Culture of Dermal Papilla Cells from Ovine Wool Follicles: An In Vitro Model for Papilla Size Determination.

Common human balding or hair loss is driven by follicle miniaturization. Miniaturization is thought to be caused by a reduction in dermal papilla size. The molecular mechanisms that regulate papilla size are poorly understood, and their elucidation would benefit from a tractable experimental model. We have found that dermal papilla cells from sheep spontaneously aggregate in culture to form papilla-like structures. Here, we describe methods for microdissecting dermal papillae from wool follicles, for initiating and maintaining cultures of ovine papilla cells, and for using these cells in an in vitro assay to measure the effect of bioactive molecules on aggregate size.

The Germinative Epithelium of Sheep Vibrissae and Wool Follicles has Extensive Proliferative Potential but is Dependent on the Dermal Papilla.

AIM: To investigate the growth potential of keratinocytes derived from the germinative epithelium (GE) of ovine hair follicles. Stem cells from the outer root sheath (ORS) of hair follicles migrate to the GE in the lower follicle where they proliferate and differentiate to form the hair fiber. It has been suggested that the GE comprises transit-amplifying cells and that the duration of anagen is determined by their limited proliferative potential. However, we show here that keratinocytes derived from the GE of ovine follicles grow extensively in vitro, arguing against this hypothesis. MATERIALS AND METHODS: Primary cultures of keratinocytes were initiated from microdissected GE tissue from ovine vibrissae and wool follicles. Clonal lines of keratinocytes were derived by limiting dilution. Their growth potential was determined by exhaustive serial passaging. Expression of differentiation markers was evaluated by real-time polymerase chain reaction. RESULTS: Initiation of these cultures required that interaction between the GE and dermal papilla was maintained. However, the keratinocytes could subsequently be cloned and were grown as pure cell populations for 26-52 cell doublings. This proliferative potential is several orders of magnitude greater than required to maintain a single anagen phase. The keratinocytes were indistinguishable from ORS keratinocytes from the same follicles, expressing K14 while undifferentiated, and upregulating the epidermal and inner root sheath markers, loricrin and KRT27 on differentiation. Thus, these cells initially depend on papilla-derived signals to grow, but can revert to an ORS-like phenotype in vitro. Their extensive proliferative capacity shows that the GE is not an exclusively transit-amplifying cell population.

Annotation of sheep keratin intermediate filament genes and their patterns of expression.

Keratin IF (KRT) and keratin-associated protein genes encode the majority of wool and hair proteins. We have identified cDNA sequences representing nine novel sheep KRT genes, increasing the known active genes from eight to 17, a number comparable to that in the human. However, the absence of KRT37 in the type I family and the discovery of type II KRT87 in sheep exemplify species-specific compositional differences in hair KRT genes. Phylogenetic analysis of hair KRT genes within type I and type II families in the sheep, cattle and human genomes revealed a high degree of consistency in their sequence conservation and grouping. However, there were differences in the fibre compartmentalisation and keratinisation zones for the expression of six ovine KRT genes compared with their human orthologs. Transcripts of three genes (KRT40, KRT82 and KRT84) were only present in the fibre cuticle. KRT32, KRT35 and KRT85 were expressed in both the cuticle and the fibre cortex. The remaining 11 genes (KRT31, KRT33A, KRT33B, KRT34, KRT36, KRT38-39, KRT81, KRT83 and KRT86-87) were expressed only in the cortex. Species-specific differences in the expressed keratin gene sets, their relative expression levels and compartmentalisation are discussed in the context of their underlying roles in wool and hair developmental programmes and the distinctive characteristics of the fibres produced.

Expression patterns of keratin intermediate filament and keratin associated protein genes in wool follicles.

The catalogue of hair keratin intermediate filaments (KIFs) and keratin-associated proteins (KAPs) present in wool follicles is incomplete. The full coding sequences for three novel sheep KIFs (KRT27, KRT35 and KRT38) and one KAP (KRTAP4-3) were established in this study. Spatial expression patterns of these and other genes (KRT31, KRT85, KRTAP6-1 and trichohyalin) were determined by in situ hybridisation in wool follicles at synchronised stages of growth. Transcription proceeded in the order: trichohyalin, KRT27, KRT85, KRT35, KRT31, KRT38, KRTAP6-1 and KRTAP4-3, as determined by increasing distance of their expression zones from the germinal matrix in anagen follicles. Expression became gradually more restricted to the lower follicle during follicle regression (catagen), and ceased during dormancy (telogen). Some genes (KRT27, KRT31, KRT85 and KRTAP6-1), but not others, were expressed in cortical cells forming the brush-end, indicating specific requirements for the formation of this anchoring structure. The resumption of keratin expression was observed only in later stages of follicle reactivation (proanagen). KIF expression patterns in primary wool follicles showed general resemblance to their human homologues but with some unique features. Consistent differences in localisation between primary and secondary wool follicles were observed. Asymmetrical expression of KRT27, KRT31, KRT35, KRT85 and trichohyalin genes in secondary follicles were associated with bulb deflection and follicle curvature, suggesting a role in the determination of follicle and fibre morphology.

Prolactin and its receptor are expressed in murine hair follicle epithelium, show hair cycle-dependent expression, and induce catagen.

Here, we provide the first study of prolactin (PRL) and prolactin receptor (PRLR) expression during the nonseasonal murine hair cycle, which is, in contrast to sheep, comparable with the human scalp and report that both PRL and PRLR are stringently restricted to the hair follicle epithelium and are strongly hair cycle-dependent. In addition we show that PRL exerts functional effects on anagen hair follicles in murine skin organ culture by down-regulation of proliferation in follicular keratinocytes. In telogen follicles, PRL-like immunoreactivity was detected in outer root sheath (ORS) keratinocytes. During early anagen (III to IV), the developing inner root sheath (IRS) and the surrounding ORS were positive for PRL. In later anagen stages, PRL could be detected in the proximal IRS and the inner layer of the ORS. The regressing (catagen) follicle showed a strong expression of PRL in the proximal ORS. In early anagen, PRLR immunoreactivity occurred in the distal part of the ORS around the developing IRS, and subsequently to a restricted area of the more distal ORS during later anagen stages and during early catagen. The dermal papilla (DP) stayed negative for both PRL and PRLR throughout the cycle. Telogen follicles showed only a very weak PRLR staining of ORS keratinocytes. The long-form PRLR transcript was shown by real-time polymerase chain reaction to be transiently down-regulated during early anagen, whereas PRL transcripts were up-regulated during mid anagen. Addition of PRL (400 ng/ml) to anagen hair follicles in murine skin organ culture for 72 hours induced premature catagen development in vitro along with a decline in the number of proliferating hair bulb keratinocytes. These data support the intriguing concept that PRL is generated locally in the hair follicle epithelium and acts directly in an autocrine or paracrine manner to modulate the hair cycle.