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Background: Human embryonic stem cells (hESCs) and human embryonal carcinoma cells (hECCs) have been extensively used for stem cell research. Although these cells are known to share many properties including high developmental capability and cell surface antigens, their origins are basically different: hECSs are derived from inner cell mass of blastocysts, while hECCs are from malignant tumors. Thus, the lack of a good method to differentiate these pluripotent cells remains a critical issue to diagnose tumorigenic potential of pluripotent stem cells for their medical applications. In this context, development of specific markers to distinguish hESCs from hECCs is also of clinical value.
Method: In this study, we focused our glycan analysis on a carbohydrate-rich glycoprotein, podocalyxin, known as a carrier of TRA-1-60 and TRA-1-81 antigens, which represent hESC glycan markers. The target glycoprotein semi-quantified by immunoblotting was enriched from the cell extracts by immunoprecipitation, and the glycosylation differences occurring between hESCs and hECCs were systematically analyzed by an advanced technology of lectin microarray, antibody-overlay lectin profiling (ALP). Profiles of human embryonic bodies (hEBs) differentiated from hESCs were also analyzed.
Results and Conclusion: A glycan profile of podocalyxin from hECCs was significantly different from that of hESCs. Lectin signals corresponding to α2-6 linked sialic acid were elevated in the hECCs, and glycosidase digestions further revealed significant difference in the non-reducing terminal and penultimate structures. These results demonstrate that the present procedure with focus on a particular glycoprotein could enhance relatively small but significant differences between closely related cells like hESCs and hECCs at the glycome level. The present finding will be helpful to develop a diagnostic method to distinguish undifferentiated stem cells from differentiated ones used for regenerative therapy.