Terminal Differentiation of Human Epidermal Stem Cells on Micro-patterned Substrates

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.