Formation of a regular neo-epidermis by cultured human outer root sheath cells grafted on nude mice.

The outer root sheath of hair follicles mainly consists of basal-like keratinocytes which can substitute for interfollicular epidermal keratinocytes, as during healing of skin wounds when outer root sheath cells migrate onto the denuded area, thus contributing to epidermal regeneration. Human outer root sheath cells represent a repeatedly available source of keratinocytes which can be easily and extensively expanded in culture. Close comparison of organotypic cultures of either outer root sheath cells or epidermal keratinocytes grafted onto nude mice demonstrated that outer root sheath cells formed a stratified epithelium resembling normal epidermis that is virtually indistinguishable from that developed by epidermal keratinocytes. Typical epidermal differentiation markers, such as the suprabasal keratins 1 and 10, involucrin, filaggrin, the basement membrane components collagen type IV and laminin, and the integrin chains alpha 2, alpha 3, alpha 6, and beta 1, were readily expressed in a mostly regular localization. These data suggest that outer root sheath cells, bearing essential advantages as compared with interfollicular keratinocytes, are suitable for skin replacement.

Phenotypic modulation of human hair matrix cells (trichocytes) by environmental influence in vitro and in vivo.

Trichocytes, i.e. precursor cells of the hair cortex and medulla, isolated from plucked human scalp hair follicles (HF) were propagated on feeder layers of post-mitotic human dermal fibroblasts (HDF). Cell isolates from five HF routinely yielded about 0.5-1 x 10(5) cells within 3 weeks. When grown as 'surface epithelia' in vitro (on dermal equivalents exposed to air), trichocytes organized into stratified epithelia largely reminiscent of epidermis with regard to both tissue architecture and localization of epidermal differentiation products (keratins K1 and K10, involucrin, filaggrin). However, when HDF in the collagen matrix were replaced by dermal papilla cells (DPC) epidermoid differentiation was largely prevented while still allowing growth and stratification. Epidermal differentiation (keratinization) was virtually complete when trichocytes grown on collagen gels with HDF were transplanted onto nude mice; this was apparent by tissue organization, expression of K1 and K10 and the nearly regular epidermal localization of involucrin. In addition, the deposition of basement membrane components (laminin, type IV collagen, bullous pemphigoid antigen) at the epithelium-collagen interface further increased and was more regular in transplants than in vitro. Cells embedded in Matrigel together with HDF developed large spheroidal structures with inward-directed differentiation and all the epidermal markers found in 'surface' cultures, while only small keratinizing spheroids formed without HDF. In this system co-culture of trichocytes with DPC suppressed almost completely epidermal keratinization. Although typical hair proteins were not detectable, our data clearly demonstrate that: (1) bona fide trichocytes inherit the options for alternative directions of differentiation, and (2) external (in part mesenchymal cell-mediated) influences play a pivotal role in this determination.