Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Generation of integration-free human induced pluripotent stem cells from postnatal blood mononuclear cells by plasmid vector expression

Nature Protocols volume 7, pages 2013–2021 (2012)Cite this article

Subjects

Abstract

Several human postnatal somatic cell types have been successfully reprogrammed to induced pluripotent stem cells (iPSCs). Blood mononuclear cells (MNCs) offer several advantages compared with other cell types. They are easily isolated from umbilical cord blood (CB) or adult peripheral blood (PB), and can be used fresh or after freezing. A short culture allows for more efficient reprogramming, with iPSC colonies forming from blood MNCs in 14 d, compared with 28 d for age-matched fibroblastic cells. The advantages of briefly cultured blood MNCs may be due to favorable epigenetic profiles and gene expression patterns. Blood cells from adults, especially nonlymphoid cells that are replenished frequently from intermittently activated blood stem cells, are short-lived in vivo and may contain less somatic mutations than skin fibroblasts, which are more exposed to environmental mutagens over time. We describe here a detailed, validated protocol for effective generation of integration-free human iPSCs from blood MNCs by plasmid vectors.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Timeline for the MNC expansion and iPSC generation protocol.
Figure 2: A diagram of the polycistronic episomal reprogramming plasmid pEB-C5.
Figure 3: Characterization of human iPSCs derived from blood cells with episomal vectors.

Similar content being viewed by others

References

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Aasen, T. et al. Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat. Biotechnol. 26, 1276–1284 (2008).

    Article  CAS  Google Scholar 

  5. Mali, P. et al. Improved efficiency and pace of generating induced pluripotent stem cells from human adult and fetal fibroblasts. Stem Cells 26, 1998–2005 (2008).

    Article  CAS  Google Scholar 

  6. Loh, Y. et al. Generation of induced pluripotent stem cells from human blood. Blood 113, 5476–5479 (2009).

    Article  CAS  Google Scholar 

  7. Ye, Z. et al. Human-induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders. Blood 114, 5473–5480 (2009).

    Article  CAS  Google Scholar 

  8. Kim, D. et al. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 4, 472–476 (2009).

    Article  CAS  Google Scholar 

  9. Warren, L. et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7, 618–630 (2010).

    Article  CAS  Google Scholar 

  10. Yu, J. et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797–801 (2009).

    Article  CAS  Google Scholar 

  11. Marchetto, M.C. et al. Transcriptional signature and memory retention of human induced pluripotent stem cells. PLoS ONE 18, e7076 (2009).

    Article  Google Scholar 

  12. Mali, P. et al. Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes. Stem Cells 28, 713–720 (2010).

    Article  CAS  Google Scholar 

  13. Chou, B.K. et al. Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures. Cell Res. 21, 518–529 (2011).

    Article  CAS  Google Scholar 

  14. Cheng, L. et al. Low incidence of DNA sequence variation in human induced pluripotent stem cells generated by nonintegrating plasmid expression. Cell Stem Cell 10, 337–344 (2012).

    Article  CAS  Google Scholar 

  15. Zou, C. et al. Efficient derivation and genetic modifications of human pluripotent stem cells on engineered human feeder cell lines. Stem Cells Dev. 21, 2298–2311 (2012).

    Article  CAS  Google Scholar 

  16. Brown, M.E. et al. Derivation of induced pluripotent stem cells from human peripheral blood T lymphocytes. PLoS ONE 5, e11373 (2010).

    Article  Google Scholar 

  17. Loh, Y.H. et al. Reprogramming of T cells from human peripheral blood. Cell Stem Cell 7, 15–19 (2010).

    Article  Google Scholar 

  18. Seki, T. et al. Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells. Cell Stem Cell 7, 11–14 (2010).

    Article  CAS  Google Scholar 

  19. Staerk, J. et al. Reprogramming of human peripheral blood cells to induced pluripotent stem cells. Cell Stem Cell 7, 20–24 (2010).

    Article  CAS  Google Scholar 

  20. Kunisato, A. et al. Direct generation of induced pluripotent stem cells from human nonmobilized blood. Stem Cells Dev 20, 159–168 (2011).

    Article  CAS  Google Scholar 

  21. Seki, T., Yuasa, S. & Fukuda, K. Generation of induced pluripotent stem cells from a small amount of human peripheral blood using a combination of activated T cells and Sendai virus. Nat. Protoc. 7, 718–728 (2012).

    Article  CAS  Google Scholar 

  22. Serwold, T. et al. T-cell receptor-driven lymphomagenesis in mice derived from a reprogrammed T cell. Proc. Natl. Acad. Sci. USA 107, 18939–18943 (2010).

    Article  CAS  Google Scholar 

  23. Hu, K. et al. Efficient generation of transgene-free induced pluripotent stem cells from normal and neoplastic bone marrow and cord blood mononuclear cells. Blood 117, e109–e119 (2011).

    Article  CAS  Google Scholar 

  24. Mali, P., Ye, Z., Chou, B.K., Yen, J. & Cheng, L. An improved method for generating and identifying human induced pluripotent stem cells. Methods Mol. Biol. 636, 191–205 (2010).

    Article  CAS  Google Scholar 

  25. Chen, G. et al. Chemically defined conditions for human iPSC derivation and culture. Nat. Methods 8, 424–429 (2011).

    Article  CAS  Google Scholar 

  26. Zhu, S. et al. Reprogramming of human primary somatic cells by OCT4 and chemical compounds. Cell Stem Cell 7, 651–655 (2010).

    Article  CAS  Google Scholar 

  27. Yu, J., Chau, K.F., Vodyanik, M.A., Jiang, J. & Jiang, Y. Efficient feeder-free episomal reprogramming with small molecules. PLoS ONE 6, e17557 (2011).

    Article  CAS  Google Scholar 

  28. Anokye-Danso, F. et al. Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell 8, 376–388 (2011).

    Article  CAS  Google Scholar 

  29. Subramanyam, D. 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).

    Article  CAS  Google Scholar 

  30. Gore, A. et al. Somatic coding mutations in human induced pluripotent stem cells. Nature 471, 63–67 (2011).

    Article  CAS  Google Scholar 

  31. Chan, E.M. et al. Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat. Biotechnol. 27, 1033–1037 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank members of the Cheng laboratory for discussion and sharing their experience. This work is supported by Johns Hopkins University and grants from the US National Institutes of Health (R01HL073781, U01HL107446, RC2HL101582 and T32HL007525).

Author information

Authors and Affiliations

  1. Division of Hematology, Department of Medicine, Stem Cell Program, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Sarah N Dowey, Xiaosong Huang, Bin-Kuan Chou, Zhaohui Ye & Linzhao Cheng

  2. Graduate Program in Cellular and Molecular Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Bin-Kuan Chou & Linzhao Cheng

Authors
  1. Sarah N Dowey
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaosong Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bin-Kuan Chou
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhaohui Ye
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Linzhao Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.N.D., B.-K.C. and Z.Y. designed and performed experiments, analyzed data and wrote the paper; X.H. designed and performed experiments; L.C. designed experiments, analyzed data and wrote the paper.

Corresponding author

Correspondence to Linzhao Cheng.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dowey, S., Huang, X., Chou, BK. et al. Generation of integration-free human induced pluripotent stem cells from postnatal blood mononuclear cells by plasmid vector expression. Nat Protoc 7, 2013–2021 (2012). https://doi.org/10.1038/nprot.2012.121

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2012.121

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing