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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
He, J., Yang, Q. & Chang, L.J. J. Virol. 79, 13497–13508 (2005).
Smith-Arica, J.R. et al. Cloning Stem Cells 5, 51–62 (2003).
Liew, C.-G., Draper, J.S., Walsh, J., Moore, H. & Andrews, P.W. Stem Cells 25, 1521–1528 (2007).
Thyagarajan, B. et al. Stem Cells 26, 119–126 (2008).
Lombardo, A. et al. Nat. Biotechnol. 25, 1298–1306 (2007).
Vieyra, D.S. & Goodell, M.A. Stem Cells 25, 2559–2566 (2007).
Wilber, A. et al. Stem Cells 25, 2919–2927 (2007).
Vallier, L., Alexander, M. & Pedersen, R. Stem Cells 25, 1490–1497 (2007).
Denning, C. et al. Int. J. Dev. Biol. 50, 27–37 (2006).
Zwaka, T.P. & Thomson, J.A. Nat. Biotechnol. 21, 319–321 (2003).
Costa, M. et al. Nat. Protoc. 2, 792–796 (2007).
Eiges, R. et al. Curr. Biol. 11, 514–518 (2001).
Draper, J.S. et al. Nat. Biotechnol. 22, 53–54 (2004).
Baker, D.E.C. et al. Nat. Biotechnol. 25, 207–215 (2007).
Passier, R. et al. Stem Cells 23, 772–780 (2005).
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
Authors and Affiliations
Contributions
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.
Corresponding author
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–5, Supplementary Table 1, Supplementary Methods. (PDF 6984 kb)
Rights and permissions
About this article
Cite this article
Braam, S., Denning, C., van den Brink, S. et al. Improved genetic manipulation of human embryonic stem cells. Nat Methods 5, 389–392 (2008). https://doi.org/10.1038/nmeth.1200
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nmeth.1200
This article is cited by
-
Genome-wide CRISPR/Cas9 screening in human iPS derived cardiomyocytes uncovers novel mediators of doxorubicin cardiotoxicity
Scientific Reports (2021)
-
CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells
Molecular Therapy - Nucleic Acids (2016)
-
Basement Membrane Matrix (BME) has Multiple Uses with Stem Cells
Stem Cell Reviews and Reports (2012)
-
Directed Differentiation of Human Embryonic Stem Cells to Interrogate the Cardiac Gene Regulatory Network
Molecular Therapy (2011)
-
Scalable expansion of human pluripotent stem cells in suspension culture
Nature Protocols (2011)