Detection and Characterization of 2-E2-Specific Surface Protein in Human Pluripotent Stem Cells

Introduction

Human embryonic stem cells (hESCs) are pluripotent stem cells that are defined by their ability to self-renew and differentiate into a variety of specialized cells.^(1,2) Therefore, hESCs may be an ideal source of therapeutic cells in many damage areas and diseases. Specific markers have been used to define the pluripotent and undifferentiated state of hESCs. Such markers include the transcription factors Oct-4 and Nanog and various cell surface markers, such as SSEA3, SSEA4, TRA-1-60, TRA-1-81, and alkaline phosphatase.^(2,3) The surface markers are associated with carbohydrate epitopes; their function in the pluripotent and undifferentiated state of hESCs has been uncertain to date.^(4–6)

In order to study the effect of the external signal on hESC self-renewal and pluripotency and to prospectively isolate hESCs from the mixed cell population, it is necessary to find the specific surface markers for hESCs. Previously, we generated a panel of monoclonal antibodies (MAbs) specific to surface molecules of undifferentiated hESCs by using a decoy immunization strategy.⁽⁷⁾ During further study of the isolated MAbs, 2-E2 was specifically shown to bind to human pluripotent stem cells, but not to mouse pluripotent stem cells. In addition, 2-E2 unexpectedly bound to human differentiated peripheral blood monocytes (PBMs). Therefore, the level of 2-E2 antigen was dissected during differentiation. The surface protein recognized by 2-E2 was also investigated.

Materials and Methods

Results and Discussion

To identify and characterize cell surface molecules on pluripotent and undifferentiated hESCs, we previously generated 70 MAbs against hESCs using a modified decoy immunization strategy.⁽⁷⁾ Most MAbs bound to undifferentiated hESCs, but only weakly or not at all to differentiated hESCs. As an attractive result, MAb 2-E2 only was shown to bind PBMCs, which was used as a differentiated primary human cell. Then 2-E2 was purified from ascitic fluid and used for further characterization in this study. In flow cytometric analysis, 2-E2 bound to human pluripotent stem cells H1, H9, CHA-hES4, NT-2/D1, and NCCIT, but not to mESC lines J1, R1, and E14Tg2a.4, and mouse embryonic fibroblasts (MEFs) (Fig. 1A). When PBMCs were gated on peripheral blood lymphocytes (PBLs) or peripheral blood monocytes (PBMs), 2-E2 did not bind to PBLs, but bound to PBMs. 2-E2 binding capability was also observed in DFCs (Fig. 1A), whereas it did not bind to RA-induced differentiated H9 cells (Fig. 1A). Therefore, we hypothesized that 2-E2 antigen expression may fluctuate during differentiation of hESCs.

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FIG. 1.

(A) 2-E2 antigen expression on hESCs and various cells. 2-E2 antigen expression was examined by flow cytometry in human pluripotent stem cells (H1, H9, CHA-hES4, NT-2/D1, NCCIT) and differentiated cells PBL, PBM, DFC, and RA-treated H9 cells. Mouse embryonic stem cells J1, R1, and E14Tg2a.4 and MEFs were also included as control cells. Cells were stained with 2-E2 followed by FITC-conjugated anti-mouse IgG. Non-viable cells identified with PI were excluded. (B) The level of 2-E2 antigen gradually increased after initial decrease during EB formation. H9 cells were differentiated for 14 days by RA treatment or EB formation. Cells were dissociated and incubated with 2-E2 or anti-TRA-1-81 at days 4, 7, 10, and 14, and then incubated with FITC-conjugated secondary antibodies. Binding percentages of TRA-1-81 and 2-E2 to undifferentiated cells (ES), EB cells, or RA-treated cells (RA) were measured by mean fluorescence intensities, normalized to control secondary antibody binding. The percentages were measured at least twice and averages of the measured percentages are presented.

In order to dissect the surface expression of 2-E2 antigen during differentiation, H9 cells were differentiated for 14 days by two different ways––RA treatment and EB formation––and the levels of 2-E2 antigen were measured by using mean fluorescence intensities from flow cytometric analyses (Fig. 1B). The levels of the hESC surface marker TRA-1-81 and 2-E2 antigens were generally maintained in undifferentiated H9 cells, although slight decreases were observed due to spontaneous differentiation over time. When H9 cells were treated with RA, the levels of TRA-1-81 and 2-E2 antigens was rapidly decreased, although the level of 2-E2 antigen was slightly decreased until day 7. The result was expected from our previous study.⁽⁷⁾ When EB cells were examined for the level of TRA-1-81, the level of TRA-1-81 was rapidly decreased over time as expected. The level of 2-E2 antigen was also decreased in EB cells, and approximately 50% of EB cells remained positive for 2-E2 antigen at day 4 (Fig. 1B). However, the level of 2-E2 antigen was gradually increased in subsequent differentiation. The results suggest that 2-E2 antigen may regulate the early stage of hESC differentiation.

In order to identify the cell surface antigen recognized by 2-E2, the surface proteins of H9 cells were biotinylated, immunoprecipitated with 2-E2, and visualized with SA-HRP. MAb 2-E2 immunoprecipitated ∼68 and 27 kDa proteins from H9 cells (Fig. 2A). The same result was obtained from NT-2/D1 cells (Fig. 2B). In Western blot analysis, however, 2-E2 was not able to detect the 68 and 27 kDa proteins (data not shown), suggesting that 2-E2 may recognize the 2-E2 antigen in a conformation-dependent manner.

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FIG. 2.

Detection of 2-E2 antigen. H9 cells (A) and NT-2/D1 (B) were biotinylated, and the cell lysates were subjected to immunoprecipitation with 2-E2 and protein-G Sepharose beads. Immunoprecipitated proteins were separated on a SDS-PAGE gel, transferred to Western membrane, and visualized with SA-HRP. The immunoprecipitated proteins are indicated by arrowheads.

As expected from our previous report,⁽⁷⁾ 2-E2 binding capacity to hESCs was downregulated during RA-induced differentiation (Fig. 1). Interestingly, when the level of 2-E2 antigen was examined during EB formation, it gradually increased after initial downregulation (Fig. 1B), indicating that 2-E2 antigen may be re-expressed in adult differentiated primary cells. Consistent with this notion, we could detect 2-E2 antigen expression in DFCs and PBMs. Therefore, 2-E2 antigen would be a surface protein that plays a role in the transition period from self-renewal phase to differentiation phase of human pluripotent stem cells. 2-E2 antigen turned out to be 68 and 27 kDa cell surface proteins (Fig. 2). However, the 27 kDa protein was not always detected in cell surface biotinylation and immunoprecipitation assay, suggesting that it may be the truncated form of 68 kDa protein (data not shown). Although we do not know the identity of 2-E2-specific 68 kDa protein at the moment, the molecular characteristics of the 2-E2-specific 68 kDa protein make the protein worthy of future study for use as an indicator of early differentiation of human pluripotent stem cells.