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First published online December 14, 2006
Stem Cells Vol. 25 No. 4 April 2007, pp. 974 -982
doi:10.1634/stemcells.2006-0224; www.StemCells.com
© 2007 AlphaMed Press

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EMBRYONIC STEM CELLS: CHARACTERIZATION SERIES

The Lectin Dolichos Biflorus Agglutinin Recognizes Glycan Epitopes on the Surface of Murine Embryonic Stem Cells: A New Tool for Characterizing Pluripotent Cells and Early Differentiation

Rodney Nasha, Lori Nevesa, Renate Faastb, Michael Piercea, Stephen Daltona

aCenter for Complex Carbohydrate Research and Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA;
bDepartment of Molecular Biosciences, University of Adelaide, Adelaide, Australia

Key Words. Embryonic stem cells • Primitive ectoderm • Lectin • Differentiation

Correspondence: Stephen Dalton, Ph.D., Coverdell Center for Biomedical and Health Sciences, Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, 30602 Telephone: 706-583-0480; Fax: 706-583-0480; e-mail: sdalton{at}uga.edu

Received April 14, 2006; accepted for publication December 5, 2006.
First published online in STEM CELLS EXPRESS   December 14, 2006.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Cell surface markers are key tools that are frequently used to characterize and separate mixed cell populations. Existing cell surface markers used to define murine embryonic stem cells (mESCs) such as stage-specific embryonic antigen 1 (SSEA1), Forssman antigen (FA), alkaline phosphatase (AP), and CD9 are limiting, however, because they do not unambiguously define the pluripotent state and are not reliable indicators of differentiation commitment. To identify glycan cell surface markers that would circumvent this problem, we used a panel of 18 lectins to identify epitopes specifically elevated on the surface of mESCs, which, during differentiation, decrease with kinetics that precede currently used markers such as CD9, SSEA1, FA, and AP. The anticipated outcome of this analysis was to identify glycans that have utility as reliable mESC markers and high-resolution readouts for early differentiation commitment. Here, we show that the lectin Dolichos biflorus agglutinin (DBA) recognizes {alpha}-N-acetylgalactosamine (GalNAc) cell surface epitopes on mESCs (CD9high SSEA1high APhigh DBAhigh). These glycan epitopes decline markedly in cells undergoing the first definable step of differentiation, the transition from mESCs to primitive ectoderm (CD9high SSEA1high APhigh DBAlow). Loss of GalNAc epitopes is, therefore, the earliest cell surface change that can be assigned to differentiating cells, and the only cell surface marker known to be tightly associated with the pluripotent state. The lectin DBA is, therefore, a useful tool to characterize mESC cultures by nondestructive approaches, an indicator of differentiation commitment, and a predictor of developmental potency.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Pluripotent murine embryonic stem cells (mESCs) are derived from the inner cell mass (ICM) of blastocyst-stage embryos [1, 2]. When cultured over extensive periods of time in the presence of leukemia inhibitory factor (LIF), mESCs retain many of the characteristics associated with pluripotent cells of the ICM, including the capacity to generate the three embryonic germ lineages. Several cell surface markers such as CD9, alkaline phosphatase (AP), and the glycolipids stage-specific embryonic antigen 1 (SSEA1) and Forssman antigen (FA) are widely used for the characterization and purification of mESCs [3–7]. These markers are problematic, however, because their expression continues well beyond the time at which mESCs become irreversibly committed to differentiation [3–7]. Although these markers are eventually extinguished, they are poor indicators of pluripotency and differentiation commitment. In contrast, marker transcripts such as Rex1, Gbx2, and c-myc [3, 5, 7] have been reliable readouts for the pluripotent state and early differentiation. Their utility is limited, however, because these markers cannot be used for the analysis and recovery of functional cells and they have severe limitations at the single-cell level of analysis. Similar problems face the characterization of human ESCs (hESCs) that express glycoepitopes such as SSEA3,4 and TRA1-60/80 [8] but again, their expression is only loosely correlated with the pluripotent state because of their delayed downregulation during differentiation. It is, therefore, important to identify new markers for human and murine ESCs that are more restricted to the pluripotent state and that can be used as indicators for the early stages of differentiation.

Lectins have frequently been used to identify, characterize, and isolate novel cell subpopulations on the basis of the presentation of specific carbohydrate groups on the cell surface. For example, peanut agglutinin (PNA), which recognizes D-(+)-galactose, has been used to fractionate murine hematopoietic stem cells [9] and, more recently, in the purification and identification of adult neural stem cells [10]. Lectins have also been used to document the repertoire of glycoepitopes on the surface of embryonal carcinoma cells and embryonic cells during vertebrate preimplantation development [11–20]. There are several examples in which lectin reactivity with cells of the early embryo shows dynamic temporal and spatial changes, indicative of functional significance.

In this report, we describe Dolichos biflorus agglutinin (DBA), which recognizes {alpha}-N-acetylgalactosamine (GalNAc) as being highly reactive toward murine ESCs. Presentation of GalNAc on the cell surface is rapidly downregulated during differentiation, preceding that of SSEA1, CD9, and FA. We determined that decreased DBA reactivity coincided with the formation of primitive ectoderm, an SSEA1high Oct4high Fgf5high (Rex1low) population that represents the first definable differentiation step involved in germ layer formation from ESCs. These findings establish utility for DBA in the characterization of pluripotent cells because it can be used as a nondestructive marker and as a reliable readout for initial differentiation events, at a level of temporal resolution that was not previously possible.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Cell Culture
D3 and R1 mESCs [21, 22] were cultured in the absence of feeders on tissue culture grade plasticware precoated with 0.1% gelatin phosphate-buffered saline (PBS), as described previously [3, 23, 24]. mESC culture medium consisted of Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com) supplemented with 10% fetal calf serum (FCS), 1 mM L-glutamine, 0.1 mM 2-mercaptoethanol, and 103 units/ml recombinant human LIF (ESGRO; Chemicon International, Temecula, CA, http://www.chemicon.com) at 37°C under 10% CO2. Primitive ectoderm (EPL) cells were formed and maintained by culturing as described previously [3, 23, 24] in mESC medium containing 50% conditioned medium (MedII) supplemented with 1 mM L-glutamine and 0.1 mM 2-mercaptoethanol, at a density of 2 x 104 cells per cm2. EPL cells were cultured for 2 days (EPL2) or passaged and cultured for a further 2 days to produce EPL4 cells. MedII conditioned medium was prepared from HepG2 cells (American Type Culture Collection [ATCC] number HB-8065) grown to confluence for 4–5 days in DMEM supplemented with 10% FCS, after being seeded at 5 x 104 cells per cm2. Conditioned medium was collected, filter sterilized, and then stored at 4°C for up to 2 weeks. Reversion of EPL4 cells was carried out as described [3, 23, 24] by reseeding cells into mESC medium containing LIF and culturing for 2 or 4 days, passaging every 2 to 3 days.

Lectin Binding, DBA Affinity Purification of Glycoproteins, and Glycan Analysis
Fluorescein-labeled lectins were purchased from Vector Laboratories (Burlingame, CA, http://www.vectorlabs.com)and are listed in Table 1. Cells were harvested to a single cell suspension by trypsinization, and then washed and resuspended in 0.1% bovine serum albumin (BSA)-PBS. Aliquots of 100 µl containing 106 cells were mixed with an equal volume of fluorescein isothiocyanate (FITC)-lectin diluted in 0.1% BSA/PBS, and incubated for 30 minutes at 4°C. Lectins were used at a final concentration of 20 µg/ml or 100 µg/ml. Cells were then washed once in 5% FCS-95% DMEM and resuspended in 500 µl of the same medium for analysis on a Beckman Coulter (Fullerton, CA, http://www.beckmancoulter.com)flow cytometer with Windows Multiple Document Interface (WinMDi, J. Trotter, Scripps Research Institute, La Jolla, CA) software. Fluorescence-activated cell sorting (FACS) analysis was performed on a MoFlo (DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com). Approximately 5 x 106 ESCs and EPL cells were harvested by trypsinization, mixed and stained with FITC-DBA (100 µg/ml), and then separated into DBAhigh and DBAlow fractions.


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  		Table 1. Summary of data obtained using antibodies raised against SSEA1, Forssman antigen, and a panel of 18 fluorescein-isothiocyanate-labelled lectins on unfixed mESCs (D3, R1), corresponding EPL cells (day 4 after addition of conditioned media), and cells grown in the absence of LIF for 6 days

 
Attached cells were prepared for lectin binding by culturing in 24-well trays to 50%–80% confluence, washed twice with PBS, and then fixed in 4% paraformaldehyde for 1 hour. Fixed cells were washed twice with PBS before the addition of lectin solution diluted to a concentration of 20 µg/ml or 100 µg/ml in PBS containing 0.1% BSA. Lectin binding was carried out for 30 minutes at 4°C, and then cells were washed with 0.1% BSA/PBS prior to analysis by fluorescence microscopy.

