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:

Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells

Nature Methods volume 2, pages 185–190 (2005)Cite this article

Abstract

Human embryonic stem cells (hESCs) are routinely cultured on fibroblast feeder layers or in fibroblast-conditioned medium (CM). Bone morphogenetic proteins (BMPs) have previously been shown to induce hESC differentiation, in apparent contrast to mouse embryonic stem (ES) cells, in which BMP4 synergizes with leukemia inhibitory factor (LIF) to maintain self-renewal. Here we demonstrate that hESCs cultured in unconditioned medium (UM) are subjected to high levels of BMP signaling activity, which is reduced in CM. The BMP antagonist noggin synergizes with basic fibroblast growth factor (bFGF) to repress BMP signaling and sustain undifferentiated proliferation of hESCs in the absence of fibroblasts or CM. These findings suggest a basic difference in the self-renewal mechanism between mouse and human ES cells and simplify the culture of hESCs.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Figure 1: UM contains differentiation-inducing activity.
Figure 2: BMP agonistic and antagonistic signals are detected in hESC culture.
Figure 3: UM containing bFGF and noggin sustains undifferentiated proliferation of hESCs.
Figure 4: hESCs cultured in UM containing bFGF and noggin retain developmental potential.
Figure 5: hESCs cultured in UM containing bFGF and noggin remain karyotypically normal.

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. 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 

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

    Article  CAS  Google Scholar 

  4. Martin, G.R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. USA 78, 7634–7638 (1981).

    Article  CAS  Google Scholar 

  5. Smith, A.G. et al. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336, 688–690 (1988).

    Article  CAS  Google Scholar 

  6. Williams, R.L. et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336, 684–687 (1988).

    Article  CAS  Google Scholar 

  7. Ying, Q.L., Nichols, J., Chambers, I. & Smith, A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115, 281–292 (2003).

    Article  CAS  Google Scholar 

  8. Humphrey, R.K. et al. Maintenance of pluripotency in human embryonic stem cells is STAT3 independent. Stem Cells 22, 522–530 (2004).

    Article  CAS  Google Scholar 

  9. 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 

  10. Xu, R.H. et al. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat. Biotechnol. 20, 1261–1264 (2002).

    Article  CAS  Google Scholar 

  11. Pera, M.F. et al. Regulation of human embryonic stem cell differentiation by BMP-2 and its antagonist noggin. J. Cell Sci. 117, 1269–1280 (2004).

    Article  CAS  Google Scholar 

  12. Price, P.J., Goldsborough, M.D. & Tilkins, M.L. Embryonic stem cell serum replacement. in International Patent Application. (1998).

    Google Scholar 

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

    Article  CAS  Google Scholar 

  14. Liu, F. et al. A human Mad protein acting as a BMP-regulated transcriptional activator. Nature 381, 620–623 (1996).

    Article  CAS  Google Scholar 

  15. Suzuki, A., Chang, C., Yingling, J.M., Wang, X.F. & Hemmati-Brivanlou, A. Smad5 induces ventral fates in Xenopus embryo. Dev. Biol. 184, 402–405 (1997).

    Article  CAS  Google Scholar 

  16. Kawai, S. et al. Mouse smad8 phosphorylation downstream of BMP receptors ALK-2, ALK-3, and ALK-6 induces its association with Smad4 and transcriptional activity. Biochem. Biophys. Res. Commun. 271, 682–687 (2000).

    Article  CAS  Google Scholar 

  17. Hsu, D.R., Economides, A.N., Wang, X., Eimon, P.M. & Harland, R.M. The Xenopus dorsalizing factor Gremlin identifies a novel family of secreted proteins that antagonize BMP activities. Mol. Cell 1, 673–683 (1998).

    Article  CAS  Google Scholar 

  18. Lopez-Rovira, T., Chalaux, E., Massague, J., Rosa, J.L. & Ventura, F. Direct binding of Smad1 and Smad4 to two distinct motifs mediates bone morphogenetic protein-specific transcriptional activation of Id1 gene. J. Biol. Chem. 277, 3176–3185 (2002).

    Article  CAS  Google Scholar 

  19. Korchynskyi, O. & ten Dijke, P. Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter. J. Biol. Chem. 277, 4883–4891 (2002).

