In the present study we prepared explant cultures of plucked total hair follicles and of fragments microdissected from the following regions: B1 (bulb region), B2 (intermediate region), B3-1 (lower central outer root sheath, ORS), B3-2 (upper central ORS) and B4 (area of fracture). The growth capacities, the start of epithelial outgrowth, the stages of differentiation and apoptosis were studied immunohistochemically in early and late explant cultures using a battery of antibodies against cytokeratins, growth factor receptors and cell adhesion molecules and proliferation markers. Whole plucked hair follicles showed epithelial outgrowths exclusively in the upper central ORS (B3-2) starting early, mostly by day 3. In microdissected fragments, in contrast, outgrowths were more widespread, mostly in B3-2 and B3-1, and started early, but were also of late onset in some cases of B2 and B4. Epithelial outgrowths exhibited a basal layer of small cuboidal cells in a low stage of differentiation and one to two suprabasal layers of large prickle-like cells expressing late differentiation markers. The former expressed the receptor of nerve growth factor (NGF) heterogeneously whereas epidermal growth factor (EGF) receptor was not detectable. This is similar to ORS cells of this area in vivo. The proliferative activity of the outgrowths was always restricted to peripheral cells. Thus no essential differences in differentiation of outgrowing cells were detected. These results suggest that keratinocytes with the highest growth capacities in plucked human hair follicles are localized in the lower central ORS (corresponding to B3-2) and some with a lower capacity in the upper central ORS (corresponding to B3-1) as established after microdissection. This is in agreement with the bulge activation theory. NGF may also play a role in hair growth.
