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

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.

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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.

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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

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.

Correspondence to Juan Carlos Izpisúa Belmonte.

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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) doi:10.1038/nprot.2010.9

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