesis, must be biochemically defined, and defined probes should be available to make detection, localization, and functional studies possible. Accordingly, it would be very useful to generate monoclonal antibodies (MAbs) specific to the cell-surface molecules because of the high specificity of MAbs.

In this study, we generated a panel of MAbs that specifically recognize the cell-surface antigens of hESCs. By using an MAb 20-202S, we found that heat shock 70-kDa protein 8 isoform 1 (HSPA8) was expressed on the cell surface of hESCs and down-regulated upon differentiation. The results suggest that the HSPA8 protein is a novel cell-surface marker for undifferentiated hESCs.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
hESC lines (Miz-hES1, Miz-hES4, Miz-hES6, and HSF6) were cultured as described previously [3, 12, 13]. Briefly, cells were cultured on a feeder cell layer of irradiated mouse embryonic fibroblast (MEF) in Dulbecco’s modified Eagle’s medium (DMEM)/ F12 medium (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), supplemented with 20% serum replacement (Invitrogen), 0.1 mM 2-mercaptoethanol, 1% nonessential amino acids, 1 mM glutamine, 100 U/ml penicillin G, 100 µg/ml streptomycin, and 4 ng/ml basic fibroblast growth factor (PeproTech Inc., Rocky Hill, NJ, http://www.peprotech.com). The hESC colonies were subcultured every 5 days by detaching the colonies with 1 mg/ml collagenase IV (Sigma, St. Louis, http://www.sigmaaldrich.com). Differentiation was induced by incorporating all-trans-retinoic acid (RA; Sigma) (10–5 M) into the medium as described for human EC cells and hESCs [14, 15]. Characterization of the cells was also carried out by the methods described previously [3].

Human embryoid body (EB) and neural progenitor cells (named hNP1) were prepared from Miz-hES1 by a previously reported method with minor modifications [16, 17]. mESC lines J1 and TC-1 (University of Connecticut Health Center, Farmington, CT, http://www.uchc.edu) were cultured on a feeder cell layer of irradiated MEF in DMEM (Invitrogen), supplemented with 15% fetal bovine serum (Invitrogen), 0.1 mM 2-mercaptoethanol, 0.1 mM nonessential aminoacids, 1 mM glutamine, 100 U/ml penicillin G, 100 µg/ml streptomycin, and 500 U/ml leukemia inhibitory factor[18]. Cancer celllines Choi-CK (cholangiocarcinoma), SCK (sarcomatoid cholangiocarcinoma), SH-J1 (sarcomatoid cholangiocarcinoma), HeLa, and HepG2 were cultured as described elsewhere [19, 20]. STO and NTERA2 cL.D1 (human EC) cells were purchased from the American Type Culture Collection (Manassas, VA, http://www.atcc.org) and cultured as described previously [3, 21]. Human peripheral blood lymphocytes (PBLs) were isolated using Ficoll density gradient centrifugation.

Generation of MAbs
BALB/c mice were cared for by the institutional guidelines of the Korea Research Institute of Bioscience and Biotechnology and were immunized triweekly by the intraperitoneal injection of approximately 2 x 106 irradiated Miz-hES1 cells. Before the immunization, each colony was divided into smaller pieces and suspended in phosphate buffered saline (PBS; pH 7.4). The mice were eye-bled 7 days after the fourth injection, and the sera were tested for the reactivity to Miz-hES1 cells by flow cytometry. Three days after the last immunization, the splenic lymphocytes were fused with the NS-1 mouse myeloma cell line, and hybridomas were selected in RPMI 1640 medium (Invitrogen) containing 10% fetal bovine serum and the HAT component (Sigma), as described previously [22]. The culture supernatants of hybridomas were tested for the reactivity for Miz-hES1 by flow cytometry, and each positive clone was isolated after two steps of subcloning. Isotype analysis of each antibody was carried out according to the supplier’s protocol (mouse immunoglobulin [Ig] isotyping ELISA [enzyme-linked immunosorbent assay] kit; BD Pharmingen, San Diego, http://www.bdbiosciences.com).

Purification and Biotinylation of MAbs
MAbs were purified from the culture supernatants of hybridomas by Protein G-Sepharose (Sigma) column chromatography as described previously [23]. Biotinylation of the purified antibody was carried out by ECL protein biotinylation module (Amersham Biosciences, Seoul, Korea, http://www.amershambiosciences.com) according to the supplier’s protocol.

