Skip to main content

Advertisement

SpringerLink
Log in

Separation of SSEA-4 and TRA-1–60 Labelled Undifferentiated Human Embryonic Stem Cells from A Heterogeneous Cell Population Using Magnetic-Activated Cell Sorting (MACS) and Fluorescence-Activated Cell Sorting (FACS)

  • Report
  • Published:
Stem Cell Reviews and Reports Aims and scope Submit manuscript

Abstract

A major concern in human embryonic stem cell (hESC)-derived cell replacement therapy is the risk of tumorigenesis from undifferentiated hESCs residing in the population of hESC-derived cells. Separation of these undifferentiated hESCs from the differentiated derivatives using cell sorting methods may be a plausible approach in overcoming this problem. We therefore explored magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) to separate labelled undifferentiated hESCs from a heterogeneous population of hESCs and hepatocellular carcinoma cells (HepG2) deliberately mixed respectively at different ratios (10:90, 20:80, 30:70, 40:60 and 50:50) to mimic a standard in vitro differentiation protocol, instead of using a hESC-differentiated cell population, so that we could be sure of the actual number of cells separated. HES-3 and HES-4 cells were labelled in separate experiments for the stem cell markers SSEA-4 and TRA-1–60 using primary antibodies. Anti-PE magnetic microbeads that recognize the PE-conjugated SSEA-4 labelled hESCs was added to the heterogeneous cell mixture and passed through the MACS column. The cells that passed through the column (‘flow-through’ fraction) and those retained (‘labelled’ fraction’) were subsequently analysed using FACS. The maximum efficacy of hESCs retention using MACS was 81.0 ± 2.9% (HES-3) and 83.6 ± 4.2% (HES-4). Using FACS, all the undifferentiated hESCs labelled with the two cell-surface markers could be removed by selective gating. Both hESCs and HepG2 cells in the ‘flow-through’ fraction following MACS separation were viable in culture whereas by FACS separation only the HepG2 cells were viable. FACS efficiently helps to eliminate the undifferentiated hESCs based on their cell-surface antigens expressed.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

References

  1. Aleckovic, M., & Simon, C. (2008). Is teratoma formation in stem cell research a characterization tool or a window to developmental biology? Reproductive Biomedicine Online, 17, 270–280.

    Article  PubMed  Google Scholar 

  2. Ben-Hur, T., Idelson, M., Khaner, H., Pera, M., Reinhartz, E., Itzik, A., et al. (2004). Transplantation of human embryonic stem cell-derived neural progenitors improves behavioral deficit in Parkinsonian rats. Stem Cells, 22, 1246–1255. doi:10.1634/stemcells.2004-0094.

    Article  PubMed  Google Scholar 

  3. Bielby, R. C., Boccaccini, A. R., Polak, J. M., & Buttery, L. D. (2004). In vitro differentiation and in vivo mineralization of osteogenic cells derived from human embryonic stem cells. Tissue Engineering, 10, 1518–1525.

    PubMed  CAS  Google Scholar 

  4. Bongso, A., Fong, C. Y., Ng, S. C., & Ratnam, S. (1994). Isolation and culture of inner cell mass cells from human blastocysts. Human Reproduction, 9, 2110–2117.

    PubMed  CAS  Google Scholar 

  5. Cao, F., Lin, S., Xie, X., Ray, P., Patel, M., Zhang, X., et al. (2006). In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery. Circulation, 113, 1005–1014. doi:10.1161/CIRCULATIONAHA.105.588954.

    Article  PubMed  Google Scholar 

  6. Choo, A. B., Tan, H. L., Ang, S. N., Fong, W. J., Chin, A., Lo, J., Zheng, L., et al. (2008). Selection against undifferentiated human embryonic stem cells by a cytotoxic antibody recognizing podocalyin-like protein-1. Stem Cells, 26, 1454–1463. doi:10.1634/stemcells.2007-0576.

    Article  PubMed  CAS  Google Scholar 

  7. Elkabetz, Y., Panagiotakos, G., Al Shamy, G., Socci, N. D., Tabar, V., & Studer, L. (2008). Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage. Genes & Development, 22, 152–165. doi:10.1101/gad.1616208.

    Article  CAS  Google Scholar 

  8. Erd, F., Buhrle, C., Blunk, J., Hoehn, M., Xia, Y., Fleischmann, B., et al. (2003). Host-dependent tumorigenesis of embryonic stem cell transplantation in experimental stroke. Journal of Cerebral Blood Flow and Metabolism, 23, 780–785. doi:10.1097/01.WCB.0000071886.63724.FB.

    Google Scholar 

  9. Fujikawa, T., Oh, S. H., Pi, L., Hatch, H. M., Shupe, T., & Petersen, B. E. (2005). Teratoma formation leads to failure of treatment for type I diabetes using embryonic stem cell-derived insulin-producing cells. American Journal of Pathology, 166, 1781–1791.

    PubMed  CAS  Google Scholar 

  10. Geens, M., Van de Velde, H., De Block, G., Goossens, E., Van Steirteghem, A., & Tournaye, H. (2006). The efficiency of magnetic-activated cell sorting and fluorescence-activated cell sorting in the decontamination of testicular cell suspensions in cancer patients. Human Reproduction, 22, 733–742. doi:10.1093/humrep/del418.

    Article  PubMed  Google Scholar 

  11. Henderson, J. K., Draper, J. S., Baillie, H. S., Fishel, S., Thomson, J. A., Moore, H., et al. (2002). Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens. Stem Cells, 20, 329–337. doi:10.1634/stemcells.20-4-329.

    Article  PubMed  CAS  Google Scholar 

  12. Adewumi, O., Aflatoonian, B., Ahrlund-Richter, L., Amit, M., Andrews, P., Beighton, G., et al., International Stem Cell Initiative. (2007). Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nature Biotechnology, 25, 803–816. doi:10.1038/nbt1318.

    Article  PubMed  CAS  Google Scholar 

  13. Laflamme, M. A., Chen, K. Y., Naumova, A. V., Muskheli, V., Fugate, J. A., Dupras, S. K., et al. (2007). Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nature Biotechnology, 25, 1015–1024. doi:10.1038/nbt1327.

    Article  PubMed  CAS  Google Scholar 

  14. Laslett, A. L., Grimmond, S., Gardiner, B., Stamp, L., Lin, A., Hawes, S. M., et al. (2007). Transcriptional analysis of early lineage commitment in human embryonic stem cells. BMC Evolutionary Biology, 7, 12. doi:10.1186/1471-213X-7-12.

    Article  CAS  Google Scholar 

  15. Lawrenz, B., Schiller, H., Willbold, E., Ruediger, M., Muhs, A., & Esser, S. (2004). Highly sensitive biosafety model for stem-cell-derived grafts. Cytotherapy, 6, 212–222. doi:10.1080/14653240410006031.

    Article  PubMed  CAS  Google Scholar 

  16. Ludwig, D. L., Pereira, D. S., Zhu, Z., Hicklin, D. J., & Bohlen, P. (2003). Monoclonal antibody therapeutics and apoptosis. Oncogene, 22, 9097–9106. doi:10.1038/sj.onc.1207104.

    Article  PubMed  CAS  Google Scholar 

  17. Narasipura, S. D., Wojciechowski, J. C., Charles, N., Liesveld, J. L., & King, M. R. (2007). P-Selectin coated microtube for enrichment of CD34+ hematopoietic stem and progenitor cells from human bone marrow. Clinical Chemistry, 54, 77–85. doi:10.1373/clinchem.2007.089896.

    Article  PubMed  CAS  Google Scholar 

  18. Nussbaum, J., Minami, E., Laflamme, M. A., Virag, J. A., Ware, C. B., Masino, A., et al. (2007). Transplantation of undifferentiated murine embryonic stem cells in the heart: teratoma formation and immune response. FASEB Journal, 21, 1345–1357. doi:10.1096/fj.06-6769com.

    Article  PubMed  CAS  Google Scholar 

  19. Peh, G. S., Lang, R., Pera, M., & Hawes, S. (2008). CD133 expression by neural progenitors derived from human embryonic stem cells and its use for their prospective isolation. Stem Cells Dev. doi:10.1089/scd.2008.0124.

  20. Prokhorova, T. A., Harkness, L. M., Frandsen, U., Ditzel, N., Burns, J. S., Schroeder, H. D., et al. (2008). Teratoma Formation by Human Embryonic Stem Cells is site-dependent and enhanced by the presence of Matrigel. Stem Cells Dev. doi:10.1089/scd.2007.0266.

  21. Pruszak, J., Sonntag, K. C., Aung, M. H., Sanchez-Pernaute, R., & Isacson, O. (2007). Markers and methods for cell sorting of human embryonic stem cell-derived neural cell populations. Stem Cells, 25, 2257–2268. doi:10.1634/stemcells.2006-0744.

    Article  PubMed  Google Scholar 

  22. Reubinoff, B. E., Pera, M. F., Fong, C. Y., Trounson, A., & Bongso, A. (2000). Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nature Biotechnology, 18, 399–404. doi:10.1038/74447.

