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Multiple targets of miR-302 and miR-372 promote reprogramming of human fibroblasts to induced pluripotent stem cells

Abstract

The embryonic stem cell–specific cell cycle–regulating (ESCC) family of microRNAs (miRNAs) enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells1. Here we show that the human ESCC miRNA orthologs hsa-miR-302b and hsa-miR-372 promote human somatic cell reprogramming. Furthermore, these miRNAs repress multiple target genes, with downregulation of individual targets only partially recapitulating the total miRNA effects. These targets regulate various cellular processes, including cell cycle, epithelial-mesenchymal transition (EMT), epigenetic regulation and vesicular transport. ESCC miRNAs have a known role in regulating the unique embryonic stem cell cycle2,3. We show that they also increase the kinetics of mesenchymal-epithelial transition during reprogramming and block TGFβ-induced EMT of human epithelial cells. These results demonstrate that the ESCC miRNAs promote dedifferentiation by acting on multiple downstream pathways. We propose that individual miRNAs generally act through numerous pathways that synergize to regulate and enforce cell fate decisions.

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Figure 1: Hsa-miR-302b and/or hsa-miR-372 enhances reprogramming efficiency of human somatic cells.
Figure 2: Hsa-miR-302b and hsa-miR-372 regulate expression of a number of targets that influence reprogramming of human somatic cells.
Figure 3: Hsa-miR-302b and hsa-miR-372 enhance reprogramming by regulating mesenchymal-epithelial transition.
Figure 4: Hsa-miR-302b, hsa-miR-372 and mmu-miR-294 inhibit TGF-β–induced epithelial-mesenchymal transition in human cells.

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Acknowledgements

We would like to thank A. Amiet at ThermoFisher for advising and kindly sharing miRNA and siRNA reagents, R. Shaw for access to fluorescence microscopy, and M. Yumoto for primers. This work was supported by funds to R.B. from Leona M. and Harry B. Helmsley Charitable Trust, US National Institutes of Health (K08 NS48118 and R01 NS057221), California Institute of Regenerative Medicine (CIRM) (Seed Grant RS1-00161, New Faculty Award RN2-00906), grant RO1-CA136690 to R.D., an American Heart Association scientist development award (no. 09SDG2280008) to S.L. and SFSU CIRM Bridges TB1-01194 to J.Y.L. Venus was a gift from K. Eggan, packaged by Harvard Gene Therapy Initiative.

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D.S. did the experiments described in Figures 1, 2 and 3. S.L. and R.L.J. did experiments described in Figure 4. J.Y.L. helped with experiments described in Figure 2a and performed experiments described in 3b. N.B. helped with experiments in Figure 1b,c. D.S. and R.B. wrote the manuscript with help from S.L., R.L.J. and R.D.

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Correspondence to Robert Blelloch.

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The authors declare no competing financial interests.

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Supplementary Tables 1 and 2 and Supplementary Figs. 1–10 (PDF 2704 kb)

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Subramanyam, D., Lamouille, S., Judson, R. et al. Multiple targets of miR-302 and miR-372 promote reprogramming of human fibroblasts to induced pluripotent stem cells. Nat Biotechnol 29, 443–448 (2011). https://doi.org/10.1038/nbt.1862

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