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Feeder-free culture of human embryonic stem cells in conditioned medium for efficient genetic modification

Abstract

Realizing the potential of human embryonic stem cells (hESCs) in research and commercial applications requires generic protocols for culture, expansion and genetic modification that function between multiple lines. Here we describe a feeder-free hESC culture protocol that was tested in 13 independent hESC lines derived in five different laboratories. The procedure is based on Matrigel adaptation in mouse embryonic fiboblast conditioned medium (CM) followed by monolayer culture of hESC. When combined, these techniques provide a robust hESC culture platform, suitable for high-efficiency genetic modification via plasmid transfection (using lipofection or electroporation), siRNA knockdown and viral transduction. In contrast to other available protocols, it does not require optimization for individual lines. hESC transiently expressing ectopic genes are obtained within 9 d and stable transgenic lines within 3 weeks.

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Figure 1: Photographs of human embryonic stem cell (hESC) at different stages.
Figure 2: Flowchart for experimental procedures.

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Acknowledgements

We are grateful to D. Ward-van Oostwaard, L. Zeinstra and S. van den Brink for expert technical assistance. We thank Drs Chad Cowan and Douglas Melton for the gift of HUES-1, -5, -7 and -15. This work is/has been supported by the Dutch Program for Tissue Engineering (S.R.B.), European Community's Sixth Framework Programme contract ('HeartRepair') LSHM-CT-2005-018630 (R.P.), the Biotechnology and Biological Sciences Research Council, British Heart Foundation and the University of Nottingham (C.D., E.M. and L.E.Y.).

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S.R.B. and C.D. contributed equally to this work.

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Correspondence to Chris Denning or Christine L Mummery.

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Braam, S., Denning, C., Matsa, E. et al. Feeder-free culture of human embryonic stem cells in conditioned medium for efficient genetic modification. Nat Protoc 3, 1435–1443 (2008). https://doi.org/10.1038/nprot.2008.140

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