Isolation and Characterization of Cutaneous Epithelial Stem Cells

  • Stephanie R. Gillespie
  • David M. OwensEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1879)


The outer layer of mammalian skin is a multilayered epithelium that perpetually renews multiple differentiated lineages. During homeostasis, the maintenance of skin epithelial turnover is ensured by regionalized populations of stem cells that largely remain dedicated to distinct epithelial lineages including squamous, follicular, sebaceous, Merkel, and sweat glands. Cutting edge developments in this field have focused on: (1) stem cell activation cues derived from a number of extrinsic sources including neurons, dermal fibroblasts and adipocyte, and immune cells; and (2) characterization of epithelial stem cell homeostasis via hierarchical versus stochastic paradigms. The techniques outlined in this chapter are designed to facilitate such studies and describe basic procedures for cutaneous stem cell isolation and purification, which are based on leveraging their unique expression of surface proteins for simultaneous targeting and purifying of multiple subpopulations in adult skin. In addition, protocols for assessment of in vitro and ex vivo progenitor capacity as well as techniques to visualize progenitor populations in whole skin are discussed.


Epithelial lineage Epithelial progenitor markers Epithelial stem cell homeostasis Skin differentiation Skin reconstitution assay 



This work was supported by Columbia University Skin DiseaseResource-Based Center (EPICURE) funded by the NIH (5P30AR069632).


