Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2

Abstract

Ectopic expression of defined sets of genetic factors can reprogram somatic cells to induced pluripotent stem (iPS) cells that closely resemble embryonic stem (ES) cells. The low efficiency with which iPS cells are derived hinders studies on the molecular mechanism of reprogramming, and integration of viral transgenes, in particular the oncogenes c-Myc and Klf4, may handicap this method for human therapeutic applications. Here we report that valproic acid (VPA), a histone deacetylase inhibitor, enables reprogramming of primary human fibroblasts with only two factors, Oct4 and Sox2, without the need for the oncogenes c-Myc or Klf4. The two factor–induced human iPS cells resemble human ES cells in pluripotency, global gene expression profiles and epigenetic states. These results support the possibility of reprogramming through purely chemical means, which would make therapeutic use of reprogrammed cells safer and more practical.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Efficient generation of iPS cells with three transcription factors (Oct4, Sox2, and Klf4).
Figure 2: Generation of iPS cells with two factors (Oct4 and Sox2) and VPA.
Figure 3: In vitro differentiation of two factor–induced iPS cells.
Figure 4: Teratomas from two factor–induced iPS cells.
Figure 5: Methylation analysis of OCT4 and NANOG promoter regions in two factor–induced iPS cells.
Figure 6: The gene expression profile of two factor–induced iPS cells closely resembles that of human ES cells.

References

  1. 1

    Gurdon, J.B., Elsdale, T.R. & Fischberg, M. Sexually mature individuals of Xenopus laevis from the transplantation of single somatic nuclei. Nature 182, 64–65 (1958).

    CAS  Article  Google Scholar 

  2. 2

    Wilmut, I. et al. Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810–813 (1997).

    CAS  Article  Google Scholar 

  3. 3

    Wakayama, T. et al. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394, 369–374 (1998).

    CAS  Article  Google Scholar 

  4. 4

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

    CAS  Article  Google Scholar 

  5. 5

    Tada, M. et al. Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells. Curr. Biol. 11, 1553–1558 (2001).

    CAS  Article  Google Scholar 

  6. 6

    Cowan, C.A., Atienza, J., Melton, D.A. & Eggan, K. Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science 309, 1369–1373 (2005).

    CAS  Article  Google Scholar 

  7. 7

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

    CAS  Article  Google Scholar 

  8. 8

    Okita, K., Ichisaka, T. & Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. Nature 448, 313–317 (2007).

    CAS  Article  Google Scholar 

  9. 9

    Maherali, N. et al. Directly Reprogrammed Fibroblasts Show Global Epigenetic Remodeling and Widespread Tissue Contribution. Cell Stem Cell 1, 55–70 (2007).

    CAS  Article  Google Scholar 

  10. 10

    Wernig, M. et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318–324 (2007).

    CAS  Article  Google Scholar 

  11. 11

    Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    CAS  Article  Google Scholar 

  12. 12

    Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

    CAS  Article  Google Scholar 

  13. 13

    Park, I.H. et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141–146 (2008).

    CAS  Article  Google Scholar 

  14. 14

    Lowry, W.E. et al. Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc. Natl. Acad. Sci. USA 105, 2883–2888 (2008).

    CAS  Article  Google Scholar 

  15. 15

    Nakagawa, M. et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat. Biotechnol. 26, 101–106 (2008).

    CAS  Article  Google Scholar 

  16. 16

    Wernig, M., Meissner, A., Cassady, J.P. & Jaenisch, R. c-Myc is dispensable for direct reprogramming of mouse fibroblasts. Cell Stem Cell 2, 10–12 (2008).

    CAS  Article  Google Scholar 

  17. 17

    Kim, J.B. et al. Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature 454, 646–650 (2008).

    CAS  Article  Google Scholar 

  18. 18

    Shi, Y. et al. A combined chemical and genetic approach for the generation of induced pluripotent stem cells. Cell Stem Cell 2, 525–528 (2008).

    CAS  Article  Google Scholar 

  19. 19

    Eminli, S. et al. Reprogramming of neural progenitor cells into iPS cells in the absence of exogenous Sox2 expression. Stem Cells, published online, doi:10.1634/stemcells.2008-0317 (17 July 2008).

  20. 20

    Nunes, M.C. et al. Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain. Nat. Med. 9, 439–447 (2003).

