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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.

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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.

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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.

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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

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  1. Ren-He Xu
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  2. Ruthann M Peck
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Correspondence to Ren-He Xu or James A Thomson.

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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)

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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

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