Skip to main content
SpringerLink
Log in

Isolation, culture and identification of epidermal stem cells from newborn mouse skin

  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

Abstract

In healthy individuals, skin integrity is maintained by epidermal stem cells which self-renew and generate daughter cells that undergo terminal differentiation. Epidermal stem cells represent a promising source of stem cells, and their culture has great potential in scientific research and clinical application. However, no single method has been universally adopted for identifying and isolating epidermal stem cells. Here, we reported the isolation and characterization of putative epidermal stem cells from newborn mouse skin. The keratinocytes were separated enzymatically. Putative epidermal stem cells were selected by rapid adherence on a composite matrix made of type I collagen and fibronectin. Unattached cells were discarded after 10 min, and the attached cells were cultured in a defined culture medium. The isolated cells showed the typical epidermal stem cell morphology. Immunofluorescence indicated that the cells were strongly stained for β1 integrin family of extracellular matrix receptors. In conclusion, mouse putative epidermal stem cells were successfully isolated from newborn mouse epidermis on the basis of high rapid adhesion to extracellular matrix proteins and cultured in vitro.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3

Similar content being viewed by others

References

  • Aberdam D. Derivation of keratinocyte progenitor cells and skin formation from embryonic stem cells. Int. J. Dev. Biol. 48: 203–206; 2004.

    Article  CAS  PubMed  Google Scholar 

  • Aberdam D. Epidermal stem cell fate: what can we learn from embryonic stem cells? Cell Tissue Res. 331: 103–107; 2008.

    Article  PubMed  Google Scholar 

  • Aberdam E.; Barak E.; Rouleau M.; de LaForest S.; Berrih-Aknin S.; Suter D. M.; Krause K. H.; Amit M.; Itskovitz-Eldor J.; Aberdam D. A pure population of ectodermal cells derived from human embryonic stem cells. Stem Cells 26: 440–444; 2008.

    Article  CAS  PubMed  Google Scholar 

  • Bickenbach J. R. Identification of label-retaining cells in oral mucosa and skin. J. Dent. Res. 60(Spec no. C): 1611–1620; 1981.

    PubMed  Google Scholar 

  • Bickenbach J. R.; Chism E. Selection and extended growth of murine epidermal stem cells in culture. Exp. Cell Res. 244: 184–195; 1998.

    Article  CAS  PubMed  Google Scholar 

  • Cotsarelis G.; Sun T. T.; Lavker R. M. 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; 1990.

    Article  CAS  PubMed  Google Scholar 

  • Dong R.; Jin Y.; Lui Y. Culture of human epidermal stem cells in different media and their biological characteristics (Zhongguo xiu fu chong jian wai ke za zhi). Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi (Chinese Journal of Reparative and Reconstructive Surgery) 19: 314–317; 2005.

    Google Scholar 

  • Dunnwald M.; Tomanek-Chalkley A.; Alexandrunas D.; Fishbaugh J.; Bickenbach J. R. Isolating a pure population of epidermal stem cells for use in tissue engineering. Exp. Dermatol. 10: 45–54; 2001.

    Article  CAS  PubMed  Google Scholar 

  • Fusenig N. E.; Worst P. K. Mouse epidermal cell cultures. II. Isolation, characterization and cultivation of epidermal cells from perinatal mouse skin. Exp. Cell Res. 93: 443–457; 1975.

    Article  CAS  PubMed  Google Scholar 

  • Germain L.; Rouabhia M.; Guignard R.; Carrier L.; Bouvard V.; Auger F. A. Improvement of human keratinocyte isolation and culture using thermolysin. Burns 19: 99–104; 1993.

    Article  CAS  PubMed  Google Scholar 

  • Hakkinen L.; Koivisto L.; Larjava H. An improved method for culture of epidermal keratinocytes from newborn mouse skin. Methods Cell Sci. 23: 189–196; 2001.

    Article  CAS  PubMed  Google Scholar 

  • Jones P. Isolation and characterization of human epidermal stem cells. Clin. Sci. 91: 141–146; 1996.

    CAS  PubMed  Google Scholar 

  • Jones P. H.; Harper S.; Watt F. M. Stem cell patterning and fate in human epidermis. Cell (Cambridge) 80: 83–93; 1995.

    CAS  Google Scholar 

  • Jones P. H.; Watt F. M. Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression. Cell 73: 713–724; 1993.

    Article  CAS  PubMed  Google Scholar 

  • Kiel M. J.; He S.; Ashkenazi R.; Gentry S. N.; Teta M.; Kushner J. A.; Jackson T. L.; Morrison S. J. Haematopoietic stem cells do not asymmetrically segregate chromosomes or retain BrdU. Nature 449: 238–242; 2007.

    Article  CAS  PubMed  Google Scholar 

  • Kim D. S.; Cho H. J.; Choi H. R.; Kwon S. B.; Park K. C. Isolation of human epidermal stem cells by adherence and the reconstruction of skin equivalents. Cell. Mol. Life Sci. 61: 2774–2781; 2004.

    Article  CAS  PubMed  Google Scholar 

  • Koster M. I.; Huntzinger K. A.; Roop D. R. Epidermal differentiation: transgenic/knockout mouse models reveal genes involved in stem cell fate decisions and commitment to differentiation. J. Investig. Dermatol. Symp. Proc. 7: 41–45; 2002.

    Article  CAS  PubMed  Google Scholar 

  • Lavker R. M.; Sun T. T. Epidermal stem cells: properties, markers, and location. Proc. Natl. Acad. Sci. 97: 13473–13475; 2000.

    Article  CAS  PubMed  Google Scholar 

  • Li A.; Simmons P. J.; Kaur P. Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype. Proc. Natl. Acad. Sci. 95: 3902–3907; 1998.

    Article  CAS  PubMed  Google Scholar 

  • Li J.; Miao C.; Guo W.; Jia L.; Zhou J.; Ma B.; Peng S.; Liu S.; Cao Y.; Duan E. Enrichment of putative human epidermal stem cells based on cell size and collagen type IV adhesiveness. Cell Res. 18: 360–371; 2008.

    Article  CAS  PubMed  Google Scholar 

  • Liang L.; Bickenbach J. R. Somatic epidermal stem cells can produce multiple cell lineages during development. Stem Cells 20: 21–31; 2002.

    Article  PubMed  Google Scholar 

  • Liu Y.; Zhou H.; Gao F. Isolation and identification of stem cells from adult cashmere goat skin. Int. J. Dermatol. 47: 551–556; 2008.

    Article  CAS  PubMed  Google Scholar 

  • Mackenzie I. C. Retroviral transduction of murine epidermal stem cells demonstrates clonal units of epidermal structure. J. Invest. Dermatol. 109: 377–383; 1997.

    Article  CAS  PubMed  Google Scholar 

  • Mackenzie I. C.; Bickenbach J. R. Label-retaining keratinocytes and Langerhans cells in mouse epithelia. Cell Tissue Res. 242: 551–556; 1985.

    Article  CAS  PubMed  Google Scholar 

  • Mackenzie I. C.; Mackenzie S. L.; Rittman G. A. Isolation of subpopulations of murine epidermal cells using monoclonal antibodies against differentiation-related cell surface molecules. Differentiation 41: 127–138; 1989.

    Article  CAS  PubMed  Google Scholar 

  • Morris R. J.; Fischer S. M.; Klein-Szanto A. J. P.; Slaga T. J. Subpopulations of primary adult murine epidermal basal cells sedimented on density gradients. Cell Prolif. 23: 587–602; 1990.

    Article  CAS  Google Scholar 

  • Papini S.; Cecchetti D.; Campani D.; Fitzgerald W.; Grivel J. C.; Chen S.; Margolis L.; Revoltella R. P. Isolation and clonal analysis of human epidermal keratinocyte stem cells in long-term culture. Stem Cells 21: 481–494; 2003.

    Article  PubMed  Google Scholar 

  • Park H. S.; Kang H. J.; Kim C. H.; Han E. S.; Han K.; Kim T. H.; Gin Y. J.; Son Y. S. Application of physical force is essential to enrich for epidermal stem cells in primary human keratinocyte isolation. Tissue Eng. 10: 343–351; 2004.

    Article  CAS  PubMed  Google Scholar 

  • Potten C. S. The epidermal proliferative unit: the possible role of the central basal cell. Cell Prolif. 7: 77–88; 1974.

    Article  CAS  Google Scholar 

  • Potten C. S. (1983) Stem cells in epidermis from the back of the mouse. Churchill Livingston, New York. pp 200-232

  • Potten C. S. Cell cycles in cell hierarchies. Int. J. Radiat. Biol. 49: 257–278; 1986.

    Article  CAS  Google Scholar 

  • Rzepka K.; Schaarschmidt G.; Nagler M.; Wohlrab J. Epidermal stem cells. JDDG 3: 962–973; 2005.

    Article  PubMed  Google Scholar 

  • Singer A. J.; Clark R. A. F. Cutaneous wound healing. N. Engl. J. Med. 341: 738–746; 1999.

    Article  CAS  PubMed  Google Scholar 

  • Spichkina O. G.; Kalmykova N. V.; Kukhareva L. V.; Voronkina I. V.; Blinova M. I.; Pinaev G. P. Isolation of human basal keratinocytes by selective adhesion to extracellular matrix proteins. Tsitologiia 48: 841–847; 2006.

    CAS  PubMed  Google Scholar 

  • Tani H.; Morris R. J.; Kaur P. Enrichment for murine keratinocyte stem cells based on cell surface phenotype. Proc. Natl. Acad. Sci. 97: 10960–10965; 2000.

    Article  CAS  PubMed  Google Scholar 

  • Taylor G.; Lehrer M. S.; Jensen P. J.; Sun T. T.; Lavker R. M. Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell 102: 451–461; 2000.

    Article  CAS  PubMed  Google Scholar 

  • Terunuma A.; Jackson K. L.; Kapoor V.; Telford W. G.; Vogel J. C. Side population keratinocytes resembling bone marrow side population stem cells are distinct from label-retaining keratinocyte stem cells. J. Invest. Dermatol. 121: 1095–1103; 2003.

    Article  CAS  PubMed  Google Scholar 

  • Terunuma A.; Kapoor V.; Yee C.; Telford W. G.; Udey M. C.; Vogel J. C. Stem cell activity of human side population and alpha 6 integrin-bright keratinocytes defined by a quantitative in vivo assay. Stem Cells 25: 664–669; 2007.

    CAS  PubMed  Google Scholar 

  • Toma J. G.; McKenzie I. A.; Bagli D.; Miller F. D. Isolation and characterization of multipotent skin-derived precursors from human skin. AlphaMed Press, pp 727–737; 2005.

  • Triel C.; Vestergaard M. E.; Bolund L.; Jensen T. G.; Jensen U. B. Side population cells in human and mouse epidermis lack stem cell characteristics. Exp. Cell Res. 295: 79–90; 2004.

    Article  CAS  PubMed  Google Scholar 

  • Watt F. M. Studies with cultured human epidermal keratinocytes: potential relevance to corneal wound healing. Eye, London, p 161; 1994.

    Google Scholar 

  • Watt F. M. Epidermal stem cells: marker, patterning and the control of stem cell fate. Philos. Trans. R Soc. Lond. 353: 831–837; 1998.

    Article  CAS  Google Scholar 

  • Watt F. M. Out of Eden: stem cells and their niches. Science 287: 1427; 2000.

    Article  CAS  PubMed  Google Scholar 

  • Yano S.; Ito Y.; Fujimoto M.; Hamazaki T. S.; Tamaki K.; Okochi H. Characterization and localization of side population cells in mouse skin. Stem Cells 23: 834–841; 2005.

    Article  CAS  PubMed  Google Scholar 

  • Yu B. D.; Mukhopadhyay A.; Wong C. Skin and hair: models for exploring organ regeneration. Hum. Mol. Genet. 17: R54–R59; 2008.

    Article  CAS  PubMed  Google Scholar 

  • Zhou J.; Chen S.; Liu W.; Cao Y.; Duan E. Enrichment and identification of human “fetal” epidermal stem cells. Hum. Reprod. 19: 968–974; 2004a.

    Article  PubMed  Google Scholar 

  • Zhou J.; Jia L.; Yang Y.; Peng S.; Cao Y.; Duan E. Enrichment and characterization of mouse putative epidermal stem cells. Cell Biol. Int. 28: 523–529; 2004b.

    Article  CAS  PubMed  Google Scholar 

  • Zhu A. J.; Haase I.; Watt F. M. Signaling via β1 integrins and mitogen-activated protein kinase determines human epidermal stem cell fate in vitro. Proc. Natl. Acad. Sci. 96: 6728–6733; 1999.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank Dr. Ebrahim Ahmadi and Dr. Hasan Nazari for their assistance during this research.

Author information

Authors and Affiliations

  1. Department of Biology, Faculty of Basic Sciences, University of Shahrekord, Shahrekord, Iran

    Somayeh Reiisi & Fariba Esmaeili

  2. Research Institute of Animal Embryo Technology, University of Shahrekord, Shahrekord, Iran

    Fariba Esmaeili & Abolfazl Shirazi

Authors
  1. Somayeh Reiisi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fariba Esmaeili
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Abolfazl Shirazi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Fariba Esmaeili.

Additional information

Editor: J. Denry Sato

Rights and permissions

Reprints and permissions

About this article

Cite this article

Reiisi, S., Esmaeili, F. & Shirazi, A. Isolation, culture and identification of epidermal stem cells from newborn mouse skin. In Vitro Cell.Dev.Biol.-Animal 46, 54–59 (2010). https://doi.org/10.1007/s11626-009-9245-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11626-009-9245-y

Keywords

Search

Navigation