We have succeeded in culturing dermal papilla (DP) cells long term and developed new techniques that enhance their hair follicle-inducing efficiency in a patch assay. The outgrowing DP cells from mouse vibrissae were markedly stimulated by 10% fetal bovine serum-Dulbecco's modified essential medium that included fibroblast growth factor-2 (FGF-2). Moreover, the potency of proliferation was maintained during serial cultivations (more than 30 passages). We combined these established DP cells with epidermal cells and implanted them subcutaneously into athymic mice to examine their hair follicle-inducing ability. New hair follicles were induced by dissociated DP cells at earlier passages (under passage 4), but the cells from later passages could not induce follicles. We next aggregated the DP cells to form spheres and then injected them with epidermal cells. Unlike the dissociated DP cells, the spheres made from the later passaged cells (more than 10 passages) did induce new hair follicles. We examined several genes specific for DP of anagen follicles and confirmed that their expression level was elevated in the spheres compared with their expression level in adherent DP cells. These results suggest that FGF-2 is essential for dermal papilla cell culture and that sphere formation partially models the intact DP, resulting in hair follicle induction, even by later passaged cells.

Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase that activates the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase signaling cascades. We report here that expression of constitutively active ASK1 (ASK1⌬N) induces neurite outgrowth in the rat pheochromocytoma cell line PC12. We found that p38 and to a lesser extent JNK, but not ERK, were activated by the expression of ASK1⌬N in PC12 cells. ASK1⌬N-induced neurite outgrowth was strongly inhibited by treatment with the p38 inhibitor SB203580 but not with the MEK inhibitors, suggesting that activation of p38, rather than of ERK, is required for the neurite-inducing activity of ASK1 in PC12 cells. We also observed that ASK1⌬N induced expression of several neuron-specific proteins and phosphorylation of neurofilament proteins, confirming that PC12 cells differentiated into mature neuronal cells by ASK1. Moreover, ASK1⌬N-expressing PC12 cells survived in serumstarved condition. ASK1 thus appears to mediate signals leading to both differentiation and survival of PC12 cells. Together with previous reports indicating that ASK1 functions as a pro-apoptotic signaling intermediate, these results suggest that ASK1 has a broad range of biological activities depending on cell types and/or cellular context.

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Hair is a mini-organ in which dermal papilla (DP) cells play important roles in hair follicle morphogenesis and formation via interactions with epithelial cells. DP cells have previously been difficult to analyze because of the lack of a specific surface marker. We have demonstrated that prominin-1/CD133 (CD133) is a useful marker for murine DP cells. DP cells express CD133 during the early anagen stage (active growth phase) not only during hair morphogenesis, but also during the growth phase of hairs after birth. Gene expression and flow cytometric analysis revealed that CD133-positive (+) cells in the skin possess the characteristics of DP cells. The CD133(+) cells isolated from embryonic or adult skin-induced new hair follicles in vivo when they were transplanted into nude mice mixed with embryonic epithelial cells, but CD133-negative (-) cells could not. We propose that the CD133 is a novel surface marker useful for collecting DP cells in the anagen stage and for analyzing the function of DP.