Flow Cytometry
Single-cell suspensions for flow cytometry were made from the undifferentiated Miz-hES1 as described previously [16]. Cell colonies were treated with collagenase IV for 1 hour in normal growth medium and treated with cell dissociation buffer (Invitrogen) for 20 minutes in a 37°C incubator. Cells were dissociated by gentle pipetting and filtered through a 40-µm cell strainer. The dissociated cells were immediately resuspended at approximately 2 x 105 cells per ml in PBA (1% bovine serum albumin, 0.02% NaN3 in PBS) and incubated with each MAb or anti-SSEA1, anti-SSEA3, or anti-SSEA4 antibody (Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/~dshbwww) for 30 minutes at 4°C. After washing twice with PBA, the cells were incubated with fluorescein isothiocyanate (FITC)–conjugated anti-mouse Ig (BD Pharmingen) for 30 minutes at 4°C. Propidium iodide (PI)–negative cells were analyzed for the antibody binding using FACSCalibur (BD Biosciences - Immunocytometry Systems, San Jose, CA, http://www.bdbiosciences.com/immunocytometry_systems) and Cell Quest software (BD Biosciences - Immunocytometry Systems). Antibodies to neural cell adhesion molecule (NCAM; Sigma) or CD133 (R&D Systems, Minneapolis, http://www.rndsytems.com) were included in flow cytometric analyses of human neural progenitor cells.

For multiple color flow cytometric analyses, cells were incubated with appropriate primary antibodies, phycoerythrin (PE)–conjugated antibody, biotin-conjugated antibody, or appropriate isotype-matched controls for 30 minutes at 4°C. Primary antibodies used were anti-SSEA3, anti–TRA-1-60 (Chemicon, Temecula, CA, http://www.chemicon.com), and anti–TRA-1-81 (Chemicon). Biotin-conjugated antibody 20-202S was also used as a primary antibody. PE-conjugated anti-SSEA4 antibody (R&D Systems) was directly used without secondary antibody conjugate. Cells were further incubated with streptavidin-FITC (Sigma) and either PE-conjugated with anti-rat IgM or anti-mouse IgM (BD Pharmingen) depending on the combination of primary antibodies. After washing twice with PBA, PI-negative cells were analyzed for the antibody binding using FACSCalibur and Cell Quest software.

Immunocytochemistry
For immunocytochemistry, the hESCs on a four-well plate were washed with Ca2+- and Mg2+-PBS and then fixed in 4% paraformaldehyde for 30 minutes at 4°C. The plate was blocked with 1.5% horse serum in Ca2+- and Mg2+-PBS for 20 minutes. The plate was incubated with each MAb diluted with blocking solution (1.5% horse serum in PBS) for 1 hour at room temperature and washed five times. The bound primary antibody was detected with biotin-labeled secondary antibody for 1 hour at room temperature. After washing five times, the plate was incubated with Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) containing a complex of avidin and horse radish peroxidase (HRP). Then, positive colonies were visualized by DAB substrate kit (Vector Laboratories).

For immunofluorescence staining, hESCs were cultured on 0.1% gelatin-coated coverslips plated with MEFs, and Choi-CK cells were cultured on 0.1% gelatin-coated coverslips. hESCs were incubated with biotin-labeled 20-202S (12 µg/ml) and/or anti–TRA-1-60 (1:100; Chemicon) for 1 hour at 4°C in the presence of normal growth medium. After washing with Ca2+- and Mg2+-PBS, cells were fixed in 4% paraformaldehyde for 30 minutes at 4°C. Then, cells were incubated with streptavidin-FITC (1:100; Sigma) and anti-mouse IgM conjugated to Texas Red (1:100; Vector Laboratories) for 1 hour at room temperature. In the case of Choi-CK cells, cells were incubated with 20-202S (12 µg/ml) and then incubated with anti-mouse IgG conjugated to Cy3 (1:400; Jackson ImmunoResearch, West Grove, PA, http://www.jacksonimmuno.com). After washing six times with Ca2+- and Mg2+-PBS, cells were stained with 4,6 diamidino-2-phenylindole (0.25 µg/ml) in PBS containing 0.1% Triton X-100. After washing four times with Ca2+- and Mg2+-PBS, cells were mounted with Vectashield (Vector Laboratories). Fluorescence images were visualized through a Zeiss 510LSM META laser-scanning microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com).

