Heat Shock 70-kDa Protein 8 Isoform 1 Is Expressed on the Surface of Human Embryonic Stem Cells and Downregulated upon Differentiation
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《干细胞学杂志》
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@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@kribb.re.kr
ABSTRACT
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 . 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 .
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 . SSEA1 is expressed on undifferentiated mESCs or differentiated hESCs, whereas SSEA3 and SSEA4 are expressed on undifferentiated hESCs but not on undifferentiated mESCs . Two human EC cell antigens, TRA-1-60 and TRA-1-81, are also used to mark undifferentiated hESCs . However, the epitopes of the surface antigens are carried by carbohydrates, and the exact functions of the carbohydrate antigens in ESCs are not known . 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
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. 1A). Of these, nine were classified into isotype IgG and 17 were IgM (Table 1). They showed various patterns of binding in flow cytometric analysis, although IgM antibodies showed higher binding activity compared with IgG MAbs (Fig. 1A). The Miz-hES1–specific binding by these MAbs was also confirmed by immunocytochemistry (Fig. 1B; Table 1). 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. 1B; Table 1). In contrast, the MAbs did not bind to MEFs, STO cells, J1, and TC-1 (Fig. 2; Table 1). 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. 2). Among 26 MAbs, 17 MAbs did not bind to the hNP1, whereas seven MAbs bound to the hNP1 partially (+/–) or strongly (+) (Fig. 2; Table 1).
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.
Table 1. Characteristics of a panel of MAbs specific to hESCs
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. 3A). 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 3B, the protein was also expressed on Choi-CK, SH-J1, and HeLa cells, but hardly on SCK, HepG2, PBL, and NTREA2 cL.D1 cells.
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. 3C), 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. 4).
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 . However, we found that the HSPA8 protein was expressed on the surface of hESC lines Miz-hES1 and Miz-hES4 (Figs. 1, 2; Table 1). 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 5A, 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. 5B). 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.
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 6, 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.
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 . 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. 6A). The result showed that the pattern of binding by 20-202S was different from those by the anti-HSP70 antibodies (Fig. 6A), 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. 2, 3B). 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. 7A). 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 7A, 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. 7B). 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.
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): ?-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 . The interaction between the peptide and HSP protein has been shown to be associated with ATP hydrolysis and adenosine diphosphate exchange . 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. 7A) 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. 7C), 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. 8A), 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 , and were subjected to flow cytometric analyses using 20-202S, anti-SSEA3, or anti-SSEA4 (Fig. 8B). 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 . 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.
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
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.
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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@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@kribb.re.kr
ABSTRACT
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 . 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 .
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 . SSEA1 is expressed on undifferentiated mESCs or differentiated hESCs, whereas SSEA3 and SSEA4 are expressed on undifferentiated hESCs but not on undifferentiated mESCs . Two human EC cell antigens, TRA-1-60 and TRA-1-81, are also used to mark undifferentiated hESCs . However, the epitopes of the surface antigens are carried by carbohydrates, and the exact functions of the carbohydrate antigens in ESCs are not known . 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
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. 1A). Of these, nine were classified into isotype IgG and 17 were IgM (Table 1). They showed various patterns of binding in flow cytometric analysis, although IgM antibodies showed higher binding activity compared with IgG MAbs (Fig. 1A). The Miz-hES1–specific binding by these MAbs was also confirmed by immunocytochemistry (Fig. 1B; Table 1). 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. 1B; Table 1). In contrast, the MAbs did not bind to MEFs, STO cells, J1, and TC-1 (Fig. 2; Table 1). 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. 2). Among 26 MAbs, 17 MAbs did not bind to the hNP1, whereas seven MAbs bound to the hNP1 partially (+/–) or strongly (+) (Fig. 2; Table 1).
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.
Table 1. Characteristics of a panel of MAbs specific to hESCs
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. 3A). 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 3B, the protein was also expressed on Choi-CK, SH-J1, and HeLa cells, but hardly on SCK, HepG2, PBL, and NTREA2 cL.D1 cells.
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. 3C), 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. 4).
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 . However, we found that the HSPA8 protein was expressed on the surface of hESC lines Miz-hES1 and Miz-hES4 (Figs. 1, 2; Table 1). 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 5A, 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. 5B). 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.
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 6, 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.
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 . 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. 6A). The result showed that the pattern of binding by 20-202S was different from those by the anti-HSP70 antibodies (Fig. 6A), 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. 2, 3B). 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. 7A). 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 7A, 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. 7B). 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.
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): ?-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 . The interaction between the peptide and HSP protein has been shown to be associated with ATP hydrolysis and adenosine diphosphate exchange . 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. 7A) 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. 7C), 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. 8A), 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 , and were subjected to flow cytometric analyses using 20-202S, anti-SSEA3, or anti-SSEA4 (Fig. 8B). 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 . 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.
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
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.
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