Hair follicle size is determined early in development by a patterning process that leads to dermal cell condensation and the formation of dermal papillae (DP). Similar events are repeated during adult follicle cycles, and are thought to be disrupted in human alopecia. We have observed that DP cells from sheep exhibit stable aggregation in culture, forming numerous, three-dimensional, papilla-like structures. We have optimised culture conditions for these cells to establish an experimental model for the investigation of molecular mechanisms regulating dermal papilla size.
Cells were grown from explants of intact DP that were dissected from the wool follicles of lambs. We determined the effects on aggregation of extensive sub-culture, different sera, and substrates coated with fibronectin, laminin, or type I collagen. Lithium chloride (a Wnt agonist and inhibitor of Src kinases and inositol phospholipid signalling) was added to the culture medium at 5-40 mM. Aggregate size was measured by edge detection using a custom image analysis program. The hair-inducing competence of aggregated cells was tested in vivo.
Cells could be propagated for approximately 90 cell doublings before senescing. Robust aggregation was maintained throughout, both for cells that were allowed to aggregate before each passage and cells that were passaged when sub-confluent. Better aggregation was achieved in the presence of 20% lamb serum than foetal calf serum. Lithium chloride at 10-30 mM induced a dose-dependent reduction in aggregate size. Aggregate size was most responsive to lithium chloride on the collagen I coated substrate. Ovine DP cells induced follicle neogenesis when grafted into glabrous rat skin.
The inductive and morphogenetic behaviour of ovine DP cells appears to be particularly stable ex vivo, compared with equivalent cells from other species. They therefore provide a useful new experimental model for investigating the size-determination mechanisms which underlie follicle morphogenesis and hair growth disorders.