Cell-Surface Biotinylation and Immunoprecipitation
Cell-surface biotinylation was performed according to the supplier’s protocol with EZ-Link Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL, http://www.piercenet.com). Biotin-labeled cells were treated with lysis buffer (25 mM Tris-HCl [pH 7.5], 250 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 2 µg/ml aprotinin, 100 µg/ ml phenylmethylsulfonyl fluoride, and 5 µg/ml leupeptin) at 4°C for 20 minutes. Nuclei were removed by centrifugation, and the cell lysates were stored at –70°C before use. The protein in the cell lysates was quantitated using a Bradford protein assay kit (Bio-Rad Laboratories, Hercules, CA, http://www.bio-rad.com). To remove the cellular proteins that nonspecifically bind to Protein G plus-Sepharose (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com), the cell lysate from approximately 1 x 107 cells was incubated with 20 µl of Protein G plus-Sepharose at 4°C for 2 hours, and the beads were recovered and extensively washed with lysis buffer to use as a negative control for the binding experiment. To immunoprecipitate the antigen recognized by an MAb, the pre-cleared lysate was incubated with approximately 1 µg of monoclonal antibody at 4°C overnight and further incubated with Protein G plus-Sepharose as described above. The beads were extensively washed with lysis buffer, and the bound proteins were eluted from the beads by heating at 100°C for 5 minutes. The precleared lysate and eluted proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 7% poly-acrylamide gel under denaturing conditions and transferred to a nitrocellulose membrane for Western blotting. The membrane was immersed in 5% skim milk in PBST (PBS containing 0.1% Tween 20) at room temperature for 1 hour. After two rinses with PBST, the membrane was incubated with HRP-conjugated streptavidin (1:1,500; Amersham Biosciences) at room temperature for 1 hour. After extensive washing, the biotinylated proteins were visualized by ECL detection reagent (Amersham Biosciences).

Target Identification of MAb 20-202S
To characterize the antigen recognized by an MAb 20-202S, the cell lysates were prepared from approximately 1 x 108 Miz-hES1 and Choi-CK cells [19] and subjected to immunoprecipitation as described above. The protein immunoprecipitated by 20-202S was resolved on SDS-PAGE gel and stained with Coo-massie G250 (Bio-Rad Laboratories) according to the supplier’s protocol. The protein band corresponding to approximately 72-kDa was excised, washed, and completely destained with 30% methanol. Then, the gel pieces were dehydrated in 100% ace-tonitrile for 10 minutes and dried for 30 minutes in a vacuum centrifuge. The protein was digested with modified porcine trypsin (Promega, Madison, WI, http://www.promega.com) in 50 mM ammonium bicarbonate for 16 hours at 37°C. The peptides extracted from the gel were concentrated using C18ZipTips (Millipore, Bedford, MA, http://www.millipore.com) and eluted with 50% (v/v) acetonitrile:water. Mass spectrometric analyses were performed using a quadrupole time-of-flight (Q-TOF) mass spectrometer (Micromass Ltd., Hertfordshire, U.K., http://www.waters.com) equipped with a nano-ESI source. The peptide solution was sprayed at a potential of approximately 2 kV, leading to the production of molecular ions. To obtain fragment ions, the collision energy was increased to 30 eV from 10 eV for collision-induced dissociation experiments. Argon was introduced as a collision gas at a pressure of 10 psi. Masslynx (Micromass) program was used for data processing, and the MS-Tag search program (Web-based: prospector.ucsf.edu/ucs-fhtml4.0/mstagfd.htm) was used to identify proteins based on the sequence of peptide fragments.

Western Blot Analysis
Cell lysates were prepared as described above without biotinylation and subjected to Western blotting with 20-202S or various anti-HSP70 antibodies such as W27 (Santa Cruz Biotechnology), SPA810 (Stressgen, Victoria, British Columbia, Canada, http://www.stressgen.com), 5G10 (BD Pharmingen), or SPA820 (Stressgen).

To separate associated peptide from HSP-peptide complex, the cell lysates were incubated with 2.5 mM ATP and 10 mM MgCl2 at 37°C for 30 minutes before SDS-PAGE. The proteins were transferred to Protran nitrocellulose membrane (Whatman, Middlesex, U.K., http://www.whatman.com). The membranes were then incubated with each MAb followed by HRP-conjugated secondary anti-body (Santa Cruz Biotechnology). Finally, the immunoblots were visualized using ECL detection reagent (Amersham Biosciences).

