Spatial Distribution and Initial Changes of SSEA-1 and Other Cell Adhesion-related Molecules on Mouse Embryonic Stem Cells Before and During Differentiation
Institute of Organ Transplants, Reconstructive Medicine and Tissue Engineering, Shinshu University Graduate School of Medicine (LC,FY,KS), and Department of Anatomy and Organ Technology, Shinshu University School of Medicine (KJ,NO,YO,KA,KS), Matsumoto, Japan
Correspondence to: Li Cui, Inst. of Organ Transplants, Reconstructive Medicine and Tissue Engineering, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan. E-mail: cui{at}sch.md.shinshu-u.ac.jp
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Summary |
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Key Words: embryonic stem cells SSEA-1 cell adhesion-related molecules immuno-SEM and -TEM flow cytometry retinoic acid
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Introduction |
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Embryonic cell surface molecules have been viewed generally as lineage markers and regulators of cellcell interactions. Cell surface carbohydrates are implicated in a number of membrane-modulated phenomena, such as cell aggregation and adhesion. They play a role in the cellular interactions of the immune system (Springer 1990) and in normal cell interactions during the embryogenesis of preimplantation mouse embryos (Eggens et al. 1989
). Stage-specific embryonic antigen-1 (SSEA-1), identified as the cell surface carbohydrate antigen Lewisx (Lex; Galß1
4[Fuc
1
3]-GlcNAcß1
3Gal), is expressed in preimplantation mouse embryos beginning at the 8-cell stage and also in teratocarcinoma stem cells and ES cells, but not in their differentiated derivatives (Solter and Knowles 1978
; Knowles et al. 1980
; Fox et al. 1981
; Eggens et al. 1989
; Kojima et al. 1994
). SSEA-1 is regarded as an excellent cell surface marker to monitor early stages of embryogenesis and ES cell differentiation (Solter and Knowles 1978
; Fox et al. 1981
; Bird and Kimber 1984
). Specific interaction of Lex with Lex has been proposed as a basis for cell adhesion in preimplantation embryos and in the aggregation of F9 teratocarcinoma cells (Kojima et al. 1994
).
Expression patterns of cell adhesion-related molecules, such as SSEA-1, ICAM-1, PECAM-1, and CD9, occur in undifferentiated and differentiated ES cells (Tian et al. 1997; Redick and Bautch 1999
; Oka et al. 2002
). PECAM-1 and ICAM-1, representative cell adhesion molecules belonging to the immunoglobulin superfamily, are known to be expressed in endothelial cells and leukocytes. CD9, a tetraspanin superfamily protein, is regarded as a surface marker on mouse and rat male germline stem cells and neural stem cells, and is associated with proliferation, migration, and differentiation of these cells (Hadjiargyrou and Patterson 1995
; Kaprielian et al. 1995
; Kanatsu-Shinohara et al. 2004
). CD9 plays a role in maintenance of undifferentiated mouse ES cells (Oka et al. 2002
). Despite high levels of expression in the cells, the spatial distribution of these cell adhesion-related molecules on undifferentiated ES cells has not been elucidated.
In this study we examined the surface ultrastructure of mouse ES cells and the spatial distribution of SSEA-1, ICAM-1, PECAM-1, and CD9 on the cells. In addition, we investigated the changes in the morphology and the expression of these CAMs on initiation of ES cell differentiation. We report for the first time the spatial distribution and expression levels of the above molecules on mouse ES cells.
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Materials and Methods |
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Differentiation was induced by adding 106 M trans-retinoic acid (RA) (Draper et al. 2002) to a differentiating medium without LIF (Johkura et al. 2003
) for 2 days.
As a control culture, ES cells were dissociated with 0.1% trypsinEDTA and resuspended in the differentiating medium without LIF for 48 hr, in which ES cells aggregated to form embryoid bodies. Aggregated embryoid bodies were collected and cultured on gelatin-coated coverslips in the same medium for 24 hr. ES cells were also cultured in the differentiating medium without LIF for 48 hr as a control culture for PCR.
