Experimental Modulation of the Differentiated Phenotype of Keratinocytes from Epidermis and Hair Follicle Outer Root Sheath and Matrix Cells

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Abstract
Follicles of human anagen hair were separated into morphologically distinct compartments (by sequential trypsinization and microdissection) for the biochemical and immunological analysis of keratins as differentiation markers to diagnose the type of epithelial differentiation. While outer root sheath contained throughout the "soft" (cyto)keratins K5, 6, 14, 16, and 17, and hair cortex contained exclusively a set of acidic and basic "hard" alpha-keratins (consistent up to the hair tip), in inner root sheath and hair cuticle peptides related or derived from suprabasal epidermal keratins K1 and 10 were detected. These keratin profiles served as in vivo correlates for the evaluation of type and degree of differentiation achieved by the respective isolated epithelial cells, comparing different growth or culture conditions. Cultures of ORS cells and hair matrix cells (PHS cells) as well as normal keratinocytes were initiated using postmitotic human dermal fibroblasts as efficient feeder cells. On lifted collagen gels populated with HDF ("surface" cultures), ORS and PHS cells formed stratified epithelial expressing epidermal differentiation markers such as keratins K1 and 10, involucrin, and filaggrin. Compared with NEK "surface" cultures, balance between growth and differentiation was better maintained by both follicular cell types. In contrast, epidermal tissue homeostasis was largely normalized in transplants on nude mice regardless of the epithelial cell type, apparent from orderly tissue structure, regular distribution of keratin K10, filaggrin, and involucrin, and distinct continuous deposition of basement membrane components at the epithelium-collagen interface. Embedded in Matrigel (on top of HDF collagen gels) ORS cells and NEK formed spheroids exhibiting inward-directed epidermoid differentiation, increasing with time. All epidermal maturation products found in "surface" cultures were likewise expressed, and again differentiation greatly outbalanced proliferation in spheroids of NEK but not of ORS cells. PHS cells embedded together with HDF in Matrigel produced similar spheroids as ORS cells. Size of spheroids and degree of epidermoid differentiation were dramatically reduced when HDF were replaced by follicular DP cells, demonstrating the crucial role of the mesenchymal "companion" cells.(ABSTRACT TRUNCATED AT 400 WORDS)