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:

A protocol describing the genetic correction of somatic human cells and subsequent generation of iPS cells

Nature Protocols volume 5, pages 647–660 (2010)Cite this article

Subjects

Abstract

The generation of patient-specific induced pluripotent stem cells (iPSCs) offers unprecedented opportunities for modeling and treating human disease. In combination with gene therapy, the iPSC technology can be used to generate disease-free progenitor cells of potential interest for autologous cell therapy. We explain a protocol for the reproducible generation of genetically corrected iPSCs starting from the skin biopsies of Fanconi anemia patients using retroviral transduction with OCT4, SOX2 and KLF4. Before reprogramming, the fibroblasts and/or keratinocytes of the patients are genetically corrected with lentiviruses expressing FANCA. The same approach may be used for other diseases susceptible to gene therapy correction. Genetically corrected, characterized lines of patient-specific iPSCs can be obtained in 4–5 months.

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: Optimal density of 293T cells for transfection.
Figure 2: Human dermal fibroblasts transduced with correcting lentivirus.
Figure 3: Reprogramming genetically corrected fibroblasts.
Figure 4: Genetically corrected FA-iPSCs are pluripotent.

Similar content being viewed by others

References

  1. 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 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Park, I.H. et al. Disease-specific induced pluripotent stem cells. Cell 134, 877–886 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Dimos, J.T. et al. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321, 1218–1221 (2008).

    Article  CAS  PubMed  Google Scholar 

  8. Ebert, A.D. et al. Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature 457, 277–280 (2009).

    Article  CAS  PubMed  Google Scholar 

  9. Raya, A. et al. Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells. Nature 460, 53–59 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Hong, H. et al. Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature 460, 1132–1135 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kawamura, T. et al. Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature 460, 1140–1144 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Marion, R.M. et al. A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity. Nature 460, 1149–1153 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  14. Takahashi, K., Okita, K., Nakagawa, M. & Yamanaka, S. Induction of pluripotent stem cells from fibroblast cultures. Nat. Protoc. 2, 3081–3089 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Park, I.H., Lerou, P.H., Zhao, R., Huo, H. & Daley, G.Q. Generation of human-induced pluripotent stem cells. Nat. Protoc. 3, 1180–1186 (2008).

    Article  CAS  PubMed  Google Scholar 

  16. Ying, Q.L. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G. & Hochedlinger, K. Induced pluripotent stem cells generated without viral integration. Science 322, 945–949 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T. & Yamanaka, S. Generation of mouse induced pluripotent stem cells without viral vectors. Science 322, 949–953 (2008).

    Article  CAS  PubMed  Google Scholar 

  19. 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  PubMed  PubMed Central  Google Scholar 

  20. Zou, J. et al. Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell 5, 97–110 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Baum, C. et al. The potent enhancer activity of the polycythemic strain of spleen focus-forming virus in hematopoietic cells is governed by a binding site for Sp1 in the upstream control region and by a unique enhancer core motif, creating an exclusive target for PEBP/CBF. J. Virol. 71, 6323–6331 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Aasen, T. & Belmonte, J.C. Isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells. Nat. Protoc. 5, 371–382 (2010).

    Article  CAS  PubMed  Google Scholar 

  23. Jacome, A. et al. Lentiviral-mediated genetic correction of hematopoietic and mesenchymal progenitor cells from Fanconi anemia patients. Mol. Ther. 17, 1083–1092 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Lerou, P.H. et al. Derivation and maintenance of human embryonic stem cells from poor-quality in vitro fertilization embryos. Nat. Protoc. 3, 923–933 (2008).

    Article  CAS  PubMed  Google Scholar 

  25. Raya, A. et al. Generation of cardiomyocytes from new human embryonic stem cell lines derived from poor-quality blastocysts. Cold Spring Harb. Symp. Quant. Biol. 73, 127–135 (2008).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are indebted to FA patients and their families for their kind cooperation. We are grateful to all the members of the laboratory for advice and discussion and to Begoña Arán, Meritxell Carrió, Yolanda Muñoz and M. Luz Lozano for their expert assistance with cell culture techniques. This work was partially supported by the Ministerio de Educación y Ciencia grants BFU2009-13277, BFU2006-12247, SAF2009-07164, PLE2009-0144, PLE2009-0100 and Genoma España (FANCOGENE), European Commission 'Marie-Curie Reintegration Grant' MIRG-CT-2007-046523 and FP7-PERSIST Rej: 222878, the Fondo de Investigaciones Sanitarias (RETIC-RD06/0010/0016, RD06/0010/0015 and PI061897), Marató de TV3 (063430), the G. Harold and Leila Y. Mathers Charitable Foundation, Fundación Marcelino Botín and Fundación Cellex.

Author information

Author notes

  1. Ángel Raya & Yvonne Richaud-Patin

    Present address: Present address: Control of Stem Cell Potency Group, Institute for Bioengineering of Catalonia (IBEC), Baldiri Reixac 15, Barcelona, Spain.,

  2. Adriana Sánchez-Danés & Antonella Consiglio

    Present address: Present address: Institute of Biomedicine of the University of Barcelona (IBUB), Baldiri Reixac 15, Barcelona, Spain.,

Authors and Affiliations

  1. Center for Regenerative Medicine in Barcelona, Barcelona, Spain

    Ángel Raya, Ignasi Rodríguez-Pizà, Yvonne Richaud-Patin, Adriana Sánchez-Danés, Antonella Consiglio & Juan Carlos Izpisúa Belmonte

  2. Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain

    Ángel Raya

  3. Networking Center of Biomedical Research in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Barcelona, Spain

    Ángel Raya & Yvonne Richaud-Patin

  4. Hematopoiesis and Gene Therapy Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Madrid, Spain

    Susana Navarro, Guillermo Guenechea & Juan Bueren

  5. Networking Center of Biomedical Research in Rare Diseases (CIBERER), Madrid, Spain

    Susana Navarro, Guillermo Guenechea & Juan Bueren

  6. Department of Biomedical Science and Biotechnology, University of Brescia, Brescia, Italy

    Antonella Consiglio

  7. Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, California, USA

    Juan Carlos Izpisúa Belmonte

Authors
  1. Ángel Raya
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ignasi Rodríguez-Pizà
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Susana Navarro
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yvonne Richaud-Patin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Guillermo Guenechea
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Adriana Sánchez-Danés
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Antonella Consiglio
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Juan Bueren
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Juan Carlos Izpisúa Belmonte
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.R. designed the overall protocol, generated and characterized the FA-iPS cells and wrote the manuscript after obtaining the material from all authors; I.R.-P., Y.R.-P., A.S.-D. and A.C. designed the reprogramming protocol and characterized the FA-iPS cells; S.N., G.G. and J.B. designed the gene therapy correction protocol and prepared the gene therapy lentiviruses; and J.C.I.B. supervised the project.

Corresponding author

Correspondence to Juan Carlos Izpisúa Belmonte.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Raya, Á., Rodríguez-Pizà, I., Navarro, S. et al. A protocol describing the genetic correction of somatic human cells and subsequent generation of iPS cells. Nat Protoc 5, 647–660 (2010). https://doi.org/10.1038/nprot.2010.9

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2010.9

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: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research