Reprogramming human fibroblasts to pluripotency using modified mRNA

Journal name:
Nature Protocols
Year published:
Published online
Corrected online


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.

At a glance


  1. Expression of reprogramming factors by modified mRNA.
    Figure 1: Expression of reprogramming factors by modified mRNA.

    (a,b) Dermal fibroblasts transfected with the indicated modified mRNA showing protein expression by (a) immunostaining (a) and western blotting (WB) (b). The antibodies used are described in MATERIALS. DAPI, 4′,6-diamidino-2-phenylindole (a DNA-binding fluorescent stain); mES, mouse ES cell lysate. Scale bars, 50 μm.

  2. Reprogramming of human fibroblasts using modified mRNA.
    Figure 2: Reprogramming of human fibroblasts using modified mRNA.

    (a) Schematic representation of a typical reprogramming experiment using modified mRNA indicating timing for key events. The medium used at each stage of the protocol is indicated: DMEM (complete) for feeder cell and fibroblast plating, Pluriton medium (complete) for modified-mRNA reprogramming, hES medium for maintenance and expansion of picked iPS cell clones on CF-1 feeder cells and mTeSR1 for adaptation and maintenance of iPS cell clones on Matrigel. The complete composition of each medium is detailed in the Reagent Setup section of the protocol. (b) Morphological changes observed during the reprogramming experiment. Areas of morphological changes characteristic of the mesenchymal to epithelial transition are marked with a yellow dashed circles, and emergent colonies are marked with a solid yellow arrows. Scale bars, 100 μm. (c) Live staining of emerging colonies at day 14 of reprogramming showing immunostaining of SSEA-4 and TRA-1-60. Low magnification showing one-fourth of a six-well plate (left) and individual colonies (right). Scale bars, 50 μm. (d) Matrigel-adapted established iPS cell colonies maintained in mTeSR1 medium, immunostained with NANOG and OCT4. Scale bars, 50 μm.

  3. IVT and quality control of modified mRNA.
    Figure 3: IVT and quality control of modified mRNA.

    (a) A schematic representation of the reprogramming constructs (OKMSL) and NDG in pcDNA3.3. Note that the T7 promoter driving mRNA synthesis is upstream of the 5′ UTR. (b) Workflow for modified-mRNA synthesis, with the estimated timeline and critical quality control measures indicated. (c) Addition of poly-(A) tail to template DNA by tail-PCR using primers Xu-F1 and Xu-T120. Gel electrophoresis image showing the size of tailed templates for L, K, M, O and S. (d) Representative NanoDrop reading of a typical IVT preparation (red line), with a poor yield reaction included for comparison (blue line marked by *). (e) Analysis of modified mRNA by Bioanalyzer. L, LIN28A; K, KLF4; M, C-MYC; O, OCT4; S, SOX2; NDG, nuclear destabilized EGFP; Mr, marker (DNA ladder in c, RNA ladder in e).

  4. Events observed during the course of reprogramming of human fibroblasts using modified mRNA.
    Figure 4: Events observed during the course of reprogramming of human fibroblasts using modified mRNA.

    (ai) Images showing the most commonly encountered events during the course of a reprogramming experiment. Their causes and possible explanations are described in the Troubleshooting section. (a) Overgrowth on plate. (b) Rare feeder cell density. (c) Holes in cell monolayer. (d) Cell clumps after passaging or transformation event. (e) Emerging colonies growing under the feeder layer (yellow arrows). Note that, in this field, a normal emerging colony is marked with a red arrow. (f) Cluster of loosely packed cells. (g) Partially reprogrammed colony. (h) Emerging colony with a sharp boundary, but probably not fully reprogrammed. (i) Typical iPS cell colony. Scale bars, 100 μm.

Change history

Corrected online 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.


  1. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663676 (2006).
  2. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861872 (2007).
  3. Park, I.H. et al. Disease-specific induced pluripotent stem cells. Cell 134, 877886 (2008).
  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, 915929 (2011).
  5. Robinton, D.A. & Daley, G.Q. The promise of induced pluripotent stem cells in research and therapy. Nature 481, 295305 (2012).
  6. Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G. & Hochedlinger, K. Induced pluripotent stem cells generated without viral integration. Science 322, 945949 (2008).
  7. Yu, J. et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797801 (2009).
  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, 89188922 (2009).
  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, e109e119 (2011).
  10. Jia, F. et al. A nonviral minicircle vector for deriving human iPS cells. Nat. Methods 7, 197199 (2010).
  11. Narsinh, K.H. et al. Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors. Nat. Protoc. 6, 7888 (2011).
  12. Okita, K. et al. A more efficient method to generate integration-free human iPS cells. Nat. Methods 8, 409412 (2011).
  13. Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T. & Yamanaka, S. Generation of mouse induced pluripotent stem cells without viral vectors. Science 322, 949953 (2008).
  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).
  15. Woltjen, K. et al. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 458, 766770 (2009).
  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).
  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, 363369 (2009).
  18. Kim, D. et al. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 4, 472476 (2009).
  19. Zhou, H. et al. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4, 381384 (2009).
  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, 348362 (2009).
  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, 1423414239 (2011).
  22. Warren, L. et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7, 618630 (2010).
  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, 15291531 (2004).
  24. Hornung, V. et al. 5′-Triphosphate RNA is the ligand for RIG-I. Science 314, 994997 (2006).
  25. Pichlmair, A. et al. RIG-I-mediated antiviral responses to single-stranded RNA bearing 5′-phosphates. Science 314, 9971001 (2006).
  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, 165175 (2005).
  27. Angel, M. & Yanik, M.F. Innate immune suppression enables frequent transfection with RNA encoding reprogramming proteins. PLoS ONE 5, e11756 (2010).
  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, 523532 (2007).
  29. Kariko, K. et al. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol. Ther. 16, 18331840 (2008).
  30. Anderson, B.R. et al. Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation. Nucleic Acids Res. 38, 58845892 (2010).
  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, 8690 (1997).
  32. Kormann, M.S. et al. Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nat. Biotechnol. 29, 154157 (2011).
  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, 948953 (2012).
  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).
  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).

Download references

Author information


  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


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.

Competing financial 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.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Data (710 KB)

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

Additional data