Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Generation of transgene-free human induced pluripotent stem cells with an excisable single polycistronic vector

Nature Protocols volume 6, pages 1251–1273 (2011)Cite this article

Subjects

Abstract

The generation of induced pluripotent stem cells (iPSCs) devoid of permanently integrated reprogramming factor genes is essential to reduce differentiation biases and artifactual phenotypes. We describe a protocol for the generation of human iPSCs using a single polycistronic lentiviral vector (pLM-fSV2A) coexpressing OCT4, SOX2, KLF4 and c-MYC; this is flanked by two loxP sites in its long terminal repeats (LTRs). Human iPSC lines are established with an efficiency of up to 1% and screened to select single or low vector copy lines. To deal with potential insertional mutagenesis, the vector integrations are then mapped to the human genome. Finally, the vector is excised by transient expression of Cre recombinase (coexpressed with mCherry) through an integrase-deficient lentiviral vector. Vector-excised iPSC lines maintain all characteristics of pluripotency. This protocol can be used to efficiently derive transgene-free iPSCs from many different starting cell types in approximately 12–14 weeks.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Overview of the strategy to generate transgene-free iPSCs described in this protocol.
Figure 2: The excisable lentiviral vector pLM-fSV2A used for reprogramming in this protocol.
Figure 3: Analysis of VCN in iPSC lines by Southern blotting.
Figure 4: Plating density of MSCs.
Figure 5: MSCs during the course of reprogramming.
Figure 6: Screening for vector excision.
Figure 7: Confirmation of vector excision.
Figure 8: Characterization of pluripotency of transgene-free iPSC lines derived from patients with β-thalassemia (thal-iPS) using this protocol.
Figure 9: Plating density of 293T cells.
Figure 10: Calcium phosphate transfection.
Figure 11: Newly forming iPSC-like colonies with atypical morphological characteristics.
Figure 12: Early passage iPSC lines.
Figure 13: Detection of Cre expression by flow cytometry.

Similar content being viewed by others

Article 01 December 2022

Zixuan Zhao, Xinyi Chen, … Hanry Yu

References

  1. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).

    Article  CAS  PubMed  Google Scholar 

  2. Papapetrou, E.P. et al. Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation. Proc. Natl. Acad. Sci. USA 106, 12759–12764 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Takahashi, K., Okita, K., Nakagawa, M. & Yamanaka, S. Induction of pluripotent stem cells from fibroblast cultures. Nat. Protoc. 2, 3081–3089 (2007).

    Article  CAS  PubMed  Google Scholar 

  4. Park, I.H., Lerou, P.H., Zhao, R., Huo, H. & Daley, G.Q. Generation of human-induced pluripotent stem cells. Nat. Protoc. 3, 1180–1186 (2008).

    Article  CAS  PubMed  Google Scholar 

  5. Raya, A. et al. A protocol describing the genetic correction of somatic human cells and subsequent generation of iPS cells. Nat. Protoc. 5, 647–660 (2010).

    Article  CAS  PubMed  Google Scholar 

  6. Aasen, T. & Belmonte, J.C. Isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells. Nat. Protoc. 5, 371–382 (2010).

    Article  CAS  PubMed  Google Scholar 

  7. Giorgetti, A. et al. Generation of induced pluripotent stem cells from human cord blood cells with only two factors: Oct4 and Sox2. Nat. Protoc. 5, 811–820 (2010).

    Article  CAS  PubMed  Google Scholar 

  8. Kim, J.B., Zaehres, H., Arauzo-Bravo, M.J. & Scholer, H.R. Generation of induced pluripotent stem cells from neural stem cells. Nat. Protoc. 4, 1464–1470 (2009).

    Article  CAS  PubMed  Google Scholar 

  9. Wernig, M. et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318–324 (2007).

    Article  CAS  PubMed  Google Scholar 

  10. Okita, K., Ichisaka, T. & Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. Nature 448, 313–317 (2007).

    Article  CAS  PubMed  Google Scholar 

  11. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. Jaenisch, R. & Young, R. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 132, 567–582 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Soldner, F. et al. Parkinson's disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell 136, 964–977 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Meissner, A., Wernig, M. & Jaenisch, R. Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat. Biotechnol. 25, 1177–1181 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Hu, B.Y. et al. Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency. Proc. Natl. Acad. Sci. USA 107, 4335–4340 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Boulting, G.L. et al. A functionally characterized test set of human induced pluripotent stem cells. Nat. Biotechnol. 29, 279–286 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Papapetrou, E.P. et al. Genomic safe harbors permit high beta-globin transgene expression in thalassemia induced pluripotent stem cells. Nat. Biotechnol. 29, 73–78 (2011).

