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Reprogramming human fibroblasts to pluripotency using modified mRNA

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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.

Change history

  • 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.

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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

Affiliations

Authors

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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