For DBA affinity precipitations, whole-cell lysates were prepared by resuspending pellets of R1 mESCs in cold lysis buffer (0.2 M Hepes, pH 7.4, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100). Cell lysate (600 µg of protein) was 1:10 diluted to 1 mg/ml protein with immunoprecipitation (IP) buffer (0.1 M Hepes, pH 7.4, 0.1 M NaCl, 5 mM CaCl2, 5 mM MgCl2, 0.1% Triton X-100 [Fisher Scientific International, Hampton, NH, http://www.fisherscientific.com]) was precleared with protein A Sepharose, tumbled with DBA agarose beads (55 µl, 1:1 slurry; Vector Laboratories) overnight at 4°C, and then washed four times in IP buffer. DBA precipitates were boiled in 10 mM sodium phosphate buffer (pH 7.5, 0.1% SDS, 0.1% vol/vol 2-mercaptoethanol) for 5 minutes, and then immediately chilled on ice. SDS was removed from eluates by addition of KCl to 100 mM followed by centrifugation at 4°C. Clarified eluates were then subject to N-glycosidase F (NEB) treatment at 37°C for 12 hours, and then protein was precipitated after addition of cold 100% ethanol and centrifugation in a microcentrifuge. Digestion of Fetuin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) was performed in parallel as a control for glycosidase F activity. Precipitated protein was then resolved on a 4%–12% Bis-Tris gradient polyacrylamide gel (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), transferred onto a filter membrane, and then probed with biotinylated DBA (Vector Laboratories) followed by streptavidin-conjugated horseradish peroxidase. Bands were then detected by ECL.

Transcript Analysis by Quantitative Real-Time Polymerase Chain Reaction
RNA was prepared using Qiagen RNeasy Mini Kits (Qiagen, Hilden, Germany, http://www1.qiagen.com). Chromosomal DNA was removed using RNase-free DNase (Qiagen). cDNA was prepared using the Superscript First Strand Synthesis System (Invitrogen) using 2 µg of total RNA. Target mRNAs were assayed using TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com; supplemental online Table 1), supplemented with Universal PCR Master Mix (Applied Biosystems). Polymerase chain reactions (PCR) were performed on an Applied Biosystems ABI 7700 Sequence Detector.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Identification of a Glycan Marker on the Surface of mESCs that Is Rapidly Downregulated During Differentiation
To identify cell surface glycan epitopes that would more precisely define the mESC state and would serve as early indicators of differentiation, we screened the surface of mESCs and differentiating cells with a panel of 18 lectins (Table 1). For a lectin to have utility, we specified that presentation of its cognate epitope must be elevated in mESCs and then decrease with kinetics that precede that of accepted markers such CD9, SSEA1, and FA. Together, this would establish the glycan epitope as having utility as a reliable mESC marker and as a high-resolution readout for early differentiation.

Quantitative PCR (Q-PCR) analysis was used to confirm the relative timing of differentiation of mESCs grown in the absence of LIF (Fig. 1A). As expected, c-myc and cyclin E1 transcript levels decreased within 2 days, whereas TERT, Oct4, and CD9 mRNAs declined between days 2 and 4. Of the 18 lectins screened by flow cytometry and immunostaining, only glycoepitopes recognized by DBA showed a clear and uniform downregulation that preceded CD9, SSEA1, or FA (Fig. 1B, 1C; Table 1). The DBAhigh mESC population declined within 2 days of LIF withdrawal, concomitant with decreases in c-myc and cyclin E1 transcripts, and was extinguished within 4 days (Fig. 1). The specificity of DBA binding was confirmed by the addition of excess amounts of GalNAc, which blocked FITC-labeled DBA binding to mESCs (Fig. 1C). In contrast, excess amounts of N-acetylglucosamine (GlcNAc) failed to block DBA binding.