    Article  CAS  PubMed  Google Scholar 

  18. Szymczak, A.L. et al. Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector. Nat. Biotechnol. 22, 589–594 (2004).

    Article  CAS  PubMed  Google Scholar 

  19. Saenz, D.T. et al. Unintegrated lentivirus DNA persistence and accessibility to expression in nondividing cells: analysis with class I integrase mutants. J. Virol. 78, 2906–2920 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G. & Hochedlinger, K. Induced pluripotent stem cells generated without viral integration. Science 322, 945–949 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Okita, K., Hong, H., Takahashi, K. & Yamanaka, S. Generation of mouse-induced pluripotent stem cells with plasmid vectors. Nat. Protoc. 5, 418–428 (2010).

    Article  CAS  PubMed  Google Scholar 

  22. Yu, J. et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797–801 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Narsinh, K.H. et al. Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors. Nat. Protoc. 6, 78–88 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Howden, S.E. et al. Genetic correction and analysis of induced pluripotent stem cells from a patient with gyrate atrophy. Proc. Natl. Acad. Sci. USA 108, 6537–6542 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Fusaki, N., Ban, H., Nishiyama, A., Saeki, K. & Hasegawa, M. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 85, 348–362 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Nishimura, K. et al. Development of defective and persistent sendai virus vector: a unique gene delivery/expression system ideal for cell reprogramming. J. Biol. Chem. 286, 4760–4771 (2011).

    Article  CAS  PubMed  Google Scholar 

  27. Warren, L. et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7, 618–630 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Zhou, H. et al. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4, 381–384 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. Kim, D. et al. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 4, 472–476 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Cho, H.J. et al. Induction of pluripotent stem cells from adult somatic cells by protein-based reprogramming without genetic manipulation. Blood 116, 386–395 (2010).

    Article  CAS  PubMed  Google Scholar 

  31. Kaji, K. et al. Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature 458, 771–775 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Chang, C.W. et al. Polycistronic lentiviral vector for 'hit and run' reprogramming of adult skin fibroblasts to induced pluripotent stem cells. Stem Cells 27, 1042–1049 (2009).

    Article  CAS  PubMed  Google Scholar 

  33. Sommer, C.A. et al. Excision of reprogramming transgenes improves the differentiation potential of iPS cells generated with a single excisable vector. Stem Cells 28, 64–74 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Somers, A. et al. Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette. Stem Cells 28, 1728–1740 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Woltjen, K. et al. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 458, 766–770 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Yusa, K., Rad, R., Takeda, J. & Bradley, A. Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon. Nat. Methods 6, 363–369 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Wang, W. et al. Chromosomal transposition of PiggyBac in mouse embryonic stem cells. Proc. Natl. Acad. Sci. USA 105, 9290–9295 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Huangfu, D. et al. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat. Biotechnol. 26, 795–797 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Schmidt, M. et al. High-resolution insertion-site analysis by linear amplification-mediated PCR (LAM-PCR). Nat. Methods 4, 1051–1057 (2007).

    Article  CAS  PubMed  Google Scholar 

  40. Laurent, L.C. et al. Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture. Cell Stem Cell 8, 106–118 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the Starr Foundation (Tri-Institutional Stem Cell Initiative, Tri-SCI-018), the New York State Stem Cell Science, NYSTEM (N08T-060) and National Heart, Blood and Lung Institute, NHLBI grant HL053750. We thank present and past members of the Sadelain, Riviere, Studer and Tomishima labs (Memorial Sloan-Kettering Cancer Center, New York) for helpful discussions and technical assistance and E. Poeschla (Mayo Clinic, Rochester, Minnesota) for providing the pCMVΔR8.91N/N plasmid.

Author information

Authors and Affiliations

  1. Center for Cell Engineering, Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Eirini P Papapetrou & Michel Sadelain

Authors
  1. Eirini P Papapetrou
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michel Sadelain
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

E.P.P. developed the protocol and wrote the paper. M.S. supervised the study and edited the paper.

Corresponding author

Correspondence to Eirini P Papapetrou.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Papapetrou, E., Sadelain, M. Generation of transgene-free human induced pluripotent stem cells with an excisable single polycistronic vector. Nat Protoc 6, 1251–1273 (2011). https://doi.org/10.1038/nprot.2011.374

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2011.374

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing