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Assaying proliferation and differentiation capacity of stem cells using disaggregated adult mouse epidermis

Nature Protocols volume 5, pages 898–911 (2010)Cite this article

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Abstract

In this protocol, we describe how to isolate keratinocytes from adult mouse epidermis, fractionate them into different sub-populations on the basis of cell surface markers and examine their function in an in vivo skin reconstitution assay with disaggregated neonatal dermal cells. We also describe how the isolated keratinocytes can be subjected to clonal analysis in vitro and in vivo and how to enrich for hair follicle-inducing dermal papilla cells in the dermal preparation. Using these approaches, it is possible to compare the capacity of different populations of adult epidermal stem cells to proliferate and to generate progeny that differentiate along the different epidermal lineages. Isolating, fractionating and grafting cells for the skin reconstitution assay is normally spread over 2 d. Clonal growth in culture is assessed after 14 d, while evaluation of the grafts is carried out after 4–5 weeks.

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Figure 1: Preparation of single cell suspension of epidermal cells from mouse back skin.
Figure 6: Measurement of epidermal colony forming ability in culture.
Figure 2: Isolation of cells based on expression of CD34, Sca1 and Integrin alpha 6.
Figure 3: Isolation of epidermal cells based on sorting for CD34 and Lrig1 expression.
Figure 4: Isolation of cells from neonatal dermis.
Figure 7: Flow cytometric analysis of dermal single cell suspension stained with APC–CD133 antibody.
Figure 5: Whole mount and histological analysis of in vivo reconstituted skin.

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Acknowledgements

We thank D. Owens and D. Roop for sharing their grafting protocols; S. Goldie for providing one of the figure panels; A. Giangreco and C. Collins for introducing the technique into our lab; and the excellent core facilities of the CRUK Cambridge Research Institute and the Wellcome Trust Centre for Stem Cell Research. This work was supported by Cancer Research UK, the Medical Research Council, the Wellcome Trust and the European Union. F. Watt gratefully acknowledges support from the University of Cambridge and Hutchison Whampoa.

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Author notes

  1. Kim B Jensen and Ryan R Driskell: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory for Epidermal Stem Cell Biology, Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK

    Kim B Jensen, Ryan R Driskell & Fiona M Watt

  2. Epithelial Cell Biology Laboratory, Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Cambridge, UK

    Fiona M Watt

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  1. Kim B Jensen
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Contributions

K.B.J. and R.R.D. tested the protocols, generated the figures and co-wrote the paper. F.M.W. co-wrote the paper.

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Correspondence to Fiona M Watt.

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The authors declare no competing financial interests.

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Jensen, K., Driskell, R. & Watt, F. Assaying proliferation and differentiation capacity of stem cells using disaggregated adult mouse epidermis. Nat Protoc 5, 898–911 (2010). https://doi.org/10.1038/nprot.2010.39

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