    Article  CAS  Google Scholar 

  20. Draper, J.S. et al. Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat. Biotechnol. 22, 53–54 (2004).

    Article  CAS  Google Scholar 

  21. Anisimov, S.V. et al. SAGE identification of gene transcripts with profiles unique to pluripotent mouse R1 embryonic stem cells. Genomics 79, 169–176 (2002).

    Article  CAS  Google Scholar 

  22. Sperger, J.M. et al. Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proc. Natl. Acad. Sci. USA 100, 13350–13355 (2003).

    Article  CAS  Google Scholar 

  23. Brandenberger, R. et al. Transcriptome characterization elucidates signaling networks that control human ES cell growth and differentiation. Nat. Biotechnol. 22, 707–716 (2004).

    Article  Google Scholar 

  24. Richards, M., Tan, S.P., Tan, J.H., Chan, W.K. & Bongso, A. The transcriptome profile of human embryonic stem cells as defined by SAGE. Stem Cells 22, 51–64 (2004).

    Article  CAS  Google Scholar 

  25. 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 

  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. Pera, E.M., Ikeda, A., Eivers, E. & De Robertis, E.M. Integration of IGF, FGF, and anti-BMP signals via Smad1 phosphorylation in neural induction. Genes Dev. 17, 3023–3028 (2003).

    Article  CAS  Google Scholar 

  28. Nakayama, K. et al. Receptor tyrosine kinases inhibit bone morphogenetic protein-Smad responsive promoter activity and differentiation of murine MC3T3–E1 osteoblast-like cells. J. Bone Miner. Res. 18, 827–835 (2003).

    Article  CAS  Google Scholar 

  29. Held, K.R. & Sonnichsen, S. The effect of oxygen tension on colony formation and cell proliferation of amniotic fluid cells in vitro. Prenat. Diagn. 4, 171–179 (1984).

    Article  CAS  Google Scholar 

  30. Brown, M. & Lawce, H. Use of Ethidium Bromide to Promote Long Chromosomes edn. 3 (Lippencott-Raven, New York, 1997).

    Google Scholar 

Download references

Acknowledgements

We thank J. Johnson for karyotyping, D. Manning and E. Mitchen for technical support, M. Piekarczyk for the teratoma assays, T. López-Rovira for pID120-Lux plasmid, W. Hu for statistical analyses, B. Becker for image editing, and M. Levenstein and T. Berggren for critical reading of the manuscript. This work was supported by WiCell Research Institute, a nonprofit subsidiary of the Wisconsin Alumni Research Foundation, National Institutes of Health grants # P20 GM069981-01 to J.A.T., and # 5P51 RR000167 to Wisconsin National Primate Research Center.

Author information

Authors and Affiliations

  1. WiCell Research Institute, Madison, 53707, Wisconsin, USA

    Ren-He Xu, Ruthann M Peck, Dong S Li & James A Thomson

  2. Department of Anatomy, University of Wisconsin-Madison Medical School, the Wisconsin National Primate Research Center and The Genome Center of Wisconsin, Madison, 53706, Wisconsin, USA

    Ren-He Xu, Xuezhu Feng, Tenneille Ludwig & James A Thomson

Authors
  1. Ren-He Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ruthann M Peck
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dong S Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xuezhu Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tenneille Ludwig
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. James A Thomson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Ren-He Xu or James A Thomson.

Ethics declarations

Competing interests

Dr. Thomson is a co-founder of Cellular Dynamics International, a company that plans to use ES cell-derived cardiomycocytes for drug screeing."

Supplementary information

Supplementary Fig. 1

Synergistic effect of bFGF and noggin in prevention of hESC differentiation. (PDF 5067 kb)

Supplementary Fig. 2

H1 and H9 cells cultured in UMFN for 7 and 6 passages, respectively, were injected into SCID-beige mice. (PDF 4089 kb)

Supplementary Table 1

Population doubling time of UMFN-cultured hESCs (PDF 48 kb)

Supplementary Table 2

Primers and cycle numbers for Q-PCR and RT-PCR (PDF 43 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Xu, RH., Peck, R., Li, D. et al. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Methods 2, 185–190 (2005). https://doi.org/10.1038/nmeth744

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth744

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