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Early role for IL-6 signalling during generation of induced pluripotent stem cells revealed by heterokaryon RNA-Seq

Nature Cell Biology volume 15, pages 1244–1252 (2013)Cite this article

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Abstract

Molecular insights into somatic cell reprogramming to induced pluripotent stem cells (iPS) would aid regenerative medicine, but are difficult to elucidate in iPS because of their heterogeneity, as relatively few cells undergo reprogramming (0.1–1%; refs 1, 2). To identify early acting regulators, we capitalized on non-dividing heterokaryons (mouse embryonic stem cells fused to human fibroblasts), in which reprogramming towards pluripotency is efficient and rapid3, enabling the identification of transient regulators required at the onset. We used bi-species transcriptome-wide RNA-seq to quantify transcriptional changes in the human somatic nucleus during reprogramming towards pluripotency in heterokaryons. During heterokaryon reprogramming, the cytokine interleukin 6 (IL6), which is not detectable at significant levels in embryonic stem cells, was induced 50-fold. A 4-day culture with IL6 at the onset of iPS reprogramming replaced stably transduced oncogenic c-Myc such that transduction of only Oct4, Klf4 and Sox2 was required. IL6 also activated another Jak/Stat target, the serine/threonine kinase gene Pim1, which accounted for the IL6-mediated twofold increase in iPS frequency. In contrast, LIF, another induced GP130 ligand, failed to increase iPS frequency or activate c-Myc or Pim1, thereby revealing a differential role for the two Jak/Stat inducers in iPS generation. These findings demonstrate the power of heterokaryon bi-species global RNA-seq to identify early acting regulators of reprogramming, for example, extrinsic replacements for stably transduced transcription factors such as the potent oncogene c-Myc.

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Figure 1: Heterokaryon global bi-species transcriptome analysis by RNA-seq identifies differentially expressed genes during somatic cell reprogramming towards pluripotency.
Figure 2: Heterokaryon bi-species RNA-seq identifies induction of GP130 signalling gene expression during somatic cell reprogramming.
Figure 3: IL6, but not LIF, enhances 4F iPS generation.
Figure 4: Transient exposure to IL6 in medium replaces viral c-Myc in reprogramming to iPS and is required only at the onset of 3F reprogramming.
Figure 5: Pim1, a pro-survival gene, acts downstream of IL6 to enhance reprogramming to iPS.

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References

  1. Vierbuchen, T. & Wernig, M. Molecular roadblocks for cellular reprogramming. Mol. Cell 47, 827–838 (2012).

    Article  CAS  PubMed  Google Scholar 

  2. Yamanaka, S. & Blau, H. M. Nuclear reprogramming to a pluripotent state by three approaches. Nature 465, 704–712 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Bhutani, N. et al. Reprogramming towards pluripotency requires AID-dependent DNA demethylation. Nature 463, 1042–1047 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5, 621–628 (2008).

    Article  CAS  PubMed  Google Scholar 

  5. Tarazona, S., Garcia-Alcalde, F., Dopazo, J., Ferrer, A. & Conesa, A. Differential expression in RNA-seq: a matter of depth. Genome Res. 21, 2213–2223 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Thomas, P. D. et al. PANTHER: a browsable database of gene products organized by biological function, using curated protein family and subfamily classification. Nucleic Acids Res. 31, 334–341 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Hirano, T., Ishihara, K. & Hibi, M. Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors. Oncogene 19, 2548–2556 (2000).

    Article  CAS  PubMed  Google Scholar 

  8. Heinrich, P. C. et al. Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem. J. 374, 1–20 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Puthier, D., Bataille, R. & Amiot, M. IL-6 up-regulates mcl-1 in human myeloma cells through JAK/STAT rather than ras/MAP kinase pathway. Eur. J. Immunol. 29, 3945–3950 (1999).

    Article  CAS  PubMed  Google Scholar 

  10. Assou, S. et al. A meta-analysis of human embryonic stem cells transcriptome integrated into a web-based expression atlas. Stem Cells 25, 961–973 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Bhattacharya, B. et al. Gene expression in human embryonic stem cell lines: unique molecular signature. Blood 103, 2956–2964 (2004).

    Article  CAS  PubMed  Google Scholar 

  12. Yang, J., Gao, C., Chai, L. & Ma, Y. A novel SALL4/OCT4 transcriptional feedback network for pluripotency of embryonic stem cells. PLoS One 5, e10766 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Sridharan, R. et al. Role of the murine reprogramming factors in the induction of pluripotency. Cell 136, 364–377 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Buganim, Y. et al. Single-cell expression analyses during cellular reprogramming reveal an early stochastic and a late hierarchic phase. Cell 150, 1209–1222 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).

