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EOS lentiviral vector selection system for human induced pluripotent stem cells

Nature Protocols volume 4, pages 1828–1844 (2009)Cite this article

Abstract

Generation of induced pluripotent stem (iPS) cells from patients has exciting applications for studying molecular mechanisms of diseases, screening drugs and ultimately for use in cell therapies. However, the low efficiency and heterogeneous nature of reprogramming is a major impediment to the generation of personalized iPS cell lines. We reported in Nature Methods (6, 370–376, 2009) the first selection system to enrich for reprogrammed human iPS cells. Using a lentiviral vector that specifically expresses the enhanced green fluorescence protein and puromycin resistance genes in pluripotent stem cells, it is now possible to mark and enrich for human iPS cell colonies expressing endogenous pluripotency markers. In this study, we describe a detailed protocol for the production of the pluripotent state-specific lentiviral vector and the selection system for the induction of healthy and disease-specific human iPS cells. Overall, preparation of the selection system takes 2 weeks, and the generation of human iPS cells takes ∼2 months.

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Figure 1: Flow diagram and timeline of the EOS selection protocol.
Figure 2: Production of EOS lentiviral vector.
Figure 3: Infection of lentiviral vectors into human fibroblasts.
Figure 4: Morphology of early human iPS cell colonies.
Figure 5: Pluripotent marker expression and differentiation abilities.
Figure 6: Characterization of iPS cell lines by teratoma formation.

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Acknowledgements

We thank P. Leboulch (Genetics Division, Department of Medicine, Brigham & Women's Hospital and Harvard Medical School) for PL lentiviral vector and packaging plasmids, T. Kitamura (Institute of Medical Science, University of Tokyo) for Plat-E cells and B. Alman for provision of human fibroblasts. We thank T. Thompson, C.A. Séguin and K. Woltjen at the Ontario Human iPS Cell Facility for infrastructure and reagents and J. Garner at the SickKids ES Facility for feeder preparation. This study was supported by grants from the Canadian Institutes of Health Research (MOP-81129, RMF-92090, and IG1-94505 to J.E. and MOP-186015 to W.L.S.), the Ontario Ministry of Research and Innovation (to J.E. and W.L.S. for the Ontario Human iPS Cell Facility), the Stem Cell Network (to J.E. and W.L.S.) and the International Rett Syndrome Foundation (to J.E.). A.H. is supported by a Restracomp Award from SickKids Hospital, A.Y.L.C. by a Graduate Scholarship from the Natural Sciences and Engineering Research Council of Canada, N.F. by an Ontario Council of Graduate Studies Master's Autism Scholars Award and the Ontario Student Opportunity Trust Funds Hayden Hantho Award and W.Y.C. by an Ontario Ministry of Research and Innovation Postdoctoral Fellowship.

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Authors and Affiliations

  1. Developmental and Stem Cell Biology Program, SickKids, Toronto, Ontario, Canada

    Akitsu Hotta, Aaron Y L Cheung, Natalie Farra, Peter Pasceri & James Ellis

  2. Ontario Human iPS Cell Facility, SickKids, Toronto, Ontario, Canada

    Akitsu Hotta, Kamal Garcha, Wing Y Chang, William L Stanford & James Ellis

  3. Department of Molecular Geneticsg, University of Toronto, Toronto, Ontario, Canada

    Aaron Y L Cheung, Natalie Farra & James Ellis

  4. Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada

    Kamal Garcha, Wing Y Chang & William L Stanford

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  1. Akitsu Hotta
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Contributions

A.H. developed the selection protocol; A.Y.L.C., N.F., K.G. and W.Y.C. derived iPS cell lines, P.P. performed teratoma experiments; W.L.S. and J.E. conceived the study; A.H. and J.E. wrote the paper; and all authors contributed to optimize the protocol and edit the paper.

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Correspondence to James Ellis.

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Hotta, A., Cheung, A., Farra, N. et al. EOS lentiviral vector selection system for human induced pluripotent stem cells. Nat Protoc 4, 1828–1844 (2009). https://doi.org/10.1038/nprot.2009.201

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