  1. 1.
    Fuchs E, Tumbar T, Guasch G (2004) Socializing with the neighbors: stem cells and their niche. Cell 116:769–778CrossRefGoogle Scholar
  2. 2.
    Yan X, Owens DM (2008) The skin: a home to multiple classes of epithelial progenitor cells. Stem Cell Rev 4:113–118CrossRefGoogle Scholar
  3. 3.
    Woo S-H, Stumpfova M, Jensen UB, Lumpkin EA, Owens DM (2010) Identification of epidermal progenitors for the Merkel cell lineage. Development 137:3965–3971CrossRefGoogle Scholar
  4. 4.
    Clayton E, Doupe DP, Klein AM, Winton DJ, Simons BD, Jones PH (2007) A single type of progenitor cell maintains normal epidermis. Nature 446:185–189CrossRefGoogle Scholar
  5. 5.
    Jensen KB, Collins CA, Nascimento E, Tan DW, Frye M, Itami S, Watt FM (2009) Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis. Cell Stem Cell 4:427–439CrossRefGoogle Scholar
  6. 6.
    Ghazizadeh S, Taichman LB (2001) Multiple classes of stem cells in cutaneous epithelium: a lineage analysis of adult mouse skin. EMBO J 20:1215–1222CrossRefGoogle Scholar
  7. 7.
    Horsley V, O’Carroll D, Tooze R, Ohinata Y, Saitou M, Obukhanych T, Nussenzweig M, Tarakhovsky A, Fuchs E (2006) Blimp1 defines a progenitor population that governs cellular input to the sebaceous gland. Cell 126:597–609CrossRefGoogle Scholar
  8. 8.
    Levy V, Lindon C, Harfe BD, Morgan BA (2005) Distinct stem cell populations regenerate the follicle and interfollicular epidermis. Dev Cell 9:855–861CrossRefGoogle Scholar
  9. 9.
    Ito M, Liu Y, Yang Z, Nguyen J, Liang F, Morris RJ, Cotsarelis G (2005) Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis. Nat Med 11:1351–1354CrossRefGoogle Scholar
  10. 10.
    Jaks V, Barker N, Kasper M, van Es JH, Snippert HJ, Clevers H, Toftgard R (2008) Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat Genet 40:1291–1299CrossRefGoogle Scholar
  11. 11.
    Snippert HJ, Haegebarth A, Kasper M, Jaks V, van Es JH, Barker N, van de Wetering M, van den Born M, Begthel H, Vries RG, Stange DE, Toftgård R, Clevers H (2010) Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin. Science 327:1385–1389CrossRefGoogle Scholar
  12. 12.
    Petersson M, Frances D, Niemann C (2013) Lineage tracing of hair follicle stem cells in epidermal whole mounts. Methods Mol Biol 989:45–60CrossRefGoogle Scholar
  13. 13.
    Doucet YS, Woo SH, Ruiz ME, Owens DM (2013) The touch dome defines an epidermal niche specialized for mechanosensory signaling. Cell Rep 3(6):1759–1765CrossRefGoogle Scholar
  14. 14.
    Maksimovic S, Nakatani M, Baba Y, Nelson AM, Marshall KL, Wellnitz SA, Firozi P, Woo S-H, Ranade S, Patapoutian A, Lumpkin EA (2014) Epidermal Merkel cells are mechanosensory cells that tune mammalian touch receptors. Nature 509:617–621CrossRefGoogle Scholar
  15. 15.
    Tani H, Morris RJ, Kaur P (2000) Enrichment for murine keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci U S A 97:10960–10965CrossRefGoogle Scholar
  16. 16.
    Trempus CS, Morris RJ, Bortner CD, Cotsarelis G, Faircloth RS, Reece JM, Tennant RW (2003) Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34. J Invest Dermatol 120:501–511PubMedGoogle Scholar
  17. 17.
    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 118:635–648CrossRefGoogle Scholar
  18. 18.
    Nijhof JG, Braun KM, Giangreco A, van Pelt C, Kawamoto H, Boyd RL, Willemze R, Mullenders LH, Watt FM, De Gruijl FR, van Ewijk W (2006) The cell-surface marker MTS24 identifies a novel population of follicular keratinocytes with characteristics of progenitor cells. Development 133:3027–3037CrossRefGoogle Scholar
  19. 19.
    Jensen UB, Yan X, Triel C, Woo SH, Christensen R, Owens DM (2008) A distinct population of clonogenic and multipotent murine follicular keratinocytes residing in the upper isthmus. J Cell Sci 121:609–617CrossRefGoogle Scholar
  20. 20.
    Soteriou D, Kostic L, Sedov E, Yosefzon Y, Steller H, Fuchs Y (2016) Isolating hair follicle stem cells and epidermal keratinocytes from dorsal mouse skin. J Vis Exp (110).
  21. 21.
    Ali N, Zirak B, Rodriguez RS, Pauli ML, Truong HA, Lai K, Ahn R, Corbin K, Lowe MM, Scharschmidt TC, Taravati K, Tan MR, Ricardo-Gonzalez RR, Nosbaum A, Bertolini M, Liao W, Nestle FO, Paus R, Cotsarelis G, Abbas AK, Rosenblum MD (2017) Regulatory T cells in skin facilitate epithelial stem cell differentiation. Cell 169(6):1119–1129CrossRefGoogle Scholar
  22. 22.
    Rahmani W, Liu Y, Rosin NL, Kline A, Raharjo E, Yoon J, Stratton JA, Sinha S, Biernaskie J (2018) Macrophages promote wound-induced hair follicle regeneration in a CX3CR1 and TGFβ1 dependent manner. J Invest Dermatol. [Epub ahead of print]CrossRefGoogle Scholar
  23. 23.
    Gay D, Kwon O, Zhang Z, Spata M, Plikus MV, Holler PD, Ito M, Yang Z, Treffeisen E, Kim CD, Nace A, Zhang X, Baratono S, Wang F, Ornitz DM, Millar SE, George C (2013) Fgf9 from dermal γδ T cells induces hair follicle neogenesis after wounding. Nat Med 19:916–923CrossRefGoogle Scholar
  24. 24.
    Xing L, Dai Z, Jabbari A, Cerise JE, Higgins CA, Gong W, de Jong A, Harel S, DeStefano GM, Rothman L, Singh P, Petukhova L, Mackay-Wiggan J, Christiano AM, Clynes R (2014) Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by JAK inhibition. Nat Med 20(9):1043–1049CrossRefGoogle Scholar
  25. 25.
    Guerrero-Juarez CF, Plikus MV (2018) Emerging nonmetabolic functions of skin fat. Nat Rev Endocrinol 14:163–173CrossRefGoogle Scholar
  26. 26.
    Zwick RK, Guerrero-Juarez CF, Horsley V, Plikus MV (2018) Anatomical, physiological, and functional diversity of adipose tissue. Cell Metab 27(1):68–83CrossRefGoogle Scholar
  27. 27.
    Plikus MV, Mayer JA, de la Cruz D, Baker RE, Maini PK, Maxson R, Chuong CM (2008) Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration. Nature 451(7176):340–344CrossRefGoogle Scholar
  28. 28.
    Xiao Y, Thoresen DT, Williams JS, Wang C, Perna J, Petrova R, Brownell I (2015) Neural Hedgehog signaling maintains stem cell renewal in the sensory touch dome epithelium. Proc Natl Acad Sci U S A 112(23):7195–7200CrossRefGoogle Scholar
  29. 29.
    Brownell I, Guevara E, Bai CB, Loomis CA, Joyner AL (2011) Nerve-derived Sonic hedgehog defines a niche for hair follicle stem cells capable of becoming epidermal stem cells. Cell Stem Cell 8(5):552–565CrossRefGoogle Scholar
  30. 30.
    Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:317–330CrossRefGoogle Scholar
  31. 31.
    Morris RJ (1994) Procedure for harvesting epidermal cells from the dorsal epidermis of adult mice for primary cell culture in “high calcium” defined medium. In: Leigh IM, Watt FM (eds) Keratinocyte methods. Cambridge University Press, Cambridge, pp 25–31Google Scholar
  32. 32.
    Weinberg WC, Goodman LV, George C, Morgan DL, Ledbetter S, Yuspa SH, Lichti U (1993) Reconstitution of hair follicle development in vivo: determination of follicle formation, hair growth, and hair quality by dermal cells. J Invest Dermatol 100:229–236CrossRefGoogle Scholar
  33. 33.
    Kamimura J, Lee D, Baden HP, Brissette J, Dotto GP (1997) Primary mouse keratinocyte cultures contain hair follicle progenitor cells with multiple differentiation potential. J Invest Dermatol 109:534–540CrossRefGoogle Scholar
  34. 34.
    Mascré G, Dekoninck S, Drogat B, Youssef KK, Broheé S, Sotiropoulou PA, Simons BD, Blanpain C (2012) Distinct contribution of stem and progenitor cells to epidermal maintenance. Nature 489(7415):257–262CrossRefGoogle Scholar
  35. 35.
    Lim X, Tan SH, Koh WL, Chau RM, Yan KS, Kuo CJ, van Amerongen R, Klein AM, Nusse R (2013) Interfollicular epidermal stem cells self-renew via autocrine Wnt signaling. Science 342(6163):1226–1230CrossRefGoogle Scholar
  36. 36.
    Hsu YC, Li L, Fuchs E (2014) Emerging interactions between skin stem cells and their niches. Nat Med 20(8):847–856CrossRefGoogle Scholar
  37. 37.
    Lyle S, Christofidou-Solomidou M, Liu Y, Elder DE, Albelda S, Cotsarelis G (1998) The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells. J Cell Sci 111:3179–3188PubMedGoogle Scholar
  38. 38.
    Greco V, Chen T, Rendl M, Schober M, Pasolli HA, Stokes N, Dela Cruz-Racelis J, Fuchs E (2009) A two-step mechanism for stem cell activation during hair regeneration. Cell Stem Cell 4:144–169CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2018

Authors and Affiliations

  1. 1.Department of Dermatology, College of Physicians & SurgeonsColumbia University Medical CenterNew YorkUSA
  2. 2.Department of Pathology and Cell Biology, College of Physicians & SurgeonsColumbia University Medical CenterNew YorkUSA
  3. 3.Russ Berrie Medical Science PavilionNew YorkUSA

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