    Article  CAS  PubMed  Google Scholar 

  16. Cheney, I. W. et al. Identification and structure-activity relationships of substituted pyridones as inhibitors of Pim-1 kinase. Bioorganic Med. Chem. Lett. 17, 1679–1683 (2007).

    Article  CAS  Google Scholar 

  17. Pierce, A. C., Jacobs, M. & Stuver-Moody, C. Docking study yields four novel inhibitors of the protooncogene Pim-1 kinase. J. Med. Chem. 51, 1972–1975 (2008).

    Article  CAS  PubMed  Google Scholar 

  18. Lee, J. et al. Activation of innate immunity is required for efficient nuclear reprogramming. Cell 151, 547–558 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Nihira, K. et al. Pim-1 controls NF-kappaB signalling by stabilizing RelA/p65. Cell Death Differ. 17, 689–698 (2010).

    Article  CAS  PubMed  Google Scholar 

  20. Pasque, V., Jullien, J., Miyamoto, K., Halley-Stott, R. P. & Gurdon, J. B. Epigenetic factors influencing resistance to nuclear reprogramming. Trends Genet. 27, 516–525 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Byrne, J. A. et al. Producing primate embryonic stem cells by somatic cell nuclear transfer. Nature 450, 497–502 (2007).

    Article  CAS  PubMed  Google Scholar 

  22. Hanna, J. et al. Direct cell reprogramming is a stochastic process amenable to acceleration. Nature 462, 595–601 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bhutani, N. et al. A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells. FASEB J. 27, 1107–1113 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Lin, C. Y. et al. Transcriptional amplification in tumor cells with elevated c-Myc. Cell 151, 56–67 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Gilbert, L. A. & Hemann, M. T. Context-specific roles for paracrine IL-6 in lymphomagenesis. Genes Dev. 26, 1758–1768 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Soufi, A., Donahue, G. & Zaret, K. S. Facilitators and impediments of the pluripotency reprogramming factors’ initial engagement with the genome. Cell 151, 994–1004 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Aho, T. L. et al. Pim-1 kinase promotes inactivation of the pro-apoptotic Bad protein by phosphorylating it on the Ser112 gatekeeper site. FEBS Lett. 571, 43–49 (2004).

    Article  CAS  PubMed  Google Scholar 

  28. Van Mourik, M. S., Macklon, N. S. & Heijnen, C. J. Embryonic implantation: cytokines, adhesion molecules, and immune cells in establishing an implantation environment. J. Leukocyte Biol. 85, 4–19 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. Tsubouchi, T. et al. DNA synthesis is required for reprogramming mediated by stem cell fusion. Cell 152, 873–883 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Foshay, K. M. et al. Embryonic stem cells induce pluripotency in somatic cell fusion through biphasic reprogramming. Mol. Cell 46, 159,–170, (2012).

    Article  CAS  Google Scholar 

  31. Holmberg, J. & Perlmann, T. Maintaining differentiated cellular identity. Nat. Rev. Genet. 13, 429–439 (2012).

    Article  CAS  PubMed  Google Scholar 

  32. Silva, J., Chambers, I., Pollard, S. & Smith, A. Nanog promotes transfer of pluripotency after cell fusion. Nature 441, 997–1001 (2006).

    Article  CAS  PubMed  Google Scholar 

  33. Pasque, V., Halley-Stott, R. P., Gillich, A., Garrett, N. & Gurdon, J. B. Epigenetic stability of repressed states involving the histone variant macroH2A revealed by nuclear transfer to Xenopus oocytes. Nucleus 2, 533–539 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Huang, X. A. & Lin, H. The miRNA regulation of stem cells. WIREs Membr. Transp. Signal. 1, 83–95 (2012).

    CAS  Google Scholar 

  35. Graf, T. Historical origins of transdifferentiation and reprogramming. Cell Stem Cell 9, 504–516 (2011).

    Article  CAS  PubMed  Google Scholar 

  36. Salero, E. et al. Adult human RPE can be activated into a multipotent stem cell that produces mesenchymal derivatives. Cell Stem Cell 10, 88–95 (2012).

