Connelly John T
Blizard Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, London, UK.
Methods Mol Biol. 2012;916:15-22. doi: 10.1007/978-1-61779-980-8_2.
Extracellular signals play a central role in coordinating the growth and differentiation of epidermal stem cells. This protocol describes a technique for quantitatively examining the influence of extracellular matrix (ECM) interactions on keratinocyte terminal differentiation through the use of micro-patterned, polymer substrates. Circular islands of type I collagen are created, first by micro-contact printing and surface-initiated polymerization of a protein resistant background. The unprotected gold is then coated with collagen by passive adsorption. When human keratinocytes are seeded onto these substrates, limited adhesion on the smallest islands induces terminal differentiation, characterized by increased involucrin, transglutaminase, and periplakin expression, as well as reduced proliferation. This platform provides a robust assay for studying the terminal differentiation of human epidermal stem cells and the regulatory roles of specific cell-matrix interactions in this process.
细胞外信号在协调表皮干细胞的生长和分化中起着核心作用。本方案描述了一种通过使用微图案化的聚合物基质定量检测细胞外基质（ECM）相互作用对角质形成细胞终末分化影响的技术。首先通过微接触印刷和对蛋白质有抗性的背景的表面引发聚合反应，创建I型胶原蛋白的圆形岛。然后通过被动吸附将未受保护的金表面涂上胶原蛋白。当将人角质形成细胞接种到这些基质上时，在最小的岛上有限的黏附会诱导终末分化，其特征是内披蛋白、转谷氨酰胺酶和周膜蛋白表达增加，以及增殖减少。该平台为研究人类表皮干细胞的终末分化以及特定细胞-基质相互作用在此过程中的调节作用提供了一个强大的检测方法。