    CAS  Article  Google Scholar 

  21. 21

    Huangfu, D. et al. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat. Biotechnol. 26, 795–797 (2008).

    CAS  Article  Google Scholar 

  22. 22

    Mikkelsen, T.S. et al. Dissecting direct reprogramming through integrative genomic analysis. Nature 454, 49–55 (2008).

    CAS  Article  Google Scholar 

  23. 23

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

    CAS  Article  Google Scholar 

  24. 24

    Meissner, A., Wernig, M. & Jaenisch, R. Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat. Biotechnol. 25, 1177–1181 (2007).

    CAS  Article  Google Scholar 

  25. 25

    Blelloch, R., Venere, M., Yen, J. & Ramalho-Santos, M. Generation of Induced Pluripotent Stem Cells in the Absence of Drug Selection. Cell Stem Cell 1, 245–247 (2007).

    CAS  Article  Google Scholar 

  26. 26

    Amit, M. et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 227, 271–278 (2000).

    CAS  Article  Google Scholar 

  27. 27

    Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    CAS  Article  Google Scholar 

  28. 28

    Kawasaki, H. et al. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 28, 31–40 (2000).

    CAS  Article  Google Scholar 

  29. 29

    Osafune, K. et al. Marked differences in differentiation propensity among human embryonic stem cell lines. Nat. Biotechnol. 26, 313–315 (2008).

    CAS  Article  Google Scholar 

  30. 30

    D'Amour, K.A. et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat. Biotechnol. 23, 1534–1541 (2005).

    CAS  Article  Google Scholar 

  31. 31

    Cowan, C.A. et al. Derivation of embryonic stem-cell lines from human blastocysts. N. Engl. J. Med. 350, 1353–1356 (2004).

    CAS  Article  Google Scholar 

  32. 32

    Deb-Rinker, P. et al. Sequential DNA methylation of the Nanog and Oct-4 upstream regions in human NT2 cells during neuronal differentiation. J. Biol. Chem. 280, 6257–6260 (2005).

    CAS  Article  Google Scholar 

  33. 33

    Freberg, C.T., Dahl, J.A., Timoskainen, S. & Collas, P. Epigenetic reprogramming of OCT4 and NANOG regulatory regions by embryonal carcinoma cell extract. Mol. Biol. Cell 18, 1543–1553 (2007).

    CAS  Article  Google Scholar 

  34. 34

    Rossant, J. Stem cells: the magic brew. Nature 448, 260–262 (2007).

    CAS  Article  Google Scholar 

  35. 35

    Zaehres, H. & Scholer, H.R. Induction of pluripotency: from mouse to human. Cell 131, 834–835 (2007).

    CAS  Article  Google Scholar 

  36. 36

    Perry, A.C. Induced pluripotency and cellular alchemy. Nat. Biotechnol. 24, 1363–1364 (2006).

    CAS  Article  Google Scholar 

  37. 37

    Leeman, B.A. & Cole, A.J. Advancements in the treatment of epilepsy. Annu. Rev. Med. 59, 503–523 (2008).

    CAS  Article  Google Scholar 

  38. 38

    Boyer, L.A. et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956 (2005).

    CAS  Article  Google Scholar 

  39. 39

    Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

D.H. and D.A.M. conceived the experiments and wrote the manuscript. D.H., K.O., R.M., W.G., A.E., S.C. and W.M. performed experiments.

Author information

Affiliations

Authors

Contributions

D.A.M. is a Howard Hughes Medical Institute Investigator. D.H. is funded by the Helen Hay Whitney Foundation and Novartis Institutes for BioMedical Research. S.C. is supported by the Juvenile Diabetics Research Foundation. The authors thank Anastasie Kweudjeu for assistance with micoarray analysis, Konrad Hochedlinger for providing probes for Southern blot analysis, Shinya Yamanaka for providing viral vectors through Addgene and Robert Weinberg for support of this study. Some monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank, which was developed under the auspices of the National Institute of Child Health and Human Development and is maintained by The University of Iowa, Department of Biological Sciences.

Corresponding author

Correspondence to Douglas A Melton.

Supplementary information

Supplementary Text and Figures

Figures 1–6, Tables 1–6 (PDF 643 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Huangfu, D., Osafune, K., Maehr, R. et al. Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nat Biotechnol 26, 1269–1275 (2008). https://doi.org/10.1038/nbt.1502

Download citation

Further reading