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Isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells

Nature Protocols volume 5, pages 371–382 (2010)Cite this article

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Abstract

The ease of generating induced pluripotent stem (iPS) cells, and possibly their properties after reprogramming, depends on the origin of the somatic cell starting population. Reprogramming of keratinocytes is both faster and more efficient compared with fibroblasts, although more care is required when isolating, culturing and infecting these cells. In this study, we describe detailed protocols using both feeder-dependent and defined serum- and feeder-free conditions for culturing human keratinocytes from foreskin samples and punch biopsies, as well as how to isolate keratinocytes from plucked hair. We further describe culture techniques and approaches to efficiently infect and reprogram these cells for the purpose of generating iPS cells. The procedure of deriving keratinocytes takes 10–14 d, whereas reprogramming and the appearance of iPS cell colonies that can be isolated and established requires another 3–4 weeks.

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Figure 1: Schematic of human skin.
Figure 2: Enzymatic isolation of the epidermis and the dermis.
Figure 3: Human keratinocytes in culture.
Figure 4: Isolation and culture of plucked human hair.
Figure 5: Infection of keratinocytes and derivation of iPS colonies.

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References

  1. Fuchs, E. Scratching the surface of skin development. Nature 445, 834–842 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Amoh, Y. et al. Human and mouse hair follicles contain both multipotent and monopotent stem cells. Cell Cycle 8, 176–177 (2009).

    Article  CAS  PubMed  Google Scholar 

  3. Ito, M. et al. Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis. Nat. Med. 11, 1351–1354 (2005).

    Article  CAS  PubMed  Google Scholar 

  4. Rheinwald, J.G. & Green, H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6, 331–343 (1975).

    Article  CAS  PubMed  Google Scholar 

  5. Tenchini, M.L., Ranzati, C. & Malcovati, M. Culture techniques for human keratinocytes. Burns 18 (Suppl 1): S11–S16 (1992).

    Article  PubMed  Google Scholar 

  6. Detmar, M., Schaart, F.M., Blume, U. & Orfanos, C.E. Culture of hair matrix and follicular keratinocytes. J. Invest. Dermatol. 101, 130S–134S (1993).

    Article  CAS  PubMed  Google Scholar 

  7. Wells, J. A simple technique for establishing cultures of epithelial cells. Br. J. Dermatol. 107, 481–482 (1982).

    Article  CAS  PubMed  Google Scholar 

  8. Moll, I. Differential epithelial outgrowth of plucked and microdissected human hair follicles in explant culture. Arch. Dermatol. Res. 288, 604–610 (1996).

    Article  CAS  PubMed  Google Scholar 

  9. Limat, A. & Noser, F.K. Serial cultivation of single keratinocytes from the outer root sheath of human scalp hair follicles. J. Invest. Dermatol. 87, 485–488 (1986).

    Article  CAS  PubMed  Google Scholar 

  10. Amoh, Y., Li, L., Katsuoka, K. & Hoffman, R.M. Multipotent nestin-expressing hair follicle stem cells. J. Dermatol. 36, 1–9 (2009).

    Article  PubMed  Google Scholar 

  11. Hunt, D.P. et al. A highly enriched niche of precursor cells with neuronal and glial potential within the hair follicle dermal papilla of adult skin. Stem Cells 26, 163–172 (2008).

    Article  CAS  PubMed  Google Scholar 

  12. Yu, H. et al. Isolation of a novel population of multipotent adult stem cells from human hair follicles. Am. J. Pathol. 168, 1879–1888 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Amoh, Y., Li, L., Katsuoka, K., Penman, S. & Hoffman, R.M. Multipotent nestin-positive, keratin-negative hair-follicle bulge stem cells can form neurons. Proc. Natl. Acad. Sci. USA 102, 5530–5534 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  15. Yamanaka, S. Elite and stochastic models for induced pluripotent stem cell generation. Nature 460, 49–52 (2009).

    Article  CAS  PubMed  Google Scholar 

  16. Muller, L.U., Daley, G.Q. & Williams, D.A. Upping the ante: recent advances in direct reprogramming. Mol. Ther. 17, 947–953 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 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 

  18. 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 

  19. Utikal, J., Maherali, N., Kulalert, W. & Hochedlinger, K. Sox2 is dispensable for the reprogramming of melanocytes and melanoma cells into induced pluripotent stem cells. J. Cell Sci. 122, 3502–3510 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 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 

  21. Gilfix, B.M. & Green, H. Bioassay of retinoids using cultured human conjunctival keratinocytes. J. Cell Physiol. 119, 172–174 (1984).

    Article  CAS  PubMed  Google Scholar 

  22. 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 

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Acknowledgements

We are grateful to Dr. Agustín Toll Abelló at the Hospital del Mar (Barcelona, Spain) for facilitating collection of skin samples. We thank Marta Berini Pérez for drawing Figure 1. This study was supported by grants from Fundacion Cellex, the G. Harold and Leila Y. Mathers Charitable Foundation, Marato de TV3, CIBER and MICINN.

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Authors and Affiliations

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

    Trond Aasen & Juan Carlos Izpisúa Belmonte

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

    Trond Aasen

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

    Juan Carlos Izpisúa Belmonte

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  1. Trond Aasen
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  2. Juan Carlos Izpisúa Belmonte
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Contributions

T.A. designed the protocol and all experimental procedures, wrote the paper and prepared figures. J.C.I.B. is a project lead and assisted with the preparation of the paper.

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Correspondence to Juan Carlos Izpisúa Belmonte.

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Aasen, T., Belmonte, J. 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). https://doi.org/10.1038/nprot.2009.241

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