Isolation and culture of epithelial stem cells.
In the skin, epithelial stem cells in the hair follicle contribute not only to the generation of a new hair follicle with each hair cycle, but also to the repair of the epidermis during wound healing. When these stem cells are isolated and expanded in culture, they can give rise to hair follicles, sebaceous glands, and epidermis when combined with dermis and grafted back onto Nude mice. In this chapter, we provide a method for isolating hair follicle epithelial stem cells from the skin of adult mice using immunofluorescent labeling to allow for the specific purification of epithelial stem cells by fluorescence-activated cell sorting (FACS). Notably, this method relies exclusively on cell surface markers, making it suitable for use with any strain of mouse and at various stages of the hair cycle. We also provide a detailed protocol for culturing epithelial stem cells isolated by FACS, allowing for analysis using a wide variety of culture assays. Additionally, we provide notes on using cultured cells for specific applications, such as viral manipulation and grafting. These techniques should be useful for directly evaluating stem cell function in normal mice and in mice with skin defects.
Nowak
Jonathan A
JA
Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA.
Fuchs
Elaine
E
D016475:3T3 Cells; D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D018859:Hair Follicle / Q000166:cytology / Q000187:drug effects; D039503:Integrin alpha6 / Q000378:metabolism; D051379:Mice; D016685:Mitomycin / Q000494:pharmacology; D013194:Staining and Labeling; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism
D018952:Antigens, CD34; D039503:Integrin alpha6; D016685:Mitomycin