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Reprogramming human fibroblasts to pluripotency using modified mRNA

Nature Protocols volume 8, pages 568–582 (2013)Cite this article

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Abstract

Induced pluripotent stem (iPS) cells hold the potential to revolutionize regenerative medicine through their capacity to generate cells of diverse lineages for future patient-specific cell-based therapies. To facilitate the transition of iPS cells to clinical practice, a variety of technologies have been developed for transgene-free pluripotency reprogramming. We recently reported efficient iPS cell generation from human fibroblasts using synthetic modified mRNAs. Here we describe a stepwise protocol for the generation of modified mRNA–derived iPS cells from primary human fibroblasts, focusing on the critical parameters including medium choice, quality control, and optimization steps needed for synthesizing modified mRNAs encoding reprogramming factors and introducing these into cells over the course of 2–3 weeks to ensure successful reprogramming. The protocol described herein is for reprogramming of human fibroblasts to pluripotency; however, the properties of modified mRNA make it a powerful platform for protein expression, which has broad applicability in directed differentiation, cell fate specification and therapeutic applications.

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Figure 1: Expression of reprogramming factors by modified mRNA.
Figure 2: Reprogramming of human fibroblasts using modified mRNA.
Figure 3: IVT and quality control of modified mRNA.
Figure 4: Events observed during the course of reprogramming of human fibroblasts using modified mRNA.

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  • 20 March 2013

     In the version of this article initially published, acknowledgment of the technical assistance of Andrew Ettenger was omitted. The error has been corrected in the HTML and PDF versions of the article.

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  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Park, I.H. et al. Disease-specific induced pluripotent stem cells. Cell 134, 877–886 (2008).

    Article  CAS  Google Scholar 

  4. Grskovic, M., Javaherian, A., Strulovici, B. & Daley, G.Q. Induced pluripotent stem cells–opportunities for disease modelling and drug discovery. Nat. Rev. Drug Discov. 10, 915–929 (2011).

    Article  CAS  Google Scholar 

  5. Robinton, D.A. & Daley, G.Q. The promise of induced pluripotent stem cells in research and therapy. Nature 481, 295–305 (2012).

    Article  CAS  Google Scholar 

  6. 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  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Gonzalez, F. et al. Generation of mouse-induced pluripotent stem cells by transient expression of a single nonviral polycistronic vector. Proc. Natl. Acad. Sci. USA 106, 8918–8922 (2009).

    Article  CAS  Google Scholar 

  9. Hu, K. et al. Efficient generation of transgene-free induced pluripotent stem cells from normal and neoplastic bone marrow and cord blood mononuclear cells. Blood 117, e109–e119 (2011).

    Article  CAS  Google Scholar 

  10. Jia, F. et al. A nonviral minicircle vector for deriving human iPS cells. Nat. Methods 7, 197–199 (2010).

    Article  CAS  Google Scholar 

  11. Narsinh, K.H. et al. Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors. Nat. Protoc. 6, 78–88 (2011).

    Article  CAS  Google Scholar 

  12. Okita, K. et al. A more efficient method to generate integration-free human iPS cells. Nat. Methods 8, 409–412 (2011).

    Article  CAS  Google Scholar 

  13. Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T. & Yamanaka, S. Generation of mouse induced pluripotent stem cells without viral vectors. Science 322, 949–953 (2008).

    Article  CAS  Google Scholar 

  14. Si-Tayeb, K. et al. Generation of human induced pluripotent stem cells by simple transient transfection of plasmid DNA encoding reprogramming factors. BMC Dev. Biol. 10, 81 (2010).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. Yu, J., Chau, K.F., Vodyanik, M.A., Jiang, J. & Jiang, Y. Efficient feeder-free episomal reprogramming with small molecules. PLoS ONE 6, e17557 (2011).

    Article  CAS  Google Scholar 

  17. 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  Google Scholar 

  18. 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  Google Scholar 

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

    Article  CAS  Google Scholar 

  20. 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  Google Scholar 

  21. Ban, H. et al. Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors. Proc. Natl. Acad. Sci. USA 108, 14234–14239 (2011).

    Article  CAS  Google Scholar 

  22. 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  Google Scholar 

  23. Diebold, S.S., Kaisho, T., Hemmi, H., Akira, S. & Reis e Sousa, C. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303, 1529–1531 (2004).

