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.

  • Article
  • Published:

Clonal isolation of hESCs reveals heterogeneity within the pluripotent stem cell compartment

Nature Methods volume 3, pages 807–815 (2006)Cite this article

Abstract

Human embryonic stem cell (hESC) lines are known to be morphologically and phenotypically heterogeneous. The functional nature and relationship of cells residing within hESC cultures, however, has not been evaluated because isolation of single hESCs is limited to drug or manual selection. Here we provide a quantitative method using flow cytometry to isolate and clonally expand hESCs based on undifferentiated markers, alone or in combination with a fluorescent reporter. This method allowed for isolation of stage-specific embryonic antigen-3–positive (SSEA-3+) and SSEA-3− cells from hESC cultures. Although both SSEA-3+ and SSEA-3− cells could initiate pluripotent hESC cultures, we show that they possess distinct cell-cycle properties, clonogenic capacity and expression of ESC transcription factors. Our study provides formal evidence for heterogeneity among self-renewing pluripotent hESCs, illustrating that this isolation technique will be instrumental in further dissecting the biology of hESC lines.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: hESC cultures are heterogeneous: comparison of SSEA-3− and SSEA-3+ subpopulations.
Figure 2: hESC-derived fibroblast-like cell (hdF) layers promote hESC culture regeneration from sorted hESCs.
Figure 3: Colonies arise from SSEA-3+–gated hESCs, not irradiated hdF layer.
Figure 4: Regeneration of hESC cultures from two distinct populations within hESC cultures.
Figure 5: SSEA-3− and SSEA-3+ sorted cells have distinct hESC marker expression.

Similar content being viewed by others

References

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

    Article  CAS  Google Scholar 

  2. Amit, M. et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 227, 271–278 (2000).

    Article  CAS  Google Scholar 

  3. Genbacev, O. et al. Serum-free derivation of human embryonic stem cell lines on human placental fibroblast feeders. Fertil. Steril. 83, 1517–1529 (2005).

    Article  Google Scholar 

  4. Lee, J.B. et al. Establishment and maintenance of human embryonic stem cell lines on human feeder cells derived from uterine endometrium under serum-free condition. Biol. Reprod. 72, 42–49 (2005).

    Article  CAS  Google Scholar 

  5. Xu, C. et al. Immortalized fibroblast-like cells derived from human embryonic stem cells support undifferentiated cell growth. Stem Cells 22, 972–980 (2004).

    Article  CAS  Google Scholar 

  6. Stojkovic, P. et al. An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells. Stem Cells 23, 306–314 (2005).

    Article  CAS  Google Scholar 

  7. Wang, Q. et al. Derivation and growing human embryonic stem cells on feeders derived from themselves. Stem Cells 23, 1221–1227 (2005).

    Article  Google Scholar 

  8. Lee, J.B. et al. Available human feeder cells for the maintenance of human embryonic stem cells. Reproduction 128, 727–735 (2004).

    Article  CAS  Google Scholar 

  9. Yoo, S.J. et al. Efficient culture system for human embryonic stem cells using autologous human embryonic stem cell-derived feeder cells. Exp. Mol. Med. 37, 399–407 (2005).

    Article  CAS  Google Scholar 

  10. Xu, C. et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19, 971–974 (2001).

    Article  CAS  Google Scholar 

  11. Li, Y., Powell, S., Brunette, E., Lebkowski, J. & Mandalam, R. Expansion of human embryonic stem cells in defined serum-free medium devoid of animal-derived products. Biotechnol. Bioeng. 91, 688–698 (2005).

    Article  CAS  Google Scholar 

  12. Amit, M., Shariki, C., Margulets, V. & Itskovitz-Eldor, J. Feeder layer- and serum-free culture of human embryonic stem cells. Biol. Reprod. 70, 837–845 (2004).

    Article  CAS  Google Scholar 

  13. Ma, Y., Ramezani, A., Lewis, R., Hawley, R.G. & Thomson, J.A. High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors. Stem Cells 21, 111–117 (2003).

    Article  CAS  Google Scholar 

  14. Costa, M. et al. The hESC line Envy expresses high levels of GFP in all differentiated progeny. Nat. Methods 2, 259–260 (2005).

    Article  CAS  Google Scholar 

  15. Vallier, L. et al. Enhancing and diminishing gene function in human embryonic stem cells. Stem Cells 22, 2–11 (2004).

    Article  CAS  Google Scholar 

  16. Carpenter, M.K. et al. Properties of four human embryonic stem cell lines maintained in a feeder-free culture system. Dev. Dyn. 229, 243–258 (2004).

    Article  CAS  Google Scholar 

  17. Mazurier, F., Gan, O.I., McKenzie, J.L., Doedens, M. & Dick, J.E. Lentivector-mediated clonal tracking reveals intrinsic heterogeneity in the human hematopoietic stem cell compartment and culture-induced stem cell impairment. Blood 103, 545–552 (2004).