Alkaline Phosphatase Staining and Immunofluorescence Labeling
ES cells cultured on the gelatin-coated coverslip were washed once with PBS and fixed in 4% paraformaldehyde/0.1 M phosphate buffer, pH 7.4, for 15 min at room temperature. After washing three times, cells were incubated with nitroblue tetrazolium/bromochloroindolyl phosphate solution (Bio-Rad; Hercules, CA) for 20 min for alkaline phosphatase staining. For immunofluorescence double staining, the following primary antibodies were used: rat monoclonal antibodies (MAbs) against mouse ICAM-1 (KAT; Antigenix America, New York, NY), PECAM-1 (MEC 13.3; Pharmingen, San Diego, CA), and CD9 (KMC8; Pharmingen), and antibody against SSEA-1 (mouse monoclonal IgM; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA). The cells were pretreated with 1.5% normal goat serum for 30 min and incubated overnight at 4C with primary antibodies diluted 1:100 in 1.5% normal goat serum. The CAMs and SSEA-1 were detected by goat anti-rat IgG conjugated with Alexa Fluor 488 and by goat anti-mouse IgM conjugated with Alexa Fluor 568 diluted 1:1000 in 1.5% normal goat serum, respectively (Molecular Probes; Eugene, OR). Specimens were incubated with DAPI (500 ng/ml; Molecular Probes) for nucleic acid staining. After washing, the cells were mounted with a ProLong Antifade Kit (Molecular Probes) and observed with an Olympus FLUOVIEW confocal laser scanning microscopy (CLSM) using Ar, He/Ne lasers, or a ZEISS LSM 510 CLSM, or a Leica TCS SP2 AOBS spectral CLSM. For negative controls, normal goat serum or PBS was used instead of the primary antibody.
Double-labeling Immunoelectron Microscopy
The cells were cultured on gelatin-coated coverslips, washed once with hypothermic UW (University of Wisconsin; Madison, WI) solution (Wahlberg et al. 1986; Cui et al. 2002
), and then reacted for 30 min with rat anti-mouse ICAM-1, PECAM-1, or CD9 each diluted 1:10 in hypothermic UW solution. After three washes with UW solution, the cells were immersed for 30 min in goat anti-rat IgG antibody conjugated with 20-nm gold particles (British Biocell International; Cardiff, UK) diluted 1:10 in hypothermic UW solution. After another wash, they were incubated with anti-mouse SSEA-1 antibody, 1:10 in hypothermic UW solution, for 30 min. This was followed by another wash and immersion for 30 min in goat anti-mouse IgM antibody conjugated with 10-nm gold particles (British Biocell International), 1:10 in hypothermic UW solution. After a final rinse, the cells were fixed in 2.5% glutaraldehyde/50 mM cacodylate-HCl, pH 7.2, for 4 hr. All procedures were carried out at 4C. Negative controls for immunostaining were obtained by substituting UW solution for the primary antibodies or by using an equivalent concentration of nonimmune rat IgG. Each experiment was repeated at least three times.
Preparation for Scanning Electron Microscopy
After several washes with 0.1 M cacodylate buffer solution for 1.5 hr, the specimens were postfixed in 1% OsO4 for 4 hr and then dehydrated and immersed in isoamyl acetate. They were dried with the CO2-critical point drying method, coated to 3-nm thickness with an osmium plasma coater (Nippon Laser and Electronics Laboratories; Nagoya, Japan), and observed with a JEOL JSM-6000F scanning electron microscope or a Hitachi S-5000 (FE-SEM) scanning electron microscope with a backscatter electron (BSE) detector at an accelerating voltage of 15 kV or 8kV, respectively.
Preparation for Transmission Electron Microscopy
The cells on coverslips were postfixed with 1% OsO4 for 1 hr, dehydrated, and embedded in epoxy resin by a standard method. Sections 1.5 µm thick were stained with 0.1% toluidine blue solution. Ultrathin sections were stained with uranyl acetate and lead citrate solution and observed with a JEM-1200 TEM at an accelerating voltage of 80 kV.
RT-PCR Analysis
Total RNA was extracted from undifferentiated ES cells and from differentiated ES cells at various stages of differentiation using TRIzol reagent (Invitrogen; Carlsbad, CA). DNase-treated total RNA was used to prepare the first-strand cDNA with SuperScript II (Invitrogen), following the protocol of the manufacturer. cDNA samples were subjected to PCR amplification with specific primers under linear conditions to approximate the original amount of the specific transcript. Amplification conditions consisted of denaturation at 95C for 3 min followed by 40 cycles of denaturation at 95C for 1 min, annealing at the temperature specified for each of the primer sets for 1 min, and elongation at 72C for 1 min. The PCR primers, predicted size of amplified products, and annealing temperature are shown in Table 1. cDNA samples from ES cells in the medium without LIF were also analyzed as a control of initial differentiation.
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Flow Cytometry
Cells for flow cytometry were resuspended in 0.5% BSA/PBS and incubated with saturating concentrations of primary antibodies for 30 min at 4C, washed twice, and then labeled with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM (Chemicon) or PECy5-conjugated goat anti-rat IgG (Cedarlane; Hornby, Canada) for 30 min at 4C. After washing three times with 0.5% BSA/PBS, stained cells were analyzed on a FACS Calibur (Becton Dickinson; Mountain View, CA). Control cells were not treated with primary antibodies. Each experiment was carried out on at least four different cultures to record variation among cultures.
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Results |
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Discussion |
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SSEA-1 (Lex) is a homophilic adhesion molecule capable of interacting with itself and was localized preferentially on cell surface projections. Carbohydratecarbohydrate interactions are important in specific recognition between cells (Eggens et al. 1989; Kojima et al. 1994
), especially during embryogenesis and organogenesis (Knowles et al. 1980
). Carbohydrate recognition requires a high density or "clustering" of molecules (Connolly et al. 1982
; Lee et al. 1983
). In ES cell compaction, cellcell contacts are maximal, suggesting strong cell adhesion. Human primordial germ cell-derived embryonic germ cell colonies, which have morphological similarity to mouse ES cell colonies, express SSEA-1 (Shamblott et al. 1998
). In contrast, the relatively flat and loosely associated primate ES cell colonies are SSEA-1-negative (Thomson et al. 1998
; Suemori et al. 2001
; Draper et al. 2002
). In our studies, SSEA-1 was present on undifferentiated mouse ES cells and diminished as cells differentiated due to the removal of LIF or the addition of retinoic acid. These changes were associated with a transition in colony morphology to that of flat colonies. On the basis of these observations, we hypothesize that SSEA-1 is involved in the formation of multilayered and tightly compacted colonies of mouse ES cells through highly specific LexLex interactions.
Immunocytochemistry and flow cytometry showed that ICAM-1 and PECAM-1 were also heterogeneously distributed on the undifferentiated ES cells. ICAM-1 was uniformly and randomly distributed on the ES cell surfaces and in the cellcell contact sites. Despite the discernible expression of ICAM-1, ligands for this molecule, i.e., LFA-1 and Mac-1, were not present in the ES cells (Tian et al. 1997). Therefore, the function of this molecule in mouse ES cells remains unclear.
As shown by CLSM, PECAM-1 is the predominant CAM at the cellcell boundaries of ES cells. This is consistent with previous findings showing that staining for PECAM-1 is specific to cellcell borders in the inner cell mass of the mouse blastocyst (Robson et al. 2001). Homophilic interaction and diffusion trapping models have been proposed to account for the characteristic distribution of PECAM-1 (Sun et al. 2000
). Movement of PECAM-1 in the cell membrane occurs relatively freely until the extracellular domain of the molecule encounters its ligand, PECAM-1, on an adjacent cell (Gingell and Owens 1992
; Singer 1992
; Sun et al. 2000
). When this occurs, the complex is captured at the cellcell interface, leading to localization at cellcell borders. Distribution at cell boundaries suggests that PECAM-1 may play a role in ES cell aggregation via its homophilic adhesion. SEM showed random distribution of PECAM-1 on the free surface of ES cells, but its accumulation at the cellcell borders could not be verified because they are inaccessible to observation by immuno-SEM. The random distribution of PECAM-1 observed by immuno-SEM may reflect the diffusion of molecules not involved in homophilic binding.
Because most undifferentiated ES cells are positive for CD9 and it quickly disappeared after initial differentiation, CD9 may be a more suitable marker for undifferentiated ES cells than SSEA-1. CD9 is a cell adhesion-related molecule and may play a role in cellextracellular matrix or cellcell interactions as a co-factor of integrin (Rubinstein et al. 1997; Berditchevski and Odintsova 1999
; Takao et al. 1999
). The preferential localization of CD9 on microvilli and protrusions of the cellular periphery suggest that it is associated with attachment of adjacent cells. It may also regulate cytoskeletal organization (Berditchevski and Odintsova 1999
; Cook et al. 2002
), thus affecting the cellECM or cellcell interactions.
The association of cell surfaces containing microdomains of adhesion molecules plays an important role in the three-dimensional cellcell interactions that affect differentiation of ES cells. The data presented here enable us to further understand the roles of these cell adhesion-related molecules in cellcell interactions and in self-renewal of ES cells. In addition, the present study indicates that these antigens may be used as markers of cell status to test the phenotypic stability of long-term ES cell cultures. Simultaneous use of immunoreactivity for multiple surface antigens will assist in the identification of positive or negative selection of target cells derived from ES cells.
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Acknowledgments |
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Mouse ES cell lines AB1 and AB2.2 were a kind gift from Dr Allan Bradley (Baylor College of Medicine). We thank Dr Kiyokazu Kametani and Ms Kayo Suzuki (Research Center for Instrumental Analysis of Shinshu University) and Mr Mitsuo Ueno (Fine Materials Engineering, Faculty of Textile Science and Technology of Shinshu University) for excellent technical assistance. We thank Dr Kei-ichi Uemura (Department of Aging Biochemistry, Neuro-aging Research Division, Research Center on Aging and Adaptation of Shinshu University School of Medicine) for advice concerning flow cytometry analysis. We also thank the Microscope Division of Carl Zeiss (Tokyo, Japan) for their support with respect to the CLSM experiment.
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Footnotes |
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