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Feeder-independent culture of human embryonic stem cells

Nature Methods volume 3, pages 637–646 (2006)Cite this article

An Erratum to this article was published on 01 October 2006

This article has been updated

Abstract

We recently reported the development of TeSR1, a serum-free, animal product–free medium that supports the derivation and long-term feeder-independent culture of human embryonic stem cells1. Although the derivation of new human embryonic stem cell lines in those defined conditions offered an important proof of principle, the costs of some of the defined components in that culture system made it impractical for everyday research use. Here we describe modifications to the medium (mTeSR1) that include the use of animal-sourced proteins (bovine serum albumin (BSA) and Matrigel) and cloned zebrafish basic fibroblast growth factor (zbFGF). We include a simple protocol that allows purification of up to 100 mg zbFGF in less than three days (Fig. 1), an amount sufficient to make 1,000 l of mTeSR1 medium. The modifications presented here make mTeSR1 practical for routine research use, and the protocols presented are those currently used in our laboratory for standard human embryonic stem cell culture.

Outline of zbFGF purification protocol.

*Note: In the version of this Protocol initially published, the references were numbered incorrectly. The error has been corrected in the HTML and PDF versions of the article.

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Figure 2: SDS-PAGE analysis of a typical zbFGF purification by HPLC.
Figure 3: Assessment of appropriate passage timing.

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Change history

  • 24 August 2006

    During production of the manuscript, the references were incorrectly numbered. The ordering within the text is fine and should remain the same, but the list of references needs to be reordered. The new reference order is attached in a Word document and is also as follows:

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Acknowledgements

The methods described in this paper are those presently used by the Thomson Laboratory at the University of Wisconsin–Madison. We thank the many laboratory colleagues contributing to the preparation of this manuscript, particularly J. Frane, D. Faupel and J. Antosiewicz for editorial review. This work was supported in part by US National Institutes of Health grants R24-RR017721 and P20-GM069981, and funds from the University of Wisconsin Foundation.

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Author notes

  1. Tenneille E Ludwig, Veit Bergendahl and Mark E Levenstein: These authors contributed equally to the development of this protocol.

Authors and Affiliations

  1. Genome Center of Wisconsin, University of Wisconsin-Madison, 425 Henry Mall, Madison, 53706, Wisconsin, USA

    Tenneille E Ludwig, Veit Bergendahl, Mitchell D Probasco & James A Thomson

  2. WiCell Research Institute, P.O. Box 7365, Madison, 53707, Wisconsin, USA

    Tenneille E Ludwig, Mark E Levenstein & James A Thomson

  3. National Primate Research Center, University of Wisconsin Graduate School, 1220 Capitol Court, Madison, 53705, Wisconsin, USA

    Tenneille E Ludwig, Junying Yu & James A Thomson

  4. Department of Anatomy, University of Wisconsin Medical School, 470 N. Charter Street, Madison, 53706, Wisconsin, USA

    James A Thomson

Authors
  1. Tenneille E Ludwig
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  2. Veit Bergendahl
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  3. Mark E Levenstein
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  4. Junying Yu
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  5. Mitchell D Probasco
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Correspondence to Tenneille E Ludwig.

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Competing interests

J.A.T. owns stock in and has served on the Board of Cellular Dynamics International (within the last two years), and owns stock in and serves on the Board of Stem Cell Products (within the last year).

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Ludwig, T., Bergendahl, V., Levenstein, M. et al. Feeder-independent culture of human embryonic stem cells. Nat Methods 3, 637–646 (2006). https://doi.org/10.1038/nmeth902

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