Isolation and Culture of Epithelial Stem Cells

  • Jonathan A. Nowak
  • Elaine Fuchs
Part of the Methods in Molecular Biology book series (MIMB, volume 482)


In the skin, epithelial stem cells in the hair follicle contribute not only to the generation of a new hair follicle with each hair cycle, but also to the repair of the epidermis during wound healing. When these stem cells are isolated and expanded in culture, they can give rise to hair follicles, sebaceous glands, and epidermis when combined with dermis and grafted back onto Nude mice. In this chapter, we provide a method for isolating hair follicle epithelial stem cells from the skin of adult mice using immunofluorescent labeling to allow for the specific purification of epithelial stem cells by fluorescence-activated cell sorting (FACS). Notably, this method relies exclusively on cell surface markers, making it suitable for use with any strain of mouse and at various stages of the hair cycle. We also provide a detailed protocol for culturing epithelial stem cells isolated by FACS, allowing for analysis using a wide variety of culture assays. Additionally, we provide notes on using cultured cells for specific applications, such as viral manipulation and grafting. These techniques should be useful for directly evaluating stem cell function in normal mice and in mice with skin defects.

Key words

Epithelial stem cells skin stem cells hair follicles bulge stem cells 


  1. 1.
    Cotsarelis, G., T.T. Sun, and R.M. Lavker, (1990) Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell, 61(7): 1329–37.CrossRefPubMedGoogle Scholar
  2. 2.
    Morris, R.J. and C.S. Potten, (1999) Highly persistent label-retaining cells in the hair follicles of mice and their fate following induction of anagen. J Invest Dermatol, 112(4): 470–5.CrossRefPubMedGoogle Scholar
  3. 3.
    Taylor, G., et al., (2000) Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell, 102(4): 451–61.CrossRefPubMedGoogle Scholar
  4. 4.
    Tumbar, T., et al., (2004) Defining the epithelial stem cell niche in skin. Science, 303(5656): 359–63.CrossRefPubMedGoogle Scholar
  5. 5.
    Morris, R.J., et al., (2004) Capturing and profiling adult hair follicle stem cells. Nat Biotechnol, 22(4): 411–7.CrossRefPubMedGoogle Scholar
  6. 6.
    Trempus, C.S., et al., (2003) Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34. J Invest Dermatol, 120(4): 501–11.CrossRefPubMedGoogle Scholar
  7. 7.
    Blanpain, C., et al., (2004) Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche. Cell, 118(5): 635–48.CrossRefPubMedGoogle Scholar
  8. 8.
    Rheinwald, J.G. and H. Green, (1977) Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature, 265(5593): 421–4.CrossRefPubMedGoogle Scholar
  9. 9.
    Rheinwald, J.G. and H. Green, (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell, 6(3): 331–43.CrossRefPubMedGoogle Scholar
  10. 10.
    Todaro, G.J. and H. Green, (1963) Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J Cell Biol, 17: 299–313.CrossRefPubMedGoogle Scholar
  11. 11.
    Shapiro, H.M., (2003) Practical flow cytometry. 4 ed.: Wiley-Liss. 736.Google Scholar
  12. 12.
    Gratzner, H.G. and R.C. Leif, (1981) An immunofluorescence method for monitoring DNA synthesis by flow cytometry. Cytometry, 1(6): 385–93.CrossRefPubMedGoogle Scholar
  13. 13.
    Weinberg, W.C., et al., (1993) Reconstitution of hair follicle development in vivo: determination of follicle formation, hair growth, and hair quality by dermal cells. J Invest Dermatol, 100(3): 229–36.CrossRefPubMedGoogle Scholar
  14. 14.
    Kuhn, U., et al., (2002) In vivo assessment of gene delivery to keratinocytes by lentiviral vectors. J Virol, 76(3): 1496–504.CrossRefPubMedGoogle Scholar
  15. 15.
    Garlick, J.A., et al., (1991) Retrovirus-mediated transduction of cultured epidermal keratinocytes. J Invest Dermatol, 97(5): 824–9.CrossRefPubMedGoogle Scholar
  16. 16.
    Wu, W.Y. and R.J. Morris, (2005) Method for the harvest and assay of in vitro clonogenic keratinocytes stem cells from mice. Methods Mol Biol, 289: 79–86.PubMedGoogle Scholar
  17. 17.
    Redvers, R.P. and P. Kaur, (2005) Serial cultivation of primary adult murine keratinocytes. Methods Mol Biol, 289: 15–22.PubMedGoogle Scholar
  18. 18.
    Yano, S. and H. Okochi, (2005) Long-term culture of adult murine epidermal keratinocytes. Br J Dermatol, 153(6): 1101–4.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Jonathan A. Nowak
    • 1
  • Elaine Fuchs
    • 1
  1. 1.Howard Hughes Medical Institute, The Rockefeller UniversityNew YorkUSA

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