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Generation of a Full-Thickness Human Skin Equivalent on an Immunodeficient Mouse

  • Nicole Diette
  • Igor Kogut
  • Ganna BilousovaEmail author
Protocol
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Part of the Methods in Molecular Biology book series (MIMB, volume 2109)

Abstract

Human skin equivalents composed of epidermal cells and fibroblasts are important for modeling human epidermal development, testing new therapeutics, and designing novel treatment strategies for human skin diseases. Here, we describe a procedure for the generation of an in vivo full-thickness human skin equivalent on an immunodeficient mouse using a grafting chamber system. The protocol involves mixing human epidermal cells and fibroblasts in a silicone grafting chamber that is surgically inserted onto the muscle fascia of a recipient immunodeficient mouse. Following the removal of the silicone chamber, the graft area is exposed to air to induce stratification of developing epidermis, resulting in the reconstitution of full-thickness human skin tissue on a live mouse. This grafting system provides a straightforward approach to study human skin diseases in an animal model and has been previously used to determine the ability of both mouse and human primary epidermal cells and cells derived from pluripotent stem cells to regenerate functional skin in vivo.

Keywords

Xenograft Human skin graft Mouse model Grafting chamber Keratinocytes Epidermal cells Fibroblasts 

Abbreviations

H&E

Hematoxylin and eosin

ID

Inner diameter

OD

Outer diameter

NOD-SCID

Nonobese diabetic/severe combined immunodeficiency

Notes

Acknowledgments

We are grateful for the funding support from the National Institutes of Health (T32 AR007411-33) and the University of Colorado Skin Diseases Research Core Center (P30 AR057212). We also thank Epidermolysis Bullosa (EB) Research Partnership, the EB Medical Research Foundation, the Cure EB Charity, Dystrophic Epidermolysis Bullosa Research Association (DEBRA) International, and the Gates Frontiers Fund.

References

  1. 1.
    Kogut I, Bilousova G (2018) Environmental influences on the development of epidermal progenitors. In: Reference module in biomedical sciences. Elsevier, AmsterdamGoogle Scholar
  2. 2.
    Fuchs E (2007) Scratching the surface of skin development. Nature 445:834–842.  https://doi.org/10.1038/nature05659 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Bremer J et al (2018) Murine type VII collagen distorts outcome in human skin graft mouse model for dystrophic epidermolysis bullosa. Exp Dermatol.  https://doi.org/10.1111/exd.13744 CrossRefGoogle Scholar
  4. 4.
    Ponec M (2002) Skin constructs for replacement of skin tissues for in vitro testing. Adv Drug Deliv Rev 54(Suppl 1):S19–S30CrossRefGoogle Scholar
  5. 5.
    de Oliveira VL et al (2012) Humanized mouse model of skin inflammation is characterized by disturbed keratinocyte differentiation and influx of IL-17A producing T cells. PLoS One 7:e45509.  https://doi.org/10.1371/journal.pone.0045509 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Landman S et al (2018) Intradermal injection of low dose human regulatory T cells inhibits skin inflammation in a humanized mouse model. Sci Rep 8:10044.  https://doi.org/10.1038/s41598-018-28346-5 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Itoh M et al (2013) Generation of 3D skin equivalents fully reconstituted from human induced pluripotent stem cells (iPSCs). PLoS One 8:e77673.  https://doi.org/10.1371/journal.pone.0077673 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Lichti U, Anders J, Yuspa SH (2008) Isolation and short-term culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice. Nat Protoc 3:799–810.  https://doi.org/10.1038/nprot.2008.50 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Shultz LD et al (1995) Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice. J Immunol 154:180–191PubMedGoogle Scholar
  10. 10.
    Shultz LD et al (2005) Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J Immunol 174:6477–6489CrossRefGoogle Scholar
  11. 11.
    Bilousova G, Roop DR (2013) Generation of functional multipotent keratinocytes from mouse induced pluripotent stem cells. Methods Mol Biol 961:337–350.  https://doi.org/10.1007/978-1-62703-227-8_22 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2019

Authors and Affiliations

  • Nicole Diette
    • 1
    • 2
  • Igor Kogut
    • 1
    • 2
  • Ganna Bilousova
    • 1
    • 2
    • 3
    Email author
  1. 1.Department of DermatologyUniversity of Colorado School of Medicine, Anschutz Medical CampusAuroraUSA
  2. 2.Charles C. Gates Center for Regenerative MedicineUniversity of Colorado School of Medicine, Anschutz Medical CampusAuroraUSA
  3. 3.Correspondence: Ganna Bilousova, Charles C. Gates Center for Regenerative MedicineUniversity of Colorado School of Medicine, Anschutz Medical CampusAuroraUSA

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