Transfection of HSPA8 Gene
Plasmid DNA containing the HSPA8 gene (Clone ID: hMU003835. pCMV-SPORT6/HSPA8) was purchased from the 21 Frontier of Human Genebank of the Korea Research Institute of Bioscience and Biotechnology, Daejon, Korea. The plasmid DNA was transfected into cholangiocarcinoma cell line SCK [19] with Lipofectamine (Invitrogen) according to the supplier’s protocol, and the HSPA8 protein was transiently expressed for 48 hours. The cell extract was prepared as described previously and subjected to Western blot analysis.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To generate the MAbs that specifically bind to the cell-surface markers for hESCs, mice were immunized with the Miz-hES1 cells. Out of a total of 252 hybridomas selected, 51 hybridomas were shown to produce MAbs binding to Miz-hES1 in flow cytometric analysis. Among them, 26 clones stably produced Miz-hES1–specific antibodies after the second or third subcloning (Fig. 1AGo). Of these, nine were classified into isotype IgG and 17 were IgM (Table 1Go). They showed various patterns of binding in flow cytometric analysis, although IgM antibodies showed higher binding activity compared with IgG MAbs (Fig. 1AGo). The Miz-hES1–specific binding by these MAbs was also confirmed by immunocytochemistry (Fig. 1BGo; Table 1Go). To confirm that the MAbs bind to the other hESC line, Miz-hES4 was used for immunocytochemistry. As expected, all the antibodies tested bound to Miz-hES4 (Fig. 1BGo; Table 1Go). In contrast, the MAbs did not bind to MEFs, STO cells, J1, and TC-1 (Fig. 2Go; Table 1Go). To further examine whether the MAbs bind to differentiated cells, we differentiated the Miz-hES1 into human neural progenitor cell hNP1. The hNP1 cells were positive for CD133 and NCAM, which are known as neural progenitor markers (Fig. 2Go). Among 26 MAbs, 17 MAbs did not bind to the hNP1, whereas seven MAbs bound to the hNP1 partially (+/–) or strongly (+) (Fig. 2Go; Table 1Go).



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  		Figure 1. Screening of MAbs specific to human embryonic stem cells by flow cytometry and immunocytochemistry. (A): Undifferentiated Miz-hES1 cells were stained with anti-SSEA1, anti-SSEA3, anti-SSEA4, or various MAbs followed by fluorescein isothiocyanate–conjugated anti-mouse IgM, anti-rat IgM, anti-mouse IgG, or anti-mouse Ig, respectively. Each panel represents one MAb. Uncolored populations indicate antibody staining, whereas red-colored populations indicate control staining in each panel. Miz-hES1 cells are positive for SSEA3 and SSEA4 but not for SSEA1. (B): Undifferentiated Miz-hES1 and Miz-hES4 colonies on four-well plates were stained with anti-SSEA1, anti-SSEA4, or various MAbs, and then detected with biotin-labeled anti-mouse IgM, anti-rat IgM, or anti-mouse IgG, respectively. Positive colonies were visualized by Vectastain immunostaining kit. Scale bar = 50 µm. Abbreviations: Ig, immunoglobulin; MAb, monoclonal antibody; SSEA, stage-specific embryonic antigen.

 

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  		Table 1. Characteristics of a panel of MAbs specific to hESCs
 


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  		Figure 2. Flow cytometric staining of various cells with the MAbs and FITC-conjugated anti-mouse Ig. Each thick line indicates antibody staining followed by FITC-conjugated anti-mouse Ig, whereas each red-colored population indicates FITC-conjugated anti-mouse Ig alone. Each panel represents one MAb. Antibodies against SSEA1 (black line) and SSEA4 (green line) were used for identification of mESCs, STOs, and MEFs, and antibodies against CD133 (black) and NCAM (green) for identification of hNP1s. Abbreviations: FITC, fluorescein isothiocyanate; hNP1, human neural progenitor; Ig, immunoglobulin; MAb, monoclonal antibody; MEF, mouse embryonic fibroblast; mESC, mouse embryonic stem cell; NCAM, neural cell adhesion molecule; SSEA, stage-specific embryonic antigen.

 
To identify the cell-surface antigens recognized by the MAbs, the surface proteins of the Miz-hES1 cells were biotinylated, and the biotinylated cell lysates were subjected to immunoprecipitation using some of the hESC-specific MAbs. We found that MAb 20-202S immunoprecipitated the cell-surface protein of 72-kDa, whereas MAbs 4-20, 4-35, and 4-38 did not immunoprecipitate any specific protein molecules (Fig. 3AGo). To more characterize the 72-kDa protein, the expression of the 72-kDa protein on the surfaces of other human cells was analyzed by flow cytometry using 20-202S. As shown in Figure 3BGo, the protein was also expressed on Choi-CK, SH-J1, and HeLa cells, but hardly on SCK, HepG2, PBL, and NTREA2 cL.D1 cells.



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  		Figure 3. Identification and immunoprecipitation of cell-surface molecules with some selected antibodies. (A): The biotinylated cell extracts from Miz-hES1 cells were immunoprecipitated and visualized as described in Materials and Methods. (B): Various human cells were stained with MAb 20-202S and fluorescein isothiocyanate–conjugated mouse immunoglobulin G. Open histograms indicate antibody staining, whereas closed histograms indicate isotype controls. (C): Proteins immunoprecipitated with MAb 20-202S were fractionated on an SDS-gel and stained with Coomassie G250. The arrowheads indicate specific protein bands immunoprecipitated with MAb 20-202S. Abbreviations: hESC, human embryonic stem cell; MAb, monoclonal antibody; MEF, mouse embryonic fibroblast; PBL, peripheral blood lymphocyte.