Figure 1
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  		Figure 1. Downregulation of DBA epitopes on the surface of mESCs precedes that of SSEA1 and CD9 after LIF withdrawal. (A): Transcript profiling of R1 mESCs by quantitative polymerase chain reaction for c-myc, TERT, Oct4, CD9 and cyclin E1 after LIF withdrawal over 4 days. Transcript levels were determined in triplicate and shown as ± SEM after being normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Levels of transcripts in mESCs were normalized against GAPDH and assigned a value of 1. (B): Flow cytometry analysis of CD9, DBA, and SSEA1 in R1 mESCs and in mESCs after LIF withdrawal for 2, 4, and 6 days. (C): mESCs were stained with fluorescein isothiocyanate (FITC)-DBA (top panel) or preincubated with 100 mM GalNAc (gray profile, bottom panel) or 100 mM GlcNAc (black profile, bottom panel) for 10 minutes before addition of FITC-DBA and analyzed by flow cytometry. (D): Immunostaining of cells probed with DBA or antibodies raised against FA, SSEA1, and CD9 (shown in green; FITC label). mESCs grown in the presence of LIF or, in the absence of LIF for 2, 4, and 6 days were simultaneously stained with 4',6-diamidino-2-phenylindole, dihydrochloride (DNA, blue). Abbreviations: d, day; DBA, Dolichos biflorus agglutinin; FA, Forssman antigen; FL1, fluorescence 1 detector; GalNAc, {alpha}-N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; LIF, leukemia inhibitory factor; mESC, murine ESC; SSEA1, stage-specific embryonic antigen 1.

 
CD9 reactivity decreased at day 4, but was elevated again by day 6 (Fig. 1B). SSEA1 reactivity decreased partially on day 2, but did not uniformly decline across the entire cell population over 6 days of differentiation. In contrast to DBA, a significant proportion of cells retained SSEA1 reactivity over the entire 6-day differentiation period. These data highlight the problems associated with using CD9, FA, and SSEA1 as markers for the mESC state because they are downregulated only after cells become irreversibly committed to differentiation, or do not uniformly change during differentiation [25]. Our results establish DBA as a more useful tool than cell surface markers currently used to monitor self-renewing mESCs and early differentiation events. All of the experiments shown were performed in R1 mESCs, but were repeated in the D3 line with similar results, indicating that these observations are a general feature of mESCs (Table 1).

DBA Distinguishes Between mESCs and Primitive Ectoderm—A High-Resolution Marker for Early Differentiation Commitment
mESCs and EPL cells represent two closely related pluripotent cell populations that, in the past, could have been discriminated at the molecular level only by transcript profiling (Fig. 2) [3, 5, 24]. Every differentiation pathway directed toward one of the three germ layers must transition through this cell type, emphasizing the importance of this lineage as an early differentiation intermediate. Our next objective was to test whether DBA could discriminate between mESCs and EPL cells as a stringent test of DBA's specificity and potential utility as an indicator for early differentiation. EPL cells were generated directly from mESCs [3, 23] by culture in the presence of MedII conditioned medium in the absence of LIF (Fig. 2). Over a period of 4 days, mESCs were uniformly converted into an early primitive ectoderm-like state [3], indicated by changes in gene expression profile and cell morphology (Figs. 2, 3A). Single cell suspensions were stained with FITC-conjugated lectins and analyzed by flow cytometry analysis. Although most lectins, such as PNA and wheat germ agglutinin, recognized carbohydrate epitopes presented on the surface of both cell populations, DBA binding was restricted to mESCs (Table 1; Figs. 2, 3). Several other lectins including Sophora japonica agglutinin (SJA) and Ulex europus agglutinin (UEA 1) recognized neither cell population. Hence, of the 18 lectins tested in this study, only DBA discriminated between mESCs and EPL cells, the latter of which represents the first definable step of differentiation.


