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


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.

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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.


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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).

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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.

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Correspondence to Irving L Weissman or Micha Drukker.

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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).

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