 
To identify the 72-kDa protein, Miz-hES1 cells and Choi-CK cells were cultured on a large scale, and the cell lysates of Miz-hES1 and Choi-CK cells were subjected to immunoprecipitation using 20-202S followed by SDS-PAGE. After Coomassie G250 staining of the gel (Fig. 3CGo), the 72-kDa protein bands were cut out and subjected to Q-TOF tandem mass spectrometry after in-gel digestion with trypsin as described in Materials and Methods. The amino acid sequences obtained by Q-TOF tandem mass spectrometry showed that the 72-kDa proteins were HSPA8 (Fig. 4Go).



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  		Figure 4. Identification of HSPA8 by Q-TOF mass spectrometry. The MS/MS spectrum of the HSPA8 protein obtained after trypsin digestion is shown by analysis with Q-TOF. The precursor ion shown in the figure is m/z 627.33, and resultant peaks were searched against NCBInr database. Eight tryptic peptides (underlined) originating from Choi-CK cells and two tryptic peptides (italics) originating from Miz-hES1 cells matched the HSPA8 protein. Abbreviations: HSPA8, heat shock 70-kDa protein 8 isoform 1; MS, mass spectrometer; Q-TOF, quadrupole time-of-flight.

 
HSP70 was originally thought to be ubiquitously expressed as a cytoplasmic protein whose function was to capture folding intermediates to prevent protein misfolding and aggregation and to facilitate proper refolding [24]. However, we found that the HSPA8 protein was expressed on the surface of hESC lines Miz-hES1 and Miz-hES4 (Figs. 1Go, 2Go; Table 1Go). To confirm the expression of the HSPA8 protein on the surface of other hESC lines, the hESC lines Miz-hES6 and HSF6 were also examined by immunocytochemistry using 20-202S under the condition that the cell colonies were fixed but not permeabilized with detergent. As shown in Figure 5AGo, 20-202S stained all the hESC lines examined as anti-SSEA3 and SSEA4 did. Also, we found by confocal microscopy that the HSPA8 protein was localized on the surface of Miz-hES1 as TRA-1-60 molecule (Fig. 5BGo). We obtained the same result with Miz-hES6 and HSF6 (data not shown), confirming that the HSPA8 protein is expressed on the surface of hESCs.



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  		Figure 5. Expression of cell-surface markers and the HSPA8 protein in various hESC lines analyzed by immunocytochemistry. (A): Cell colonies from various hESC lines were stained positive for SSEA3, SSEA4, and the HSPA8 protein in immunostaining using Vectastain immunostaining kit. Antibody staining is in red. Scale bar = 50 µm. (B): Cell colonies from undifferentiated Miz-hES1 or Choi-CK were incubated with 20-202S and anti–TRA-1-60 and then fixed as described in Materials and Methods. Antibody staining is in green or red, and nuclear 4,6 diamidino-2-phenylindole staining is in blue. The white arrowheads in the right panels indicate that the immunostaining is on the cell surface. Scale bars = 50 µm in Miz-hES1 and Choi-CK. Abbreviations: hESC, human embryonic stem cell; HSPA8, heat shock 70-kDa protein 8 isoform 1; SSEA, stage-specific embryonic antigen.

 
Next, phenotyping of the HSPA8-positive cells was accomplished using multicolor flow cytometric analysis with hESC-specific MAbs anti-SSEA3, anti-SSEA4, anti–TRA-1-60, or anti–TRA-1-81. To get rid of cross-reactivity between the antibodies used, 20-202S was biotinylated and detected with streptavidin-FITC. As shown in Figure 6Go, approximately 97%, 94%, 60%, and 74% of the HSPA8-positive Miz-hES1 cells are positive in the expression of SSEA3, SSEA4, TRA-1-60, and TRA-1-81, respectively. Approximately 96%, 97%, 80%, and 84% of the HSPA8-positive HSF6 cells are also positive in the expression of SSEA3, SSEA4, TRA-1-60, and TRA-1-81, respectively (data not shown). These results indicate that most of the HSPA8-positive cells are also positive in the expression of those hESC-specific surface markers.



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  		Figure 6. Flow cytometric analysis of Miz-hES1 cells with MAb 20-202S and hESC-specific antibodies. Miz-hES1 cells were simultaneously stained with biotin-labeled MAb 20-202S and one of hESC-specific antibodies, SSEA3, SSEA4-PE, TRA-1-60, or TRA-1-81. Cells were further incubated with streptavidin-fluorescein isothio-cyanate and one of the secondary antibodies, PE-conjugated anti-rat IgM (rIgM) or anti-mouse IgM (mIgM) as appropriate. The appropriate isotype-matched control staining was also done to prove no cross-reactivity between the antibodies used. Values in each quadrant indicate the percentage of positive cells. Abbreviations: hESC, human embryonic stem cell; MAb, monoclonal antibody; PE, phyco-erythrin; SSEA, stage-specific embryonic antigen.

 
Previous studies showed that HSP70 proteins were found on the surface of many cancer cells, monocytes, and the umbilical vein endothelial cell line [25–27]. Because the HSPA8 protein belongs to an HSP70 family that has significant homology among the members, 20-202S could be one of many anti-HSP70 MAbs. Therefore, we compared the expression pattern of the HSPA8 protein recognized by 20-202S in many cells, including hESCs, mESCs, human and mouse primary cells, and human cancer cells, with those of the HAP70 proteins recognized by four other anti-HSP70 MAbs (5G10, W27, SPA810, and SPA820) by Western blot analysis (Fig. 6AGo). The result showed that the pattern of binding by 20-202S was different from those by the anti-HSP70 antibodies (Fig. 6AGo), indicating that 20-202S is different from those anti-HSP70 antibodies. MAb 20-202S showed positive binding in Miz-hES1, Choi-CK, SH-J1, and HeLa but not in MEF, STO, hNP1, mESC, SCK, and HepG2. This result was consistent with that from the flow cytometric analyses (Figs. 2Go, 3BGo). The other four anti-HSP70 antibodies, however, could not bind to all the cell lines examined in flow cytometric analyses (data not shown), although they bound to the HSP70 proteins in the Western blot analysis (Fig. 7AGo). This indicates that the HSPA8 protein is expressed on the cell surface, whereas the other HSP70 proteins recognized by the four antibodies are not. To further demonstrate that 20-202S recognizes the HSPA8 protein, the HSPA8 expression plasmid was transfected into the SCK cells that did not show any binding reactivity for 20-202S in the Western blot analysis shown in Figure 7AGo, and the transfected cells were subjected to Western blot analysis using 20-202S. As expected, a strong binding signal was detected from the transfected SCK cells, but not from the mock-transfected cells (Fig. 7BGo). Taken together, 20-202S recognizes the HSPA8 protein that is expressed on the surface of hESCs and some cancer cell lines but not on mESCs and differentiated cells.



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  		Figure 7. (A): Characterization of MAb 20-202S. Cell extracts from various cells were analyzed with MAb 20-202S and four different anti-HSP70 antibodies by Western blotting. (B): ß-Actin was used for loading control. SCK cells were transfected for 48 hours with pCMV-SPORT6/HSPA8 (HSPA8) or without DNA (Mock). (C): Cell extracts from various cells were preincubated with ATP and MgCl2 (+) or without ATP and MgCl2 (–) before running on a gel. The HSPA8 protein was detected with MAb 20-202S and horse radish peroxidase–conjugated mouse immunoglobulin G. Abbreviations: HSPA8, heat shock 70-kDa protein 8 isoform 1; HSP70, heat shock protein 70; MAb, monoclonal antibody; MEF, mouse embryonic fibroblast; mESC, mouse embryonic stem cell.

 
HSPs interact with a wide range of peptides, and HSP-peptide complexes play an important role in innate and adaptive immune responses [28–30]. The interaction between the peptide and HSP protein has been shown to be associated with ATP hydrolysis and adenosine diphosphate exchange [31]. Therefore, to examine whether the binding of 20-202S to the HSPA8 protein is affected by the presence of ATP, the lysates of Miz-hES1, Choi-CK, SH-J1, and HeLa cells that were shown to express the HSPA8 protein (Fig. 7AGo) were incubated with or without ATP, then the mixtures were subjected to Western blot analysis using 20-202S. Interestingly, 20-202S did not bind to the HSPA8 protein in the presence of ATP, whereas it did bind to the HSPA8 protein in the absence of ATP (Fig. 7CGo), indicating that the binding of MAb 20-202S to the HSPA8 protein is affected by the presence of ATP.

