Skip to main content

Thank you for visiting 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.

Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension


Undifferentiated human embryonic stem cells (hESCs) are currently propagated on a relatively small scale as monolayer colonies1,2,3,4,5,6,7. Culture of hESCs as floating aggregates is widely used for induction of differentiation into embryoid bodies8. Here we show that hESC lines can be derived from floating inner cell masses in suspension culture conditions that do not involve feeder cells or microcarriers. This culture system supports prolonged propagation of the pluripotent stem cells as floating clusters without their differentiation into embryoid bodies. HESCs cultivated as aggregates in suspension maintain the expression of pluripotency markers and can differentiate into progeny of the three germ layers both in vitro and in vivo. We further show the controlled differentiation of hESC clusters in suspension into neural spheres. These results pave the way for large-scale expansion and controlled differentiation of hESCs in suspension, which would be valuable in basic and applied research.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


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

Figure 1: Human ESCs remain pluripotent after 10 weeks propagation in suspension.
Figure 2: Derivation of hESCs in suspension.
Figure 3: Controlled conversion of the hESC clusters in suspension into neural precursor spheres.


  1. 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 

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

    Article  CAS  Google Scholar 

  3. Ludwig, T.E. et al. Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnol. 24, 185–187 (2006).

    Article  CAS  Google Scholar 

  4. Richards, M., Fong, C.Y., Chan, W.K., Wong, P.C. & Bongso, A. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat. Biotechnol. 20, 933–936 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Braam, S.R. et al. Feeder-free culture of human embryonic stem cells in conditioned medium for efficient genetic modification. Nat. Protoc. 3, 1435–1443 (2008).

    Article  CAS  Google Scholar 

  7. Wang, L. et al. Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling. Blood 110, 4111–4119 (2007).

    Article  CAS  Google Scholar 

  8. Kurosawa, H. Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells. J. Biosci. Bioeng. 103, 389–398 (2007).

    Article  CAS  Google Scholar 

  9. McDevitt, T.C. & Palecek, S.P. Innovation in the culture and derivation of pluripotent human stem cells. Curr. Opin. Biotechnol. 19, 527–533 (2008).

    Article  CAS  Google Scholar 

  10. Lock, L.T. & Tzanakakis, E.S. Expansion and differentiation of human embryonic stem cells to endoderm progeny in a microcarrier stirred-suspension culture. Tissue Eng. Part A 15, 2051–2063 (2009).

    Article  CAS  Google Scholar 

  11. Nie, Y., Bergendahl, V., Hei, D.J., Jones, J.M. & Palecek, S.P. Scalable culture and cryopreservation of human embryonic stem cells on microcarriers. Biotechnol. Prog. 25, 20–31 (2009).

    Article  CAS  Google Scholar 

  12. Oh, S.K. et al. Long-term microcarrier suspension cultures of human embryonic stem cells. Stem Cell Res. (Amst.) 4, 4 (2009).

    Google Scholar 

  13. Krawetz, R. et al. Large-scale expansion of pluripotent human embryonic stem cells in stirred suspension bioreactors. Tissue Eng. Part C Methods 8, 8 (2009).

    Google Scholar 

  14. Itsykson, P. et al. Derivation of neural precursors from human embryonic stem cells in the presence of noggin. Mol. Cell. Neurosci. 30, 24–36 (2005).

    Article  CAS  Google Scholar 

  15. Hoover, C.S. & Martin, R.L. Antibody production and growth of mouse hybridoma cells in Nutridoma media supplements. Biotechniques 8, 76–82 (1990).

    CAS  PubMed  Google Scholar 

  16. Stockinger, H. Serum-free medium for mammalian cells. US patent 5,063,157 (1991).

  17. 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 

  18. Xu, C. et al. Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium. Stem Cells 23, 315–323 (2005).

    Article  CAS  Google Scholar 

  19. Pyle, A.D., Lock, L.F. & Donovan, P.J. Neurotrophins mediate human embryonic stem cell survival. Nat. Biotechnol. 24, 344–350 (2006).

    Article  CAS  Google Scholar 

  20. Beattie, G.M. et al. Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers. Stem Cells 23, 489–495 (2005).

    Article  CAS  Google Scholar 

  21. Furue, M.K. et al. Heparin promotes the growth of human embryonic stem cells in a defined serum-free medium. Proc. Natl. Acad. Sci. USA 105, 13409–13414 (2008).

    Article  CAS  Google Scholar 

  22. Turetsky, T. et al. Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis. Hum. Reprod. 23, 46–53 (2008).

    Article  CAS  Google Scholar 

  23. Herszfeld, D. et al. CD30 is a survival factor and a biomarker for transformed human pluripotent stem cells. Nat. Biotechnol. 24, 351–357 (2006).

    Article  CAS  Google Scholar 

  24. Baker, D.E. et al. Adaptation to culture of human embryonic stem cells and oncogenesis in vivo. Nat. Biotechnol. 25, 207–215 (2007).

    Article  CAS  Google Scholar 

  25. Mitalipova, M.M. et al. Preserving the genetic integrity of human embryonic stem cells. Nat. Biotechnol. 23, 19–20 (2005).

    Article  CAS  Google Scholar 

  26. Lyons, A.B. & Parish, C.R. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171, 131–137 (1994).

    Article  CAS  Google Scholar 

  27. Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).

    Article  CAS  Google Scholar 

  28. Lee, S.H., Lumelsky, N., Studer, L., Auerbach, J.M. & McKay, R.D. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat. Biotechnol. 18, 675–679 (2000).

    Article  CAS  Google Scholar 

  29. Yan, Y. et al. Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells. Stem Cells 23, 781–790 (2005).

    Article  CAS  Google Scholar 

  30. Gropp, M. & Reubinoff, B. Lentiviral vector-mediated gene delivery into human embryonic stem cells. Methods Enzymol. 420, 64–81 (2006).

    Article  CAS  Google Scholar 

Download references


We are grateful to the following members of the Hadassah Human Embryonic Stem Cell Research Center: M. Gropp, M. Aharonowiz and O. Singer for technical assistance; S. Tennenbaum for editing the manuscript. We thank K.M. Yamada (National Institute of Dental and Craniofacial Research, National Institutes of Health) for providing anti-fibronectin antibody, N. Benvenisty for the QPCR primers and WiCell Research Institute for providing H7 hESCs. This research was supported by a gift from Judy and Sidney Swartz, the Sidney Swartz Chair in Human Embryonic Stem Cell Research and Legacy Heritage Fund.

Author information

Authors and Affiliations



D.S. designed and performed the experiments, analyzed the data and wrote the manuscript; H.K. and M.C. performed the neural differentiation study; S.E.-R. conducted immunostainings and confocal analysis; Y.G. performed the teratoma studies; P.I. contributed to developing the concept of suspension culture; T.T. contributed to the experiments; M.I. performed PCR analysis; E.A. contributed to embryo recruitment, culture and isolation of inner cell masses; R.R. and Y.B.-Z. conducted karyotype analysis. B.R. conceived the study and wrote the paper.

Corresponding author

Correspondence to Benjamin Reubinoff.

Ethics declarations

Competing interests

B.R. is the CSO and holds shares in CellCure Neurosciences Ltd. However, the project was not funded by CellCure Neurosciences Ltd. and the company has no rights in its results.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–10 (PDF 795 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Steiner, D., Khaner, H., Cohen, M. et al. Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension. Nat Biotechnol 28, 361–364 (2010).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research