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Stem Cell Protocols pp 147–164Cite as

Isolation and Functional Assessment of Cutaneous Stem Cells

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1235))

Abstract

The epidermis and associated appendages of the skin represent a multi-lineage tissue that is maintained by perpetual rounds of renewal. During homeostasis, turnover of epidermal lineages is achieved by input from regionalized keratinocytes stem or progenitor populations with little overlap from neighboring niches. Over the last decade, molecular markers selectively expressed by a number of these stem or progenitor pools have been identified, allowing for the isolation and functional assessment of stem cells and genetic lineage tracing analysis within intact skin. These advancements have led to many fundamental observations about epidermal stem cell function such as the identification of their progeny, their role in maintenance of skin homeostasis, or their contribution to wound healing. In this chapter, we provide a methodology to identify and isolate epidermal stem cells and to assess their functional role in their respective niche. Furthermore, recent evidence has shown that the microenvironment also plays a crucial role in stem cell function. Indeed, epidermal cells are under the influence of surrounding fibroblasts, adipocytes, and sensory neurons that provide extrinsic signals and mechanical cues to the niche and contribute to skin morphogenesis and homeostasis. A better understanding of these microenvironmental cues will help engineer in vitro experimental models with more relevance to in vivo skin biology. New approaches to address and study these environmental cues in vitro will also be addressed.

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References

  1. Fuchs E, Tumbar T, Guasch G (2004) Socializing with the neighbors: stem cells and their niche. Cell 116:769–778

    Article  CAS  PubMed  Google Scholar 

  2. Yan X, Owens DM (2008) The skin: a home to multiple classes of epithelial progenitor cells. Stem Cell Rev 4:113–118

    Article  PubMed Central  PubMed  Google Scholar 

  3. Lobitz C, Dobson L (1961) Dermatology: the eccrine sweat glands. Annu Rev Med 12;289–298

    Google Scholar 

  4. Cotsarelis G, Sun TT, Lavker RM (1990) Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61:1329–1337

    Article  CAS  PubMed  Google Scholar 

  5. Trempus CS, Morris RJ, Bortner CD et al (2003) Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34. J Invest Dermatol 120:501–511

    Article  CAS  PubMed  Google Scholar 

  6. Morris RJ, Liu Y, Marles L et al (2004) Capturing and profiling adult hair follicle stem cells. Nat Biotechnol 22:411–417

    Article  CAS  PubMed  Google Scholar 

  7. Horsley V, O’Carroll D, Tooze R et al (2006) Blimp1 defines a progenitor population that governs cellular input to the sebaceous gland. Cell 126:597–609

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Nijhof JGW, Braun KM, Giangreco A et al (2006) The cell-surface marker MTS24 identifies a novel population of follicular keratinocytes with characteristics of progenitor cells. Development 133:3027–3037

    Article  CAS  PubMed  Google Scholar 

  9. Ghazizadeh S, Taichman LB (2001) Multiple classes of stem cells in cutaneous epithelium: a lineage analysis of adult mouse skin. EMBO J 20:1215–1222

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Levy V, Lindon C, Harfe BD et al (2005) Distinct stem cell populations regenerate the follicle and interfollicular epidermis. Dev Cell 9:855–861

    Article  CAS  PubMed  Google Scholar 

  11. Clayton E, Doupé DP, Klein AM et al (2007) A single type of progenitor cell maintains normal epidermis. Nature 446:185–189

    Article  CAS  PubMed  Google Scholar 

  12. Jensen KB, Collins CA, Nascimento E et al (2009) Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis. Cell Stem Cell 4:427–439

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. Woo S-H, Stumpfova M, Jensen UB et al (2010) Identification of epidermal progenitors for the Merkel cell lineage. Development 139:622

    Article  Google Scholar 

  14. Doucet YS, Woo S-H, Ruiz ME et al (2013) The touch dome defines an epidermal niche specialized for mechanosensory signaling. Cell Rep 3:1759–1765

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Morrison SJ, Kimble J (2006) Asymmetric and symmetric stem-cell divisions in development and cancer. Nature 441:1068–1074

    Article  CAS  PubMed  Google Scholar 

  16. Lechler T, Fuchs E (2005) Asymmetric cell divisions promote stratification and differentiation of mammalian skin. Nature 437:275–280

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  17. Weinberg W, Goodman L (1993) Reconstitution of hair follicle development in vivo: determination of follicle formation, hair growth and hair quality by dermal cells. J Investig Dermatol 100:229–236

    Article  CAS  PubMed  Google Scholar 

  18. Kamimura J, Lee D, Baden H (1997) Primary mouse keratinocyte cultures contain hair follicle progenitor cells with multiple differentiation potential. J Investig Dermatol 109:534–540

    Article  CAS  PubMed  Google Scholar 

  19. Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331–343

    Article  CAS  PubMed  Google Scholar 

  20. McNairn AJ, Doucet Y, Demaude J et al (2013) TGFβ signaling regulates lipogenesis in human sebaceous glands cells. BMC Dermatol 13:2

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Gledhill K, Gardner A, Jahoda C (2013) Isolation and establishment of hair follicle dermal papilla cell cultures. In: Turksen K (ed) Skin Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol 989. Springer, New York, pp 285–292

    Chapter  Google Scholar 

  22. 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 97:10960–10965

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Rendl M, Lewis L, Fuchs E (2005) Molecular dissection of mesenchymal-epithelial interactions in the hair follicle. PLoS Biol 3:e331

    Article  PubMed Central  PubMed  Google Scholar 

  24. Birgersdotter A, Sandberg R, Ernberg I (2005) Gene expression perturbation in vitro–a growing case for three-dimensional (3D) culture systems. Semin Cancer Biol 15:405–412

    Article  PubMed  Google Scholar 

  25. Kishimoto J, Burgeson RE, Morgan BA (2000) Wnt signaling maintains the hair-inducing activity of the dermal papilla Wnt signaling maintains the hair-inducing activity of the dermal papilla. Genes Dev 14:1181–1185

    CAS  PubMed Central  PubMed  Google Scholar 

  26. Rendl M, Polak L, Fuchs E (2008) BMP signaling in dermal papilla cells is required for their hair follicle-inductive properties. Genes Dev 22:543–557

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Ouji Y, Nakamura-Uchiyama F, Yoshikawa M (2013) Canonical Wnts, specifically Wnt-10b, show ability to maintain dermal papilla cells. Biochem Biophys Res Commun 438:493–499

    Article  CAS  PubMed  Google Scholar 

  28. Ohyama M, Kobayashi T, Sasaki T et al (2012) Restoration of the intrinsic properties of human dermal papilla in vitro. J Cell Sci 125:4114–4125

    Article  CAS  PubMed  Google Scholar 

  29. Kandyba E, Leung Y, Chen Y-B et al (2013) Competitive balance of intrabulge BMP/Wnt signaling reveals a robust gene network ruling stem cell homeostasis and cyclic activation. Proc Natl Acad Sci U S A 110:1351–1356

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Nolte SV, Xu W, Rennekampff H-O et al (2008) Diversity of fibroblasts—a review on implications for skin tissue engineering. Cells Tissues Organs 187:165–176

    Article  PubMed  Google Scholar 

  31. Festa E, Fretz J, Berry R et al (2011) Adipocyte lineage cells contribute to the skin stem cell niche to drive hair cycling. Cell 146:761–771

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  32. Watt FM, Hogan BL (2000) Out of Eden: stem cells and their niches. Science 287:1427–1430

    Article  CAS  PubMed  Google Scholar 

  33. Itoh M, Umegaki-Arao N, Guo Z et al (2013) Generation of 3D skin equivalents fully reconstituted from human induced pluripotent stem cells (iPSCs). PLoS One 8:e77673

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Millar SE (2002) Molecular mechanisms regulating hair follicle development. J Invest Dermatol 118:216–225

    Article  CAS  PubMed  Google Scholar 

  35. Rompolas P, Deschene ER, Zito G et al (2012) Live imaging of stem cell and progeny behaviour in physiological hair-follicle regeneration. Nature 487:496–499

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  36. Kishimoto J, Ehama R, Wu L et al (1999) Selective activation of the versican promoter by epithelial- mesenchymal interactions during hair follicle development. Proc Natl Acad Sci U S A 96:7336–7341

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  37. Higgins CA, Chen JC, Cerise JE, et al (2013) Microenvironmental reprogramming by three-dimensional culture enables dermal papilla cells to induce de novo human hair-follicle growth. Proc Natl Acad Sci U S A [Epub ahead of print] PMID: 24145441

    Google Scholar 

  38. Gangatirkar P, Paquet-Fifield S, Li A et al (2007) Establishment of 3D organotypic cultures using human neonatal epidermal cells. Nat Protoc 2:178–186

    Article  CAS  PubMed  Google Scholar 

  39. Carroll JM, Molès JP (2000) A three-dimensional skin culture model for mouse keratinocytes: application to transgenic mouse keratinocytes. Exp Dermatol 9:20–24

    Article  CAS  PubMed  Google Scholar 

  40. Goldstein J, Horsley V (2012) Home sweet home: skin stem cell niches. Cell Mol Life Sci 69:2573–2582

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  41. Chan RK, Zamora DO, Wrice NL et al (2012) Development of a vascularized skin construct using adipose-derived stem cells from debrided burned skin. Stem Cells Int 2012:841203

    Article  PubMed Central  PubMed  Google Scholar 

  42. Liu Y, Suwa F, Wang X et al (2007) Reconstruction of a tissue-engineered skin containing melanocytes. Cell Biol Int 31:985–990

    Article  CAS  PubMed  Google Scholar 

  43. Blanpain C, Lowry W, Geoghegan A (2004) Self-renewal, multipotency and the existence of two cell populations within an epithelial stem cell niche. Cell 118:635–648

    Article  CAS  PubMed  Google Scholar 

  44. Barrandon Y, Green H (1987) Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci U S A 84:2302–2306

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  45. Lu CP, Polak L, Rocha AS et al (2012) Identification of stem cell populations in sweat glands and ducts reveals roles in homeostasis and wound repair. Cell 150:136–150

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  46. Jensen U, Yan X, Triel C et al (2008) A distinct population of clonogenic and multipotent murine follicular keratinocytes residing in the upper isthmus. J Cell Sci 121:609–617

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  47. Yuspa SH, Kilkenny AE, Steinert PM et al (1989) Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro. J Cell Biol 109:1207–1217

    Article  CAS  PubMed  Google Scholar 

  48. Yano S, Komine M, Fujimoto M et al (2004) Mechanical stretching in vitro regulates signal transduction pathways and cellular proliferation in human epidermal keratinocytes. J Invest Dermatol 122:783–790

    Article  CAS  PubMed  Google Scholar 

  49. Reichelt J (2007) Mechanotransduction of keratinocytes in culture and in the epidermis. Eur J Cell Biol 86:807–816

    Article  CAS  PubMed  Google Scholar 

  50. Fliniaux I, Viallet JP, Dhouailly D et al (2004) Transformation of amnion epithelium into skin and hair follicles. Differentiation 72:558–565

    Article  CAS  PubMed  Google Scholar 

  51. Toyoshima K, Asakawa K, Ishibashi N, Toki H, Ogawa M, Hasegawa T, Irié T, Tachikawa T, Sato A, Takeda A, Tsuji T (2012) Fully functional hair follicle regeneration through the rearrangement of stem cells and their niches. Nat Commun 3:784

    Article  PubMed Central  PubMed  Google Scholar 

  52. Clavel C, Grisanti L, Zemla R et al (2012) Sox2 in the dermal papilla niche controls hair growth by fine-tuning BMP signaling in differentiating hair shaft progenitors. Dev Cell 23:981–994

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  53. Ito Y, Hamazaki TS, Ohnuma K et al (2007) Isolation of murine hair-inducing cells using the cell surface marker prominin-1/CD133. J Invest Dermatol 127:1052–1060

    Article  CAS  PubMed  Google Scholar 

  54. Greco V, Chen T, Rendl M et al (2009) A two-step mechanism for stem cell activation during hair regeneration. Cell Stem Cell 4:155–169

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  55. Jaks V, Barker N, Kasper M et al (2008) Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat Genet 40:1291–1299

    Article  CAS  PubMed  Google Scholar 

  56. Vidal VPI, Chaboissier M-C, Lützkendorf S et al (2005) Sox9 is essential for outer root sheath differentiation and the formation of the hair stem cell compartment. Curr Biol 15:1340–1351

    Article  CAS  PubMed  Google Scholar 

  57. Nowak JA, Polak L, Pasolli HA et al (2008) Hair follicle stem cells are specified and function in early skin morphogenesis. Cell Stem Cell 3:33–43

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  58. Horsley V, Aliprantis AO, Polak L et al (2008) NFATc1 balances quiescence and proliferation of skin stem cells. Cell 132:299–310

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  59. Nguyen H, Merrill BJ, Polak L et al (2009) Tcf3 and Tcf4 are essential for long-term homeostasis of skin epithelia. Nat Genet 41:1068–1075

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  60. Rhee H, Polak L, Fuchs E (2006) Lhx2 maintains stem cell character in hair follicles. Science 312:1946–1949

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  61. Youssef KK, Van Keymeulen A, Lapouge G et al (2010) Identification of the cell lineage at the origin of basal cell carcinoma. Nat Cell Biol 12:299–305

    CAS  PubMed  Google Scholar 

  62. Lapouge G, Youssef KK, Vokaer B et al (2011) Identifying the cellular origin of squamous skin tumors. Proc Natl Acad Sci U S A 108:7431–7436

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  63. Ohyama M, Terunuma A, Tock CL et al (2006) Characterization and isolation of stem cell—enriched human hair follicle bulge cells. J Clin Invest 116:249–260

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  64. Snippert HJ, Haegebarth A, Kasper M et al (2010) Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin. Science 327:1385–1389

    Article  CAS  PubMed  Google Scholar 

  65. Leung Y, Kandyba E, Chen Y-B et al (2013) Label retaining cells (LRCs) with myoepithelial characteristic from the proximal acinar region define stem cells in the sweat gland. PLoS One 8:e74174

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Dermatology, College of Physicians and Surgeons, Columbia University, New York, NY, 10032, USA

    Yanne S. Doucet & David M. Owens

  2. Ecole Doctorale des Sciences de la Vie et de la Santé, Université Aix-Marseille, Marseille, France

    Yanne S. Doucet

  3. Department of Pathology and Cell Biology, Columbia University, 1150 St. Nicholas Avenue, New York, NY, 10032, USA

    David M. Owens

Authors
  1. Yanne S. Doucet
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  2. David M. Owens
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Corresponding author

Correspondence to David M. Owens .

Editor information

Editors and Affiliations

  1. HemoGenix, Inc., Colorado Springs, Colorado, USA

    Ivan N. Rich

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Doucet, Y.S., Owens, D.M. (2015). Isolation and Functional Assessment of Cutaneous Stem Cells. In: Rich, I. (eds) Stem Cell Protocols. Methods in Molecular Biology, vol 1235. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1785-3_13

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  • DOI: https://doi.org/10.1007/978-1-4939-1785-3_13

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1784-6

  • Online ISBN: 978-1-4939-1785-3

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