Studies of developing human hair shaft cells in vitro

Methods for studying aspects of hair formation in vitro have been devised on the basis of isolating developing hair shaft cells. These cells were obtained using a sterile microdissection technique. Plucked anagen follicles were dissected free of surrounding tissues (inner and outer root sheaths), and presumptive hair shaft cells (including germinal epithelia) were cultured directly on mammalian fibroblasts or in media preconditioned by fibroblasts. Specimens were cultured either as dispersions or in whole tissue pieces. Trypsinized whole tissue specimens in culture were sometimes observed to form increased bulk, while dispersed cells appeared to elongate and form larger colonies. In sections of these colonies examined by transmission electron microscopy, intracellular hard keratin intermediate filaments (IFs) together with IF-matrix hard keratin complexes were observed. Radiolabelled cysteine [35S] was added to cultures (3-20 days), showing a continuing but reduced synthesis of hard keratin IF proteins (low-sulfur) over the period of study. Matrix protein (high-sulfur) production was drastically reduced after 3 days. Monoclonal antibodies directed against hair keratin IF components were used in Western transfers and immunofluorescent studies to help assess the specificity of proteins synthesized in culture. Our observations indicate that, with some refinement, the presently described methods enable preparation of hair shaft precursor cells suitable for observing certain hair-forming processes in vitro.