Interfollicular Epidermal Stem Cells: Boosting and Rescuing from Adult Skin

  • Mariana T. Cerqueira
  • Ana M. Frias
  • Rui L. Reis
  • Alexandra P. Marques
Part of the Methods in Molecular Biology book series (MIMB, volume 989)


Epidermal stem cells isolation struggle remains, mainly due to the yet essential requirement of well-defined approaches and markers. The herein proposed methodology integrates an assemblage of strategies to accomplish the enrichment of the interfollicular epidermal stem cells multipotent fraction and their subsequent separation from the remaining primary human keratinocytes culture. Those include rapid adherence of freshly isolated human keratinocytes to collagen type IV through the β1-integrin ligand and Rho-Associated Protein Kinase Inhibitor Y- 27632 administration to the cultures, followed by an immunomagnetic separation to obtain populations based in the combined CD49fbri/CD71dim expression. Flow cytometry is the supporting method to analyze the effect of the treatments over the expression rate of early epidermal markers keratins19/5/14 and in correlation to CD49fbri/CD71dim subpopulations. The step-by-step methodology herein described indulges the boosting and consecutive purification and separation of interfollicular epidermal stem cells from human keratinocytes cultures.

Key words

Epidermal stem cells Collagen IV Rock inhibitor Immunomagnetic separation Flow cytometry 


  1. 1.
    Blanpain C, Lowry WE, Geoghegan A, Polak L, Fuchs E (2004) Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche. Cell 118:635–648PubMedCrossRefGoogle Scholar
  2. 2.
    Lau K, Paus R, Tiede S, Day P, Bayat A (2009) Exploring the role of stem cells in cutaneous wound healing. Exp Dermatol 18:921–933PubMedCrossRefGoogle Scholar
  3. 3.
    Watt FM, Lo Celso C, Silva-Vargas V (2006) Epidermal stem cells: an update. Curr Opin Genet Dev 16:518–524PubMedCrossRefGoogle Scholar
  4. 4.
    Blanpain C, Fuchs E (2006) Epidermal stem cells of the skin. Annu Rev Cell Dev Biol 22:339–373PubMedCrossRefGoogle Scholar
  5. 5.
    Li A, Simmons PJ, Kaur P (1998) Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci USA 95:3902–3907PubMedCrossRefGoogle Scholar
  6. 6.
    Taylor G, Lehrer MS, Jensen PJ, Sun TT, Lavker RM (2000) Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell 102:451–461PubMedCrossRefGoogle Scholar
  7. 7.
    Pellegrini G, Dellambra E, Golisano O, Martinelli E, Fantozzi I, Bondanza S, Ponzin D, McKeon F, De Luca M (2001) p63 identifies keratinocyte stem cells. Proc Natl Acad Sci USA 98:3156–3161PubMedCrossRefGoogle Scholar
  8. 8.
    Pontiggia L, Biedermann T, Meuli M, Widmer D, Bottcher-Haberzeth S, Schiestl C, Schneider J, Braziulis E, Montano I, Meuli-Simmen C, Reichmann E (2009) Markers to evaluate the quality and self-renewing potential of engineered human skin substitutes in vitro and after transplantation. J Invest Dermatol 129:480–490PubMedCrossRefGoogle Scholar
  9. 9.
    Alonso L, Fuchs E (2003) Stem cells of the skin epithelium. Proc Natl Acad Sci USA 100(Suppl 1):11830–11835PubMedCrossRefGoogle Scholar
  10. 10.
    Kaur P, Li A (2000) Adhesive properties of human basal epidermal cells: an analysis of keratinocyte stem cells, transit amplifying cells, and postmitotic differentiating cells. J Invest Dermatol 114:413–420PubMedCrossRefGoogle Scholar
  11. 11.
    Schluter H, Paquet-Fifield S, Gangatirkar P, Li J, Kaur P (2011) Functional characterization of quiescent keratinocyte stem cells and their progeny reveals a hierarchical organization in human skin epidermis. Stem Cells 29:1256–1268PubMedCrossRefGoogle Scholar
  12. 12.
    McMullan R, Lax S, Robertson VH, Radford DJ, Broad S, Watt FM, Rowles A, Croft DR, Olson MF, Hotchin NA (2003) Keratinocyte differentiation is regulated by the Rho and ROCK signaling pathway. Curr Biol 13:2185–2189PubMedCrossRefGoogle Scholar
  13. 13.
    Terunuma A, Limgala RP, Park CJ, Choudhary I, Vogel JC (2010) Efficient procurement of epithelial stem cells from human tissue specimens using a rho-associated protein kinase inhibitor Y-27632. Tissue Eng Part A 16:1363–1368PubMedCrossRefGoogle Scholar
  14. 14.
    Chapman S, Liu X, Meyers C, Schlegel R, McBride AA (2010) Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor. J Clin Invest 120:2619–2626PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Mariana T. Cerqueira
    • 1
    • 2
  • Ana M. Frias
    • 1
    • 2
  • Rui L. Reis
    • 1
    • 2
  • Alexandra P. Marques
    • 3
    • 2
  1. 1.3B’s Research Group—Biomaterials, Biodegradables, and BiomimeticsUniversity of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative MedicineGuimarãesPortugal
  2. 2.ICVS/3B’s - PT Government Associate LaboratoryBraga/GuimarãesPortugal
  3. 3.1- 3B’s Research Group—Biomaterials, Biodegradables, and BiomimeticsUniversity of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative MedicineGuimarãesPortugal

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