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Faster generation of hiPSCs by coupling high-titer lentivirus and column-based positive selection

Nature Protocols volume 6, pages 701–714 (2011)Cite this article

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Abstract

The protocols described here address methods used in two crucial stages in the retroviral reprogramming of somatic cells to produce human induced pluripotent stem cell (hiPSC) lines. The first is an optimized method for producing lentivirus at an efficiency 600-fold greater than previously published, and it includes conjugation of the lentivirus to streptavidin superparamagnetic particles; this process takes 8 d. The second method enables the isolation of true hiPSCs immediately after somatic cell reprogramming and involves column-based positive selection of cells expressing the pluripotency marker TRA-1-81. This process takes 2 h and, as it is directly compatible with feeder-free culture, the time burden of manually identifying and mechanically propagating hiPSC colonies is reduced drastically. Taken together, these methods accelerate the production of hiPSCs and enable lines to be isolated, expanded to ∼107 cells and cryopreserved within 6–8 weeks.

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Figure 1
Figure 2: Transduction of fibroblasts and anticipated results from hiPSC derivation using column enrichment.
Figure 3: Characterization of hiPSCs.

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Acknowledgements

This work was supported by the Medical Research Council, the Biotechnology and Biological Sciences Research Council and the British Heart Foundation. We would like to thank our collaborators at the University of Newcastle International Centre for Life who provided fibroblast samples from the BioBank in Newcastle, which is part of the MRC centre for Neuromuscular Diseases and EuroBioBank.

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Authors and Affiliations

  1. Wolfson Centre for Stem Cells, Tissue Engineering & Modelling, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, UK

    Emily Dick, Elena Matsa, Lorraine E Young & Chris Denning

  2. Department of Haematological Medicine, King's College, Guy's, King's and St. Thomas' School of Medicine, The Rayne Institute, London, UK

    David Darling

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Contributions

E.D. designed the experiments, carried out the work, analyzed the results and prepared the manuscript. E.M. derived the HUES-Fib line used for control experiments. L.E.Y. was involved in hiPSC setup. D.D. derived the modified 293T line, BL15, and provided methods for the calcium precipitation of lentiviral supernatants. C.D. is the principal investigator on the project and coordinates research activity and funding.

Corresponding author

Correspondence to Chris Denning.

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The authors declare no competing financial interests.

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Dick, E., Matsa, E., Young, L. et al. Faster generation of hiPSCs by coupling high-titer lentivirus and column-based positive selection. Nat Protoc 6, 701–714 (2011). https://doi.org/10.1038/nprot.2011.320

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