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MicroRNA-based discovery of barriers to dedifferentiation of fibroblasts to pluripotent stem cells

Nature Structural & Molecular Biology volume 20, pages 1227–1235 (2013)Cite this article

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Abstract

Individual microRNAs (miRNAs) can target hundreds of mRNAs forming networks of presumably cooperating genes. To test this presumption, we functionally screened miRNAs and their targets in the context of dedifferentiation of mouse fibroblasts to induced pluripotent stem cells (iPSCs). Along with the miR-302–miR-294 family, the miR-181 family arose as a previously unidentified enhancer of the initiation phase of reprogramming. Endogenous miR-181 miRNAs were transiently elevated with the introduction of Pou5f1 (also known as Oct4), Sox2 and Klf4 (referred to as OSK), and miR-181 inhibition diminished iPSC colony formation. We tested the functional contribution of 114 individual targets of the two families, revealing 25 genes that normally suppress initiation. Coinhibition of targets cooperatively promoted both the frequency and kinetics of OSK-induced reprogramming. These data establish two of the largest functionally defined networks of miRNA-mRNA interactions and reveal previously unidentified relationships among genes that act together to suppress early stages of reprogramming.

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Figure 1: A genome-wide screen identifies known and unknown miRNA enhancers of OSK-induced reprogramming.
Figure 2: The miR-181 family is an OSK-activated positive regulator of reprogramming.
Figure 3: miR-294 and miR-181 enhance reprogramming during the early initiation phase.
Figure 4: An unbiased screen reveals previously unidentified direct targets of miR-294 and miR-181 that inhibit initiation of reprogramming.
Figure 5: miR-294 and miR-181 alter both distinct and common properties of reprogramming.
Figure 6: miRNA-targeted genes cooperate to reduce both frequency and rate of reprogramming.
Figure 7: MiR-294 and miR-181 targets converge on cooperating pathways and processes to enhance reprogramming.

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Acknowledgements

We thank C. Belair, M. Cook, R. Krishnakumar, M. La Russa, M. Shveygert and other members of the Blelloch laboratory for critical reading of the manuscript, A. Amiet (Dharmacon Thermo Scientific) for providing miRNA and siRNA libraries, M. McMahon (University of California, San Francisco) for AKT expression constructs, H. Zhang for assistance with the high-content analysis, J. Paquette, R. Bell and A. Diaz for advice concerning our statistical methods, A. Shenoy for assistance with bioinformatics, and M. Kissner for assistance with flow cytometry. This work was supported by funds to R.B. from US National Institutes of Health (R01:GM101180), the Leona M. and Harry B. Helmsley Charitable Trust (09PG-T1D002) and the California Institute of Regenerative Medicine (RN2-00906-1). R.L.J. was supported by a US National Science Foundation graduate research fellowship.

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Authors and Affiliations

  1. The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences, Program in Biomedical Sciences, University of California San Francisco, San Francisco, California, USA

    Robert L Judson, Tobias S Greve, Ronald J Parchem & Robert Blelloch

  2. Department of Urology, University of California San Francisco, San Francisco, California, USA

    Robert L Judson, Tobias S Greve, Ronald J Parchem & Robert Blelloch

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  1. Robert L Judson
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  4. Robert Blelloch
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Contributions

R.L.J. contributed to data in Figures 1,2,3,4a–e,5,6,7a,f,g and Supplementary Figures 2–6. T.S.G. contributed to data in Figures 4d,f and 7b–e and Supplementary Figures 1, 2, 4 and 7. R.J.P. contributed to data in Figures 2b and Supplementary Figures 1 and 2. R.B. and R.L.J. conceived the experiments, analyzed the data and wrote the manuscript.

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Correspondence to Robert Blelloch.

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The authors declare no competing financial interests.

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Judson, R., Greve, T., Parchem, R. et al. MicroRNA-based discovery of barriers to dedifferentiation of fibroblasts to pluripotent stem cells. Nat Struct Mol Biol 20, 1227–1235 (2013). https://doi.org/10.1038/nsmb.2665

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