Improved genetic manipulation of human embryonic stem cells

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Abstract

Low efficiency of transfection limits the ability to genetically manipulate human embryonic stem cells (hESCs), and differences in cell derivation and culture methods require optimization of transfection protocols. We transiently transferred multiple independent hESC lines with different growth requirements to standardized feeder-free culture, and optimized conditions for clonal growth and efficient gene transfer without loss of pluripotency. Stably transfected lines retained differentiation potential, and most lines displayed normal karyotypes.

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Figure 1: Procedure and characterization of cells adapted to trypsin-based Matrigel culture.
Figure 2: Low-density Matrigel culture results in greatly enhanced transfection efficiencies of hESCs.
Figure 3: Functional validation of transfection methods.

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Acknowledgements

We are grateful to J. Braam for assistance creating Figure 1a, D. Ward-van Oostwaard and J. Monshouwer-Kloots for expert technical assistance. We thank J. Thomson and T. Zwaka (University of Wisconsin Medical School and the National Primate Research Center, Madison) for providing the POU5F1 targeting vector and C. Cowan and D. Melton (Harvard Stem Cell Institute) for the gift of HUES-1,5,7 and -15. This work was supported by the Dutch Program for Tissue Engineering and European Commission Sixth Framework Programme contract ('Heart Repair') LSHM-CT-2005-018630. C. Denning is supported by the Biotechnology and Biological Sciences Research Council.

Author information

S.R.B., C.D., R.P. and C.L.M. designed and planned the study. S.R.B. performed the majority of the laboratory experiments. S.v.d.B. maintained hESC lines on feeders and C.D. contributed to experiments with NOTT1, 2 and HUES7. P.K. and R.H. performed karyotype analyses. S.B., R.P. and C.M. prepared the manuscript.

Correspondence to Christine L Mummery.

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Supplementary Figures 1–5, Supplementary Table 1, Supplementary Methods. (PDF 6984 kb)

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