Isolation and culture of epithelial stem cells.
Isolation and culture of epithelial stem cells.
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DOI：
10.1007/978-1-59745-060-7_14
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发表时间：
2009
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在皮肤中，毛囊中的上皮干细胞不仅有助于每个毛发周期产生新的毛囊，而且有助于伤口愈合期间表皮的修复。当这些干细胞被分离并在培养中扩增时，当与真皮结合并移植回裸鼠时，它们可以产生毛囊、皮脂腺和表皮。在这一章中，我们提供了一种方法，用于分离毛囊上皮干细胞从成年小鼠的皮肤使用免疫荧光标记，允许上皮干细胞的特异性纯化荧光激活细胞分选（FACS）。值得注意的是，这种方法完全依赖于细胞表面标记物，使其适用于任何品系的小鼠和毛发周期的各个阶段。我们还提供了一个详细的协议，培养上皮干细胞分离的流式细胞仪，允许使用各种各样的培养分析。此外，我们还提供了关于将培养细胞用于特定应用的说明，例如病毒操作和移植。这些技术应该是有用的，直接评估干细胞在正常小鼠和皮肤缺损小鼠的功能。
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.