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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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.
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