Finally, to examine whether the expression of HSPA8 is downregulated upon differentiation, EBs were derived from Miz-hES1 and HSF6 cells, cultured in bacterial Petri dishes for 4 or 6 days, and then examined for the expression of HSPA8. Approximately 98%, 94%, and 83% of undifferentiated Miz-hES1 cells expressed the SSEA3, SSEA4, HSPA8 antigens, respectively (Fig. 8AGo), whereas only 59%, 54%, and 48% of the EBs expressed the SSEA3, SSEA4, and HSPA8 antigens, respectively. Similar data were obtained, irrespective of the culture day and cell line (data not shown). These data indicate that the HSPA8 protein on the surface is downregulated in the EB cells as the SSEA3 and SSEA4 antigens. To further confirm the downregulation of HSPA8 in differentiated hESCs, the Miz-hES1 cells were treated for 8 days with RA, which was known to induce the differentiation of human ECs and hESCs [14, 15], and were subjected to flow cytometric analyses using 20-202S, anti-SSEA3, or anti-SSEA4 (Fig. 8BGo). When the Miz-hES1 cells were cultured in the presence of RA, SSEA3 was markedly downregulated, whereas SSEA4 was less downregulated after initial transient upregulation, which was consistent with the previous results observed with the hESC lines H7 and H17 [15, 32]. In the case of the HSPA8 protein, the protein was transiently upregulated, similar to SSEA4, and then downregulated more rapidly than SSEA3. Taken together, these results suggest that the HSPA8 protein is a novel cell-surface marker for undifferentiated hESCs.



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  		Figure 8. Surface expression of HSPA8 in Miz-hES1 cells upon differentiation. Miz-hES1 cells were differentiated into embryoid bodies for 4 days in a bacterial Petri dish. Miz-hES1 (upper panel) and embryoid bodies (lower panel) were incubated with anti-SSEA3, anti-SSEA4, or 20-202S and further incubated with fluorescein isothiocyanate–conjugated anti-rat IgM or anti-mouse IgG (A). The fluorescence was compared with that of control (closed histogram) in each panel. Note the decreased percentages of all three antigens in the embryoid bodies. To study the surface expression of HSPA8 in the presence (RA) or absence (control) of RA, Miz-hES1 cells were cultured for 8 days in the presence of RA 2 days after plating. (B): Then the cells were detached and stained with the indicated antibodies every 2 days. Each point is an average derived from two independent experiments. Abbreviations: HSPA8, heat shock 70-kDa protein 8 isoform 1; Ig, immunoglobulin; RA, retinoic acid; SSEA, stage-specific embryonic antigen.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
MAbs specific to the cell-surface molecules of hESCs will be very useful in the identification and analysis of specific cell populations of undifferentiated hESCs, the study of the pluripotency and self-renewal of hESCs, and the purification of cells for cell transplantation therapy. Until now, many MAbs directed against the cell-surface antigens of mouse embryos, mouse ECs, or human ECs have been generated to study embryonic development. Some of the MAbs also detect the cell-surface antigens of hESCs. However, most of the cell-surface markers are associated with carbohydrate epitopes whose functions are uncertain. Besides, several studies have shown that protein molecules on the cell surface are different between hESCs and mESCs [32–34]. So far, no one has reported that hESCs were used as an immunogen to generate MAbs specific to the cell-surface molecules on hESCs. In this study, we immunized mice with Miz-hES1 cells and generated 26 MAbs that bound to the hESCs but not to mESCs. Although we did not identify most of the antigens recognized by the MAbs, these antibodies will be very useful to identify and analyze the differential expression of surface molecules between hESCs and mESCs.

By using MAb 20-202S, we have identified HSPA8 (also known as HSP73) as a cell-surface protein of all hESC lines tested. Originally, HSPs were known to play an important role in chaperoning by transiently associating with nascent polypeptides to facilitate correct folding in the cytoplasm and nucleus [35]. However, recently it has been demonstrated that HSP70 does occur on the surface of a number of cell types [25, 26, 36–42]. In most studies, the extracellular presence of HSP70 was considered to be the outcome of necrotic or apoptotic cell death of neighboring cells, but recent evidence strongly supports active release of HSP70s from cells in response to stresses, including cytokines, acute psychological stress, and exercise [43, 44]. Recent microarray analyses of hESCs showed that HSP70 proteins belong to the group of highly expressed genes in all the cell lines examined [4, 13, 45, 46]. HSPA4 was highly expressed in BG01, BG02, GE01, GE09, and TE06, and HSPA8 was highly expressed in HSF1, HSF6, H9, BS01, and BS02, although the cell-surface expression of the molecules was not shown [13, 46]. In this study, we proved by flow cytometry and immunocytochemistry that the HSPA8 protein was expressed on the surface of hESCs and the expression of the HSPA8 protein was markedly downregulated upon differentiation in culture. On the other hand, to test whether other HSP70 proteins are present on the surface of Miz-hES1 and Miz-hES6, we did flow cytometric analyses with commercially available four anti-HSP70 antibodies. None of them bound to both hESC lines (data not shown). Taking these findings into account, we propose that the HSPA8 protein is a novel cell-surface marker for undifferentiated hESCs.

