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Protocol

Nature Methods - 3, 637 - 646 (2006)
Published online: 21 July 2006; Corrected online: 24 August 2006 | doi:10.1038/nmeth902


There is an Erratum (October 2006) associated with this Protocol.

Feeder-independent culture of human embryonic stem cells

Tenneille E Ludwig1, 2, 3, 5, Veit Bergendahl1, 5, Mark E Levenstein2, 5, Junying Yu3, Mitchell D Probasco1 & James A Thomson1, 2, 3, 4

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

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

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

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

5  These authors contributed equally to the development of this protocol.

Correspondence should be addressed to Tenneille E Ludwig tludwig@primate.wisc.edu

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.
*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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Nature Methods
ISSN: 1548-7091
EISSN: 1548-7105
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