Figure 2
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  		Figure 2. DBA discriminates between mESCs and early primitive ectoderm. (A): mESCs can be converted into a slightly more developmentally advanced population, known as EPL by withdrawal of leukemia inhibitory factor (LIF) in the presence of conditioned media (CM) from HepGII cells (MedII). The transition from mESCs to EPL cells is fully reversible by readdition of LIF and withdrawal of MedII-CM [3]. (B): Bright-field images of D3 mESCs and EPL cells generated by addition of CM for two (EPL2) or four (EPL4) days. mESC + EPL4 represents analysis performed on equal numbers of mESCs and d4 EPL cells mixed together. After 4 days of treatment with CM, EPL cells were reverted back to mESCs (EPL revert), as described previously. Scale bars = 50 µm. Gene expression states (mRNA), cell surface marker expression (SSEA1, alkaline phosphatase, flow cytometry, and immunostaining) and blastocyst colonization capacity for corresponding cell states have been documented previously [3, 7, 23, 24]. (C): Flow cytometry profiles for the corresponding cell populations shown in (B). Cells were analyzed without lectin or, after staining with fluorescein isothiocyanate (FITC)-DBA or FITC-peanut agglutinin. (D): High DBA reactivity is restored after the reversion of EPL cells to ESCs. FITC-DBA flow profiles for EPL cells (day 4) and EPL reverted cells (4 days). Abbreviations: AP, alkaline phosphatase; d, day; DBA, Dolichos biflorus agglutinin; EPL, primitive ectoderm; FL1, fluorescence 1 detector; LIF, leukemia inhibitory factor; mESC, murine ESC; PNA, peanut agglutinin; SSEA1, stage-specific embryonic antigen 1.

 


Figure 3
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  		Figure 3. DBA recognizes glycan epitopes that discriminate between R1 mESCs and the early stages of differentiation. (A): Transcript profiling of R1 mESCs and d2 and d4 EPL cells by quantitative polymerase chain reaction for c-myc, TERT, Oct4, CD9, and Fgf5. Transcript levels were determined in triplicate and shown as ± SEM after being normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Levels of transcripts in mESCs were normalized against GAPDH and assigned a value of 1. (B): Flow cytometry analysis of CD9, DBA, and SSEA1 in R1 mESCs; d2 and d4 EPL cells and d2 and d4 EPL reverted cells. (C): Immunostaining of cells probed with DBA or antibodies raised against SSEA1 and CD9 (shown in green). Cells were simultaneously stained with 4',6-diamidino-2-phenylindole, dihydrochloride (DNA, blue). (D): DBA epitopes in mESCs are insensitive to N-glycanase treatment. Glycoproteins were precipitated with DBA-beads from R1 mESC lysates (600 µg of total protein). Precipitates were resolved on a polyacrylamide gel, transferred to a filter membrane, and probed with biotinylated DBA followed by streptavidin-conjugated horseradish peroxidase (HRP). Lane 1, DBA precipitate; lane 2, N-glycanase F-treated DBA precipitate; lane 3, DBA precipitate probed only with streptavidin-HRP; lane 4, as for lane 3, but precipitate treated with N-glycanase F; lane 5, Fetuin (500 ng) untreated; lane 6, Fetuin (500 ng) treated with N-glycanase F. Arrows point to Fetuin in lanes 5 and 6, which shifts in mobility after N-glycanase F treatment. Abbreviations: d, day; DBA, Dolichos biflorus agglutinin; EPL, primitive ectoderm; FL1, fluorescence 1 detector; mESC, murine ESC; SSEA1, stage-specific embryonic antigen 1.

 
EPL cells can be reverted to mESCs by withdrawal of MedII conditioned medium and by the addition of LIF [3]. After 4 days, these cells exhibit a distinct dome-shaped mESC colony morphology, an mESC gene expression profile, and an mESC differentiation potential, including the ability to colonize a blastocyst and contribute to chimerism in adult mice [3, 23]. This program of dedifferentiation, or reprogramming, is homogeneous and complete within 4 days. To determine whether changes in lectin-binding epitopes on the surface were also a reversible event, we grew EPL cells in the absence of conditioned medium, supplemented with LIF. Within 4 days, these cells had reassumed a dome-shaped mESC morphology, and the DBA binding characteristics of mESCs was restored (Figs. 2, 3). Interconversion between the two cell states is, therefore, associated with distinct and reversible changes in cell surface glycoepitopes. To confirm these results, we characterized DBA binding to paraformaldehyde-fixed mESC, EPL, and reverted EPL cells while still attached to culture dishes. Consistent with flow cytometry data, DBA reactivity was high in mESCs and low in EPL cells, and was restored when EPL cells were reverted to mESCs (data not shown). Other lectins tested in this assay corroborated the flow cytometry data.