NTREA2 cL.D1 cells are pluripotent stem cells derived from teratocarcinoma and are considered the malignant counterparts of hESCs. They closely resemble hESCs in terms of the expression of cell-surface markers. Expectedly, the NTERA2 cL.D1 cells were stained with anti-SSEA3, anti-SSEA4, anti–TRA1-60, and anti–TRA1-81, as HSF6 and Miz-hES1 cells, but not with anti-SSEA1 (data not shown). However, NTERA2 cL.D1 was not stained with 20-202S at all (Fig. 3BGo), suggesting that the HSPA8 protein is a surface marker that can discriminate Miz-hES1 and HSF6 from NTERA2 cL.D1.

As HSP70 was expressed on the surface of several cancer cell lines [25, 26, 36–42], we examined the surface expression of the HSPA8 protein in some cancer cell lines by flow cytometry. As in Miz-hES1, we could observe its surface expression in Choi-CK, SH-J1, and HeLa cells. Western blot analysis using the whole cell extracts of the cancer cell lines showed the same expression pattern as the flow cytometric analysis. Although we did not know the mechanism by which HSPs were physiologically exposed to the cell surface, we thought that HSP-peptide complexes might be expressed on the surface of Miz-hES1, Choi-CK, SH-J1, and HeLa cells because the cancer-derived HSPs were known to be noncovalently associated with peptides derived from tumor-specific antigens [28], and the HSP-peptide complexes were shown to be stable even under denaturing conditions of SDS-PAGE [47]. Because we used whole cell clumps as an immunogen to generate the MAbs, 20-202S would recognize native conformation of the HSPA8 protein on the surface. This means that 20-202S would recognize the HSPA8 protein that might be associated with a certain peptide. To take a close look at whether 20-202S binds to the HSPA8 protein itself, we stripped the HSPA8 protein by ATP treatment before SDS-PAGE. The result showed that 20-202S did not bind to ATP-treated HSPA8 but did bind to untreated HSPA8 in the Western blot analysis. Although this result cannot confirm that 20-202S recognizes the HSPA8-peptide complex itself, it indicates that 20-202S recognizes the epitope on the HSPA8 protein in an ATP-sensitive manner. We are now studying the mechanism by which 20-202S recognizes the HSPA8 protein on the surface of hESCs.

HSPs evolved to bind peptides at the beginning of life as a consequence of their chaperoning functions. Their ability to interact with a wide range of proteins and peptides has made the HSPs uniquely suited to an important role in organismal survival by their participation in innate and adaptive immune responses. Actually, the HSPs are emerging as key "danger" signals of the innate immune system. Exogenous HSP70 stimulated the secretion of proinflammatory cytokines through a CD14-dependent pathway as bacterial lipopolysaccharide does [26, 48]. CD14 and toll-like receptors were known to be solely responsible for the lipopolysaccharide (LPS)-binding in the beginning, but the HSPs, including HSPA8 and HSP90A, were also identified on the cell surface of monocytes that form an activation cluster after LPS ligation and were shown to be involved in LPS signal transduction [27]. Anti-body inhibition analysis suggested that disruption of cluster formation abrogates tumor necrosis factor release. Based on these results, the authors proposed that HSPs, which are highly conserved from bacteria to eukaryotic cells, play a crucial role in the host’s innate defense against microbial pathogen. Several HSPs have also shown the ability to carry tumor- or virus-derived peptides and generate protective immunity [28]. HSPs derived from tumor cells are recognized by T cells with either T-cell receptor {alpha}, ß or {gamma}, {delta} [49]. The biological roles of HSPA8 on the surface of hESCs are unknown at the moment. However, based on the immunological properties of HSPs, one could speculate that the role of HSP70 on the surface of hESCs might be associated with immune responses under certain circumstances. Actually, several studies provided evidence that immune sensitization to HSP protein is associated with unsuccessful embryo development and implantation failure in a mouse embryo culture and in vitro fertilization patients [50, 51], suggesting that HSP70s are present on the surface of human and mouse embryos under certain circumstances. Because hESCs do not express the major histocompatibility complex (MHC) proteins enough before differentiation [52], it is conceivable that HSP proteins, and not MHC proteins, play a certain role in innate and adaptive immunity in hESCs during embryonic development.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We are grateful to Jeong Hwa Lee and Dr. Jin Young Kim of the Korean Basic Science Institute for their excellent technical assistance in protein analysis. This research was supported by a grant from KRIBB Research Initiative Program and a grant (SC12023) from Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology of Korea and partly by a grant (BGM0700511) from the Ministry of Health and Welfare of Korea.

DISCLOSURES
The authors indicate no potential conflicts of interest.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received November 8, 2004; accepted for publication June 7, 2005.



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