    Article  CAS  Google Scholar 

  24. Hornung, V. et al. 5′-Triphosphate RNA is the ligand for RIG-I. Science 314, 994–997 (2006).

    Article  Google Scholar 

  25. Pichlmair, A. et al. RIG-I-mediated antiviral responses to single-stranded RNA bearing 5′-phosphates. Science 314, 997–1001 (2006).

    Article  CAS  Google Scholar 

  26. Kariko, K., Buckstein, M., Ni, H. & Weissman, D. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23, 165–175 (2005).

    Article  CAS  Google Scholar 

  27. Angel, M. & Yanik, M.F. Innate immune suppression enables frequent transfection with RNA encoding reprogramming proteins. PLoS ONE 5, e11756 (2010).

    Article  Google Scholar 

  28. Kariko, K. & Weissman, D. Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development. Curr. Opin Drug Discov. Devel. 10, 523–532 (2007).

    CAS  PubMed  Google Scholar 

  29. Kariko, K. et al. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol. Ther. 16, 1833–1840 (2008).

    Article  CAS  Google Scholar 

  30. Anderson, B.R. et al. Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation. Nucleic Acids Res. 38, 5884–5892 (2010).

    Article  CAS  Google Scholar 

  31. Liptakova, H., Kontsekova, E., Alcami, A., Smith, G.L. & Kontsek, P. Analysis of an interaction between the soluble vaccinia virus-coded type I interferon (IFN)-receptor and human IFN-α1 and IFN-α2. Virology 232, 86–90 (1997).

    Article  CAS  Google Scholar 

  32. Kormann, M.S. et al. Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nat. Biotechnol. 29, 154–157 (2011).

    Article  CAS  Google Scholar 

  33. Kariko, K., Muramatsu, H., Keller, J.M. & Weissman, D. Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin. Mol. Ther. 20, 948–953 (2012).

    Article  CAS  Google Scholar 

  34. Warren, L., Ni, Y., Wang, J. & Guo, X. Feeder-free derivation of human induced pluripotent stem cells with messenger RNA. Scientific Reports 2, 657 doi:10.1038/srep00657 (2012).

    Article  Google Scholar 

  35. McElroy, S.L. & Reijo Pera, R.A. Culturing human embryonic stem cells in feeder-free conditions. Cold Spring Harb. Protoc. 2008, doi:10.1101/pdb.prot5044 (2008).

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Acknowledgements

We thank T. Schlaeger, L. Daheron, W. Ebina and L. Zhangi for their valuable suggestions and discussion, and L. Warren and P. Manos for past contributions. We thank A. Ettenger for technical assistance. This work was funded in part by a grant from the Harvard Stem Cell Institute. D.J.R. is a New York Stem Cell Foundation Robertson Investigator.

Author information

Authors and Affiliations

  1. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA

    Pankaj K Mandal & Derrick J Rossi

  2. Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA

    Pankaj K Mandal & Derrick J Rossi

  3. Division of Hematology/Oncology, Boston Children's Hospital, Boston,, Massachusetts, USA

    Derrick J Rossi

  4. Department of Pediatrics, Harvard Medical School, Boston,, Massachusetts, USA

    Derrick J Rossi

  5. Harvard Stem Cell Institute, Cambridge, Massachusetts, USA

    Derrick J Rossi

Authors
  1. Pankaj K Mandal
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  2. Derrick J Rossi
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Contributions

P.K.M. and D.J.R. designed the experiments. P.K.M. performed the experiments. P.K.M. and D.J.R. analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Derrick J Rossi.

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Competing interests

D.J.R. is a cofounder of ModeRNA Therapeutics, a Cambridge, Massachusetts–based biotechnology company that is exploring the therapeutic potential of modified mRNA.

Supplementary information

Supplementary Data

Annotated sequence files of reprogramming factors (KLF4, c-MYC, OCT4, SOX2 and LIN28A) and NDG (PDF 710 kb)

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Mandal, P., Rossi, D. Reprogramming human fibroblasts to pluripotency using modified mRNA. Nat Protoc 8, 568–582 (2013). https://doi.org/10.1038/nprot.2013.019

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