Reprogramming human fibroblasts to pluripotency using modified mRNA

Journal name:
Nature Protocols
Volume:
8,
Pages:
568–582
Year published:
DOI:
doi:10.1038/nprot.2013.019
Published online
Corrected online

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.

At a glance

Figures

  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.

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Author information

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

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.

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:

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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