To confirm the resolution by which DBA distinguishes between mESCs and EPL cells, relative to currently used markers, we compared DBA, SSEA1, and CD9 epitopes on the surface of EPL cells. Whereas DBA was virtually extinguished in day-4 EPL cells, the CD9 profile had not shifted appreciably during this period (Fig. 3). Although there was a slight shift in the SSEA1 profile in day-4 EPL cells, it was heterogeneous, indicating that this marker does not accurately discriminate between mESCs and EPL cells. We conclude that DBA reliably discriminates between mESC and EPL cells in contrast to currently used cell surface markers such as CD9 and SSEA1. In total, these results validate the use of DBA as a marker for mESCs and as a means to evaluate differentiation events as early as the transition from mESCs to primitive ectoderm. Because EPL cells do not have the capacity colonize blastocyst embryos [3, 23], it is possible that DBA could be used as one criterion to predict the pluripotency of mESCs.

As an initial characterization of DBA epitopes, we asked whether mESC glycan structures recognized by this lectin were glycoprotein associated and whether they were N- or O-linked. Lectin blots of DBA precipitates from whole-cell lysates reveal the complexity of GalNAc-conjugated glycoproteins in mESCs (Fig. 3D). Although some nuclear and cytoplasmic proteins are GlcNAc modified on serines and threonines, GalNAc modifications are exclusively associated with secreted or cell surface glycans [26], indicating that the DBA epitopes detected in lectin blots correspond to those detected on the cell surface in intact cells. We then asked whether these glycoproteins were N- or O- linked by digesting DBA precipitates with N-glycanase F, an enzyme that specifically cleaves N-linked, but not O-linked, glycan structures[27, 28]. This analysis showed that DBA-precipitated proteins were insensitive to digestion, whereas Fetuin, a serum protein with well-characterized N-linked structures, was sensitive to N-glycanase F treatment (Fig. 3D). This indicates that epitopes recognized by DBA in our studies are O-linked glycoproteins, although we cannot rule out the possibility that GalNAc glycolipid epitopes recognized by DBA are also present on the surface of mESCs.

As proof of concept that DBA can be used to discriminate between mESCs and early primitive ectoderm, we mixed both populations, probed them with FITC-DBA, and separated DBAhigh and DBAlow cells by FACS (Fig. 4A). Rex1, Fgf5, and Gbx2 transcript levels in DBAhigh and DBAlow populations were then evaluated by Q-PCR. As expected, DBAhigh cells expressed high levels of Gbx2 and Rex1 transcripts, but, in contrast, DBAlow cells expressed low levels of Gbx2 and Rex1 mRNA but elevated levels of Fgf5 transcript (Fig. 4B). DBA can, therefore, be used to identify and discriminate between ESCs and EPL cells by FACS.


Figure 4
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  		Figure 4. Separation of murine embryonic stem cells (mESCs) and early primitive ectoderm (EPL) based on Dolichos biflorus agglutinin (DBA) reactivity by fluorescence-activated cell sorting (FACS) analysis. (A): 2.5 x 106 R1 ESCs and R1-derived EPL cells (day 4) were mixed, stained with DBA-fluorescein isothiocyanate (100 µg/ml) and then sorted into DBAhigh and DBAlow populations by FACS. The high and low selected populations in the FACS profile are indicated by brackets. (B): Gene expression in both populations was then evaluated in triplicate by quantitative polymerase chain reaction using TaqMan probes (Applied Biosystems) for Rex1, Fgf5 and Gbx2. Abbreviation: FL1, fluorescence 1 detector.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
This work highlights the deficiencies associated with current cell surface markers used to characterize pluripotent mESCs. Here, we report that the lectin DBA will be a more reliable indicator of the pluripotent state and as readout for cells undergoing early differentiation. The resolution of DBA as a marker is highlighted by its ability to discriminate between mESCs and primitive ectoderm (EPL). Because EPL cells have lost the capacity to colonize blastocyst-stage embryos [3], DBA is also likely to have utility as an indicator of developmental pluripotency. None of the currently used cell surface markers can be used for this purpose. DBA will also have utility as a sensitive, high-resolution tool for monitoring the early stages of differentiation in living cells that was not previously possible using other markers such as SSEA1, CD9, and FA. Interestingly, the only sugar implicated to change dramatically on the surface of mESCs during differentiation was GalNAc. It will be of interest to determine how GalNAc epitopes are regulated on the surface of mESCs as they differentiate and to determine the identity of proteins/lipids marked with the sugar.

