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

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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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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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.

Corresponding author

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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