    Article  PubMed  CAS  Google Scholar 

  23. Servida, F., Soligo, D., Caneva, L., Bertolini, F., de Harven, E., Campiglio, S., et al. (1996). Functional and morphological characterization of immunomagnetically selected CD34+ hematopoietic progenitor cells. Stem Cells, 14, 430–438.

    Article  PubMed  CAS  Google Scholar 

  24. Shevinsky, L. H., Knowles, B. B., Damjanov, I., & Solter, D. (1982). Monoclonal antibody to murine embryos defines a stage-specific embryonic antigen expressed on mouse embryos and human teratocarcinoma cells. Cell, 30, 697–705. doi:10.1016/0092-8674(82)90274-4.

    Article  PubMed  CAS  Google Scholar 

  25. Shibata, H., Ageyama, N., Tanaka, Y., Kishi, Y., Sasaki, K., Nakamura, S., et al. (2006). Improved safety of hematopoietic transplantation with monkey embryonic stem cells in the allogeneic setting. Stem Cells, 24, 1450–1457. doi:10.1634/stemcells.2005-0391.

    Article  PubMed  Google Scholar 

  26. Shih, C. C., Forman, S. J., Chu, P., & Slovak, M. (2007). Human embryonic stem cells are prone to generate primitive, undifferentiated tumors in engrafted human fetal tissues in severe combined immunodeficient mice. Stem Cells Development, 16, 893–902. doi:10.1089/scd.2007.0070.

    Article  CAS  Google Scholar 

  27. Shim, J. H., Kim, S. E., Woo, D. H., Kim, S. K., Oh, C. H., McKay, R., et al. (2007). Directed differentiation of human embryonic stem cells towards a pancreatic cell fate. Diabetologia, 50, 1228–1238. doi:10.1007/s00125-007-0634-z.

    Article  PubMed  CAS  Google Scholar 

  28. Steff, A. M., Fortin, M., Philippoussis, F., Lesage, S., Arguin, C., Johnson, P., et al. (2003). A cell death pathway induced by antibody-mediated cross-linking of CD45 on lymphocytes. Critical Reviews in Immunology, 23, 421–440. doi:10.1615/CritRevImmunol.v23.i56.40.

    Article  PubMed  CAS  Google Scholar 

  29. Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., et al. (1998). Embryonic stem cell lines derived from human blastocysts. Science, 282, 1145–1147. doi:10.1126/science.282.5391.1145.

    Article  PubMed  CAS  Google Scholar 

  30. Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., et al. (2007). A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nature Biotechnology, 25, 681–686. doi:10.1038/nbt1310.

    Article  PubMed  CAS  Google Scholar 

  31. Wojciechowski, J. C., Narasipura, S. D., Charles, N., Mickelsen, D., Rana, K., Blair, M. L., et al. (2008). Capture and enrichment of CD34-positive haematopoietic stem and progenitor cells from blood circulating using P-selectin in an implantable device. British Journal of Haematology, 140, 673–681. doi:10.1111/j.1365-2141.2007.06967.x.

    Article  PubMed  Google Scholar 

  32. Yang, D., Zhang, Z. J., Oldenburg, M., Ayala, M., & Zhang, S. C. (2008). Human embryonic stem cell-derived dopaminergic neurons reverse functional deficit in parkinsonian rats. Stem Cells, 26, 55–63. doi:10.1634/stemcells.2007-0494.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

The authors acknowledge the grant support provided by the National University of Singapore (R-174-000-089-133) and National Medical Research Council, Singapore (R-174-000-103-213). The technical assistance of Miss Manasi and Mr Arjunan Subramanian is greatly appreciated.

Author information

Authors and Affiliations

  1. Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Lower Kent Ridge Road, Singapore, 119074, Singapore

    Chui Yee Fong, Gary S. L. Peh, Kalamegam Gauthaman & Ariff Bongso

Authors
  1. Chui Yee Fong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gary S. L. Peh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kalamegam Gauthaman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ariff Bongso
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ariff Bongso.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fong, C.Y., Peh, G.S.L., Gauthaman, K. et al. Separation of SSEA-4 and TRA-1–60 Labelled Undifferentiated Human Embryonic Stem Cells from A Heterogeneous Cell Population Using Magnetic-Activated Cell Sorting (MACS) and Fluorescence-Activated Cell Sorting (FACS). Stem Cell Rev and Rep 5, 72–80 (2009). https://doi.org/10.1007/s12015-009-9054-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12015-009-9054-4

Keywords

Search

Navigation