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Isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells
Trond Aasen1, 2 & Juan Carlos Izpisa Belmonte1, 3
1Center of Regenerative Medicine in Barcelona, Barcelona, Spain. 2Networking Center of Biomedical Research in Bioengineering, Biomaterials and Nanomedicine (CIBERBBN), Zaragoza, Spain. 3Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, California, USA. Correspondence should be addressed to J.C.I.B. ([email protected] and [email protected]).
Published online 4 February 2010; doi:10.1038/nprot.2009.241
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 1014 d, whereas reprogramming and the appearance of iPS cell colonies that can be isolated and established requires another 34 weeks.
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: INTRODUCTIONKeratinocytesThe epidermis is a thin nonvascular layer (Fig. 1) consisting mainly of stratifying epithelial keratinocytes. These cells undergo continuous and rapid proliferation and are thought to be continually regenerated from a pool of multipotent stem cells1. As keratinocytes leave the basal layer and move upward, they undergo terminal differentiation forming a continually shedding protective barrier at the surface of the skin. Skin fibroblasts are situated in the thicker dermis (Fig. 1), which is situated directly below the epidermis (separated by the basement membrane), and provide, among other functions, growth factors for keratinocytes, a feature that has proved important for early keratinocyte culture models. Thus, it has been possible for many years to efficiently isolate both keratinocytes and fibroblasts from the same skin sample. Several other cell types can also be isolated from skin, including endothelial cells and epidermal melanocytes. In addition, keratinocytes...