    Article  CAS  Google Scholar 

  18. Evans, M.J. & Kaufman, M.H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).

    Article  CAS  Google Scholar 

  19. Hope, K.J., Jin, L. & Dick, J.E. Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity. Nat. Immunol. 5, 738–743 (2004).

    Article  CAS  Google Scholar 

  20. Enver, T. et al. Cellular differentiation hierarchies in normal and culture-adapted human embryonic stem cells. Hum. Mol. Genet. 14, 3129–3140 (2005).

    Article  CAS  Google Scholar 

  21. 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 

  22. 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 

  23. Dean, P.N. & Jett, J.H. Mathematical analysis of DNA distributions derived from flow microfluorometry. J. Cell Biol. 60, 523–527 (1974).

    Article  CAS  Google Scholar 

  24. Fox, M.H. A model for the computer analysis of synchronous DNA distributions obtained by flow cytometry. Cytometry 1, 71–77 (1980).

    Article  CAS  Google Scholar 

  25. Martin, G.R. & Evans, M.J. The morphology and growth of a pluripotent teratocarcinoma cell line and its derivatives in tissue culture. Cell 2, 163–172 (1974).

    Article  CAS  Google Scholar 

  26. Sato, N., Meijer, L., Skaltsounis, L., Greengard, P. & Brivanlou, A.H. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat. Med. 10, 55–63 (2004).

    Article  CAS  Google Scholar 

  27. 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 

  28. 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 

  29. Eiges, R. et al. Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells. Curr. Biol. 11, 514–518 (2001).

    Article  CAS  Google Scholar 

  30. Andrews, P.W. et al. A pluripotent human stem-cell clone isolated from the TERA-2 teratocarcinoma line lacks antigens SSEA-3 and SSEA-4 in vitro, but expresses these antigens when grown as a xenograft tumor. Differentiation 29, 127–135 (1985).

    Article  CAS  Google Scholar 

  31. Chadwick, K. et al. Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells. Blood 102, 906–915 (2003).

    Article  CAS  Google Scholar 

  32. Wang, L., Li, L., Menendez, P., Cerdan, C. & Bhatia, M. Human embryonic stem cells maintained in the absence of mouse embryonic fibroblasts or conditioned media are capable of hematopoietic development. Blood 105, 4598–4603 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We acknowledge R. Mondeh, A. Rouleau and J. Yang for outstanding technical assistance, and D. Sheerar for cell isolation. We thank K. Vijayaragavan for critical review of the manuscript. This research was funded by a grant from the Canadian Institutes of Health Research (CIHR) and the National Centres of Excellence–Stem Cell Network Program and postgraduate scholarship award from the Stem Cell Network to M.H.S., and CIHR Canadian Graduate Scholarships to S.C.B. and K.C.

Author information

Authors and Affiliations

  1. Department of Biochemistry, McMaster Stem Cell and Cancer Research Institute, Hamilton, Ontario L9G 4L6, Canada and McMaster University, Faculty of Health Sciences, 1200 Main Street West, Hamilton, Ontario, Canada

    Morag H Stewart, Pablo Menendez, Sean C Bendall & Mickie Bhatia

  2. Department of Microbiology and Immunology, The University of Western Ontario, 1151 Richmond Street, Suite 2, London, Ontario, Canada

    Morag H Stewart, Marc Bossé, Kristin Chadwick, Sean C Bendall & Mickie Bhatia

Authors
  1. Morag H Stewart
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marc Bossé
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kristin Chadwick
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pablo Menendez
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sean C Bendall
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mickie Bhatia
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.H.S. performed experiments and assisted in writing the paper; M.B., K.C., P.M. and S.C.B. assisted with experiments; M.B. wrote and edited the paper.

Corresponding author

Correspondence to Mickie Bhatia.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Feeder-free hESC cultures are heterogeneous. (PDF 939 kb)

Supplementary Fig. 2

Irradiated hdFs allow for hESC culture regeneration from FACS isolated hESC populations. (PDF 592 kb)

Supplementary Fig. 3

eGFP and DsRed Lentiviral vectors. (PDF 257 kb)

Supplementary Fig. 4

Sorted hESC cultures retain pluripotency demonstrated by in vivo differentiation. (PDF 1832 kb)

Supplementary Fig. 5

hESC cultures regenerated from sorted hESC populations are karyotypically normal. (PDF 452 kb)

Supplementary Fig. 6

Unlike sorted SSEA-3- cells derived from hESC cultures (Figure 5b) hdFs do not express mRNA for POU5F1 or NANOG. (PDF 324 kb)

Supplementary Protocol 1

Generation of hESC derived fibroblast-like cell (hdF) cultures. (PDF 181 kb)

Supplementary Protocol 2

Sorting of feeder free hESCs and regeneration of sorted hESC cultures. (PDF 125 kb)

Supplementary Methods (PDF 84 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Stewart, M., Bossé, M., Chadwick, K. et al. Clonal isolation of hESCs reveals heterogeneity within the pluripotent stem cell compartment. Nat Methods 3, 807–815 (2006). https://doi.org/10.1038/nmeth939

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth939

This article is cited by

Search

Advanced 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