Even though the sugar recognized by DBA, GalNAc, is part of the Forssman antigen [29] our results indicate that their pattern of reactivity is not completely overlapping. Flow cytometry and immunostaining shows that, during ESC differentiation, DBA epitopes decrease before those recognized by antibodies raised against FA. This could be explained by differences in the affinity of lectins and antibodies for their targets. For example, the association constant of DBA for different GalNAc ligands is in the 1 x 103 mol–1 range, whereas the association constant for an antibody usually ranges between 105 and 1012 mol–1 [30]. This higher affinity may explain why, during the early stages of differentiation, when the number of epitopes is decreasing, residual FA is detected with an antibody, whereas reactivity against the lectin DBA is lost. Our analysis indicates that epitopes recognized by DBA in mESCs are O-linked glycoproteins. If the anti-Forssman antibody and DBA are recognizing the same epitopes, this would be one of the few cases in which FA is part of a glycoprotein instead of a glycolipid [29, 32–34]. Further characterization of the DBA epitopes in mESCs should help resolve this question.

Figure 5 illustrates the temporal expression of different epitopes during mESC differentiation. Cell surface epitopes previously used as mESC markers (SSEA1, CD9, AP, FA) can be placed into two broad categories: CD9 and FA, which are elevated in mESCs and EPL cells but rapidly downregulated during lineage commitment, and SSEA1 and AP, which are generally lost in cells after CD9 and FA become downregulated [3, 5, 6]. Our observations that DBA epitopes are downregulated before any of these markers now allows us to define cell populations with greater temporal resolution. In combination with these markers, DBA can be used to define in detail the early stages of differentiation in living cell populations. 5T4 oncofetal antigen was recently reported to have utility as an indicator of mESC differentiation and as a means of signifying loss of blastocyst colonization potential [6]. 5T4, however, appears on the cell surface after the formation of primitive ectoderm, at approximately the time when CD9 and FA are downregulated. Our data indicate that DBA will have additional utility in this area because downregulation of DBA coincides with primitive ectoderm formation (as described herein) and loss of blastocyst colonization capacity [3].


Figure 5
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  		Figure 5. Temporal expression of cell surface markers used for the characterization and purification of mESCs. Although CD9, FA, and SSEA1 are expressed on the surface of mESCs they are downregulated after the formation of primitive ectoderm, sometimes coinciding with the appearance of a general cell surface differentiation markers such as 5T4 [6]. DBA, however, is downregulated at the time of the earliest definable differentiation transition (primitive ectoderm), coinciding with loss of blastocyst colonization capacity [3, 5, 23], establishing it as a high resolution marker for the mESC state, pluripotency and the onset of differentiation. Abbreviations: AP, alkaline phosphatase; DBA, Dolichos biflorus agglutinin; FA, Forssman antigen; mESC, murine embryonic stem cell; SSEA1, stage-specific embryonic antigen 1.

 
Although the role of GalNAc residues on the surface of pluripotent mESCs is not currently understood, this glycoepitope is also present on the surface of ICM cells in the preimplantation embryo. Sato and Muramatsu [31] reported that high DBA binding in the ICM of mouse blastocysts is lost, however, by the late egg cylinder stage, although the exact timing of this event was not determined at high resolution. It is of interest that Forssman antigen shows a similar developmental profile to that described for DBA by Sato and Muramatsu [31, 35]. Together with our analysis of DBA epitopes in mESCs, this suggests that Forssman antigen and DBA epitopes in the early embryo may be glycoprotein associated. It is possible that our in vitro data reflect cell surface changes equivalent to those described during early development. If so, this would indicate that changes in carbohydrate composition on the cell surface are, in fact, similar between embryonic cells and their in vitro equivalents, pointing toward some biological function associated with pluripotent cell biology.

The cell surface markers currently used to characterize hESCs (SSEA3,4 and TRA1-60/80) have similar limitations to those of the conventional mESC markers described in this report. Therefore, there is a clear requirement to identify new cell surface markers that can more reliably assess the hESC state and to monitor early differentiation events. Although DBA epitopes are not enriched on the surface of hESCs (unpublished data), it is possible that, through glycoepitope screening, new tools that can be used for their characterization will be identified.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Gerardo Alvarez-Manilla, Karen Abbott, and members of the Dalton laboratory for useful comments throughout the course of this work. We also thank the faculty of the Center for Complex Carbohydrate Research at the University of Georgia for their support and advice. This work was funded by the Georgia Cancer Coalition, the Georgia Research Alliance, and the NIH through the Program for Integrated Biomedical Technology Research Resources for Proteomics and Glycomics at the University of Georgia.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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