    Article  CAS  PubMed  Google Scholar 

  37. Marson, A. et al. Wnt signaling promotes reprogramming of somatic cells to pluripotency. Cell Stem Cell 3, 132–135 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Jiang, H. & Wong, W. H. SeqMap: mapping massive amount of oligonucleotides to the genome. Bioinformatics 24, 2395–2396 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank M. Wernig and S. Marro for providing expert guidance with iPS protocols and for the STEMCAA 4F lentiviral construct and R. Tran-Bussat for isolating MEFs. We thank S. L. Oliver, S. Corbel and D. Burns for their insightful comments on the manuscript, the ENCODE Project, in particular Barbara Wold who was very helpful in the initiation of this work, and the Stanford FACS Facility, PAN Facility, and Stem Cell Institute Genome Facility. This work was supported by NSF and Bio-X Graduate Research Fellowships to J.J.B., by NIH grant R01 HG005717 and HG006018 to W.H.W., and by CIRM grant RB1-01292, NIH U01 HL100397, NIH R01 AG009521 and the Baxter Foundation to H.M.B.

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  1. Hui Jiang

    Present address: Present address: Department of Biostatistics, University of Michigan, Ann Arbor, Michigan 48109, USA,

Authors and Affiliations

  1. Department of Microbiology and Immunology, Baxter Laboratory for Stem Cell Biology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California 94305, USA

    Jennifer J. Brady, Silpa Suthram & Helen M. Blau

  2. Institute for Computational and Mathematical Engineering, Stanford University, Stanford, California 94305, USA

    Mavis Li & Hui Jiang

  3. Department of Statistics, Stanford University, Stanford, California 94305, USA

    Mavis Li, Hui Jiang & Wing H. Wong

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Contributions

J.J.B. and H.M.B. designed the experiments, interpreted the results and wrote the manuscript. J.J.B. performed all of the experiments, H.J. designed SeqMap and M.L. generated the RPKM values under the direction of W.H.W. S.S. performed the NOISeq analysis.

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Correspondence to Helen M. Blau.

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Integrated supplementary information

Supplementary Figure 1 Expression of IL6 type cytokine family members, Il6r, and Lifr in heterokaryon or 4F virus induced reprogramming.

a, Relative transcript levels of IL6 type cytokine family members OSM, IL11, CNTF, CT-1, LIF, and IL6 during heterokaryon reprogramming, normalized to unfused human fibroblasts. Data are represented as mean with range (n = 2). b, MEFs were transduced with 4F virus and Il6 and Lif receptor transcript levels were monitored over a 6-day reprogramming time course by qPCR. Il6r was maximally induced 6.6 ± 0.7-fold while Lifr was maximally induced 1.4 ± 0.2-fold (mean ± s.d. for n = 4 experiments). Gene expression was calculated relative to Day 0 and Hprt levels.

Supplementary Figure 2 Characterization of iPS colonies and clones.

a,b, iPS colonies were stained for Hoechst and the secondary antibody indicated. Scale bar = 200 μm. c, RT-PCR for mouse pluripotency genes from 3F + IL6 and 4F derived mouse iPS clones. Mouse ES cells and untreated MEFs are shown as positive and negative controls for gene expression, respectively. Full gels are shown in Supplementary Fig. S5. d, Genomic PCR for viral integration of the reprogramming factors Oct4, Klf4, Sox2 and c-Myc from 3F + IL6, 3F + c-Myc, and 4F virus derived iPS clones. Full gels are shown in Supplementary Fig. S5.

Supplementary Figure 3 Pim1 inhibitors used in this study.

a, 3-Cyano-4-phenyl-6-(3-bromo-6-hydroxy) phenyl-2(1 H)-pyridone. b, 4-(3-(4-Chlorophenyl)-2,1-benzisoxazol-5-yl)-2-pyrimidinamine. c, Western blot for Bad phosphorylation (a Pim1 substrate) upon IL6 stimulation in the presence of DMSO or Pim1 inhibitors. Quantification is presented as levels of p-Bad normalized to levels of total Bad. Pim1 and α-Tubulin are shown as a reference and loading control. Full blots are shown in Supplementary Fig. S5.

Supplementary Figure 4 Il6r loss of function and validation.

a, Diagram of the mouse Il6 receptor transcript and shRNA target regions. b, Target sequences of Il6r shRNAs. c, Knockdown of mIl6r in MEFs. Transcript levels of Il6r mRNA are shown relative to Actin and the shControl, assessed 3 days post transduction (mean ± s.d., n = 3).

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Brady, J., Li, M., Suthram, S. et al. Early role for IL-6 signalling during generation of induced pluripotent stem cells revealed by heterokaryon RNA-Seq. Nat Cell Biol 15, 1244–1252 (2013). https://doi.org/10.1038/ncb2835

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