No abstract

Cells from the dermal papilla and dermal sheath of hair follicles exhibit pronounced plasticity in vitro, being capable of adopting fat, bone, hematopoietic, and nerve cell phenotypes. In this study, we show that bovine dermal papilla cells (DPC) are also capable of undergoing skeletal muscle differentiation. DiI labeled DPC incorporated into myotubes when co-cultured with differentiating C(2)C(12) myoblasts. Bovine-specific PCR assays showed that the muscle markers MyoD and myogenin were up-regulated, confirming that the DPC had adopted a myogenic gene expression program. Nine clonal lines of DPC underwent both adipogenic and myogenic differentiation, demonstrating the multipotency of individual cells. Primary populations of both DPC and extra-follicular dermal fibroblasts were also capable of both adipogenic and myogenic differentiation. However, on myogenic differentiation, cells derived from dermal papillae expressed higher levels of myogenin than primary fibroblasts derived from extra-follicular dermis, suggesting that papilla cells undergo myogenesis more efficiently. This result shows that populations of fibroblastic cells derived from different anatomical sites within the skin are not equivalent with respect to their plasticity. Cultured DPC and dermal fibroblasts both expressed Pax3, a marker for the dermomyotome which represents a common embryological origin of muscle and dermis. Quantitative PCR showed that Pax3 expression levels before myogenic induction correlated with myogenin expression levels after myogenesis. These results suggest that a degree of dedifferentiation may underlie the plasticity of dermal cells in vitro, and that this plasticity may be predicted, at least in part, by levels of Pax3 expression.

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.