Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells

Abstract

An important risk in the clinical application of human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), is teratoma formation by residual undifferentiated cells. We raised a monoclonal antibody against hESCs, designated anti–stage-specific embryonic antigen (SSEA)-5, which binds a previously unidentified antigen highly and specifically expressed on hPSCs—the H type-1 glycan. Separation based on SSEA-5 expression through fluorescence-activated cell sorting (FACS) greatly reduced teratoma-formation potential of heterogeneously differentiated cultures. To ensure complete removal of teratoma-forming cells, we identified additional pluripotency surface markers (PSMs) exhibiting a large dynamic expression range during differentiation: CD9, CD30, CD50, CD90 and CD200. Immunohistochemistry studies of human fetal tissues and bioinformatics analysis of a microarray database revealed that concurrent expression of these markers is both common and specific to hPSCs. Immunodepletion with antibodies against SSEA-5 and two additional PSMs completely removed teratoma-formation potential from incompletely differentiated hESC cultures.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Anti-SSEA-5 mAb is specific for hPSCs.
Figure 2: Anti-SSEA-5 mAb enables partial removal of teratoma-initiating cells.
Figure 3: Depletion of cells concurrently expressing three PSMs eliminates teratoma-initiation potential.

References

  1. Blum, B. & Benvenisty, N. The tumorigenicity of human embryonic stem cells. Adv. Cancer Res. 100, 133–158 (2008).

    Article  Google Scholar 

  2. Tang, C. & Drukker, M. Potential barriers to therapeutics utilizing pluripotent cell derivatives: intrinsic immunogenicity of in vitro maintained and matured populations. Semin. Immunopathol. published online, doi: 10.1007/s00281-011-0269-5 (11 April 2011).

  3. Schuldiner, M., Itskovitz-Eldor, J. & Benvenisty, N. Selective ablation of human embryonic stem cells expressing a “suicide” gene. Stem Cells 21, 257–265 (2003).

    Article  CAS  Google Scholar 

  4. Cao, F. et al. Molecular imaging of embryonic stem cell misbehavior and suicide gene ablation. Cloning Stem Cells 9, 107–117 (2007).

    Article  CAS  Google Scholar 

  5. Choo, A.B. et al. Selection against undifferentiated human embryonic stem cells by a cytotoxic antibody recognizing podocalyxin-like protein-1. Stem Cells 26, 1454–1463 (2008).

    Article  CAS  Google Scholar 

  6. Tan, H.L., Fong, W.J., Lee, E.H., Yap, M. & Choo, A. mAb 84, a cytotoxic antibody that kills undifferentiated human embryonic stem cells via oncosis. Stem Cells 27, 1792–1801 (2009).

    Article  CAS  Google Scholar 

  7. Drukker, M., Muscat, C. & Weissman, I.L. Generation of a monoclonal antibody library against human embryonic stem cells. Methods Mol. Biol. 407, 63–81 (2007).

    Article  CAS  Google Scholar 

  8. Andrews, P.W., Banting, G., Damjanov, I., Arnaud, D. & Avner, P. Three monoclonal antibodies defining distinct differentiation antigens associated with different high molecular weight polypeptides on the surface of human embryonal carcinoma cells. Hybridoma 3, 347–361 (1984).

    Article  CAS  Google Scholar 

  9. Shevinsky, L.H., Knowles, B.B., Damjanov, I. & Solter, D. Monoclonal antibody to murine embryos defines a stage-specific embryonic antigen expressed on mouse embryos and human teratocarcinoma cells. Cell 30, 697–705 (1982).

    Article  CAS  Google Scholar 

  10. Kannagi, R. et al. Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells. EMBO J. 2, 2355–2361 (1983).

    Article  CAS  Google Scholar 

  11. Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    Article  CAS  Google Scholar 

  12. Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A. & Bongso, A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat. Biotechnol. 18, 399–404 (2000).

    Article  CAS  Google Scholar 

  13. Behr, B. et al. Blastocyst-ET and monozygotic twinning. J. Assist. Reprod. Genet. 17, 349–351 (2000).

    Article  CAS  Google Scholar 

  14. Stevens, L.C. & Little, C.C. Spontaneous testicular teratomas in an inbred strain of mice. Proc. Natl. Acad. Sci. USA 40, 1080–1087 (1954).

    Article  CAS  Google Scholar 

  15. Hentze, H. et al. Teratoma formation by human embryonic stem cells: Evaluation of essential parameters for future safety studies. Stem Cell Res. 2, 198–210 (2009).

    Article  Google Scholar 

  16. Wu, J.C., Sundaresan, G., Iyer, M. & Gambhir, S.S. Noninvasive optical imaging of firefly luciferase reporter gene expression in skeletal muscles of living mice. Mol. Ther. 4, 297–306 (2001).

    Article  CAS  Google Scholar 

  17. Lee, A.S. et al. Effects of cell number on teratoma formation by human embryonic stem cells. Cell Cycle 8, 2608–2612 (2009).

    Article  CAS  Google Scholar 

  18. Vodyanik, M.A. & Slukvin, II. Hematoendothelial differentiation of human embryonic stem cells. Curr. Protoc. Cell Biol. 36, 23.6.1–23.6.28 (2007).

    Article  Google Scholar 

  19. Nagano, K., Yoshida, Y. & Isobe, T. Cell surface biomarkers of embryonic stem cells. Proteomics 8, 4025–4035 (2008).

    Article  CAS  Google Scholar 

  20. Raman, R. et al. Advancing glycomics: implementation strategies at the consortium for functional glycomics. Glycobiology 16, 82R–90R (2006).

    Article  CAS  Google Scholar 

  21. Torrado, J., Gutierrez Hoyos, A., Blasco, E., Larraz, J. & Fernandez Rivas, J.L. Immunohistological patterns of blood group ABO and type 1 chain (Lewis a Lewis b) and type 2 chain (H-2, Y) antigens in normal uterine cervix. Tissue Antigens 36, 8–11 (1990).

    Article  CAS  Google Scholar 

  22. Rosler, E.S. et al. Long-term culture of human embryonic stem cells in feeder-free conditions. Dev. Dyn. 229, 259–274 (2004).

    Article  CAS  Google Scholar 

  23. Mateizel, I. et al. Characterization of CD30 expression in human embryonic stem cell lines cultured in serum-free media and passaged mechanically. Hum. Reprod. 24, 2477–2489 (2009).

    Article  CAS  Google Scholar 

  24. Draper, J.S., Pigott, C., Thomson, J.A. & Andrews, P.W. Surface antigens of human embryonic stem cells: changes upon differentiation in culture. J. Anat. 200, 249–258 (2002).

    Article  CAS  Google Scholar 

  25. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

    Article  CAS  Google Scholar 

  26. Sahoo, D. et al. MiDReG: a method of mining developmentally regulated genes using Boolean implications. Proc. Natl. Acad. Sci. USA 107, 5732–5737 (2010).

    Article  CAS  Google Scholar 

  27. Inlay, M.A. et al. Ly6d marks the earliest stage of B-cell specification and identifies the branchpoint between B-cell and T-cell development. Genes Dev. 23, 2376–2381 (2009).

    Article  CAS  Google Scholar 

  28. Solter, D. & Knowles, B.B. Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1). Proc. Natl. Acad. Sci. USA 75, 5565–5569 (1978).

    Article  CAS  Google Scholar 

  29. Hakomori, S. Glycosphingolipids in cellular interaction, differentiation, and oncogenesis. Annu. Rev. Biochem. 50, 733–764 (1981).

    Article  CAS  Google Scholar 

  30. Liang, Y.J. et al. Switching of the core structures of glycosphingolipids from globo- and lacto- to ganglio-series upon human embryonic stem cell differentiation. Proc. Natl. Acad. Sci. USA 107, 22564–22569 (2010).

    Article  CAS  Google Scholar 

  31. Sahoo, D., Dill, D.L., Gentles, A.J., Tibshirani, R. & Plevritis, S.K. Boolean implication networks derived from large scale, whole genome microarray datasets. Genome Biol. 9, R157 (2008).

    Article  Google Scholar 

  32. Goldman, J.P. et al. Enhanced human cell engraftment in mice deficient in RAG2 and the common cytokine receptor gamma chain. Br. J. Haematol. 103, 335–342 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors acknowledge C. Contag for providing luciferase constructs, M. van de Rijn and K. Montgomery for their assistance scanning fetal array slides and providing online access to these slides, P. Chu for assistance with hematoxylin and eosin staining, C. Muscat and T. Naik for assistance with hybridoma culture, W. Zhang for assistance in cell culturing, the Consortium for Functional Glycomics for providing and testing glycan arrays, and T. Serwold and C. Bertozzi for critical advice. This work was supported by funds provided by the California Institute of Regenerative Medicine (CIRM) (Comprehensive grant RC1-00354-1). C.T. and A.S.L. are supported by the Howard Hughes Medical Institute Medical Fellows and the Stanford Medical Scholars Program, J.-P.V. is supported by the Deutsche Forschungsgemeinschaft, C.T., M.A.I., R.A. and M.D. are supported by CIRM (Comprehensive grant RC1-00354-1).

Author information

Authors and Affiliations

Authors

Contributions

C.T., J.-P.V., I.L.W. and M.D. designed the experiments and wrote the manuscript. C.T., A.S.L., J.-P.V., D.S., A.R.M., D.N., M.A.I. and M.D. performed the experiments and analyzed data. R.A., S.L.C., R.R.P., B.B. and J.C.W. provided samples and reagents. All authors endorse the full content of this work.

Corresponding authors

Correspondence to Irving L Weissman or Micha Drukker.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Table 1 and Supplementary Figures 1–7 (PDF 1998 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Tang, C., Lee, A., Volkmer, JP. et al. An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells. Nat Biotechnol 29, 829–834 (2011). https://doi.org/10.1038/nbt.1947

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.1947

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing