C Roh, Q. Tao and S. Lyle

Department of Pathology, Beth Israel Deaconess Medical Center, Boton, MA, USA

The epithelial-mesenchymal interactions between keratinocyte stem cells and dermal papilla cells are crucial for normal development of the hair follicle as well as during hair cycling. In order to characterize the events occurring during the process of epithelial stem cell fate determination, we utilized a co-culture system by incubating human hair follicle keratinocyte stem cells with dermal papilla cells. Using Genechip microarrays, we analyzed changes in gene expression profiles within the stem cells upon induction by dermal papilla over a 1, 2 and 5 day time-course. After normalization and filtration, 272 genes were up- or down-regulated by >3-fold. Among up-regulated genes, the hair-specific keratin 6 hair follicle (K6hf) gene increased 7.9 fold, with a resulting 2-3 fold increase in the protein levels. The up-regulation of K6hf was unique to dermal papilla-induced differentiation since expression of K6hf was not induced by increased calcium. We also found that the more committed transit-amplifying cells of the hair matrix are more readily differentiated by the dermal papilla than epithelial stem cells.  For matrix cells the expression of K6hf protein peaked at day 2, while for stem cells it took 3-4 days.  Since the beta-catenin signaling pathway has been implicated in the initiation of hair follicle development, we also examined the status of beta-catenin in the co-culture system. Although no changes are seen in beta-catenin gene expression, there was a significant increase in nuclear localization indicating that beta-catenin-induced gene expression is occurring.

Our results show that the co-culture system recapitulates the epithelial-mesenchymal interactions which induce hair differentiation of the multi-potent keratinocyte stem cells and identify markers of differentiation as well as pathways involved in fate determination.  Our findings also show that K6hf can be used as a marker for the initiation of dermal papilla-induced hair follicle differentiation of keratinocyte stem cells.