Identification of Epithelial Stem Cells In Vivo and In Vitro Using Keratin 19 and BrdU

  • Danielle Larouche
  • Amélie Lavoie
  • Claudie Paquet
  • Carolyne Simard-Bisson
  • Lucie Germain
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 585)

Abstract

Progress in the identification of skin stem cells and the improvement of culture methods open the possibility to use stem cells in regenerative medicine. Based on their quiescent nature, the development of label retention assays allowed the localization of skin stem cells in the bulge region of the pilosebaceous units and in the bottom of rete ridges in glabrous skin. The development of markers such as keratin 19 also permits their study in human tissues. In this chapter, protocols to identify skin stem cells based on their slow-cycling property and their expression of keratin 19 will be described in detail. The methods include the labeling of skin stem cells within mouse or rat tissues in vivo, the labeling of proliferative human cells in vitro using 5-bromo-2-deoxyuridine (BrdU), and the detection of keratin 19 and BrdU by immunofluorescence or immunoperoxidase staining.

Key words

Cell culture Epidermis Hair follicle Human Keratinocytes Keratin 19 Mouse Proliferation Rat Regenerative medicine Stem cells Vibrissa 

Notes

Acknowledgments

The authors would like to thank current and former members of the LOEX laboratory who have contributed to develop the foregoing protocols, particularly Claudia Fugère and Israël Martel. We are thankful to Nathalie Tremblay for her help regarding the research of technical information.

References

  1. 1.
    Michel, M., Torok, N., Godbout, M. J., Lussier, M., Gaudreau, P., Royal, A., and Germain, L. (1996) Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage.J Cell Sci, 109 (Pt 5), 1017–28.PubMedGoogle Scholar
  2. 2.
    Larouche, D., Tong, X., Fradette, J., Coulombe, P. A., and Germain, L. (2008) Vibrissa hair bulge houses two populations of skin epithelial stem cells distinct by their keratin profile.FASEB J, 22, 1404–15.PubMedCrossRefGoogle Scholar
  3. 3.
    Bickenbach, J. R., McCutecheon, J., and Mackenzie, I. C. (1986) Rate of loss of tritiated thymidine label in basal cells in mouse epithelial tissues. Cell Tissue Kinet, 19, 325–33.PubMedGoogle Scholar
  4. 4.
    Larouche, D., Hayward, C., Cuffley, K., and Germain, L. (2005) Keratin 19 as a stem cell marker in vivo and in vitro.Methods Mol Biol, 289, 103–10.PubMedGoogle Scholar
  5. 5.
    Bickenbach, J. R., and Chism, E. (1998) Selection and extended growth of murine epidermal stem cells in culture. Exp Cell Res, 244, 184–95.PubMedCrossRefGoogle Scholar
  6. 6.
    Lussier, M., Ouellet, T., Lampron, C., Lapointe, L., and Royal, A. (1989) Mouse keratin 19: complete amino acid sequence and gene expression during development. Gene, 85, 435–44.PubMedCrossRefGoogle Scholar
  7. 7.
    Larouche, D., Paquet, C., Fradette, J., Carrier, P., Auger, F. A., and Germain, L. (2009) Stem cells in regenerative medicine. Methods Mol Biol, 482, 233–56.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Danielle Larouche
    • 1
  • Amélie Lavoie
    • 1
  • Claudie Paquet
    • 1
  • Carolyne Simard-Bisson
    • 1
  • Lucie Germain
    • 1
  1. 1.Laboratoire d’Organogénèse Exprimentale/LOEX and Departments of SurgeryOto-Rhino-Laryngology and Ophthalmology, Laval UniversitySainte-FoyCanada

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