Murine Vibrissae Cultured in Serum-Free Medium Reinitiate Anagen

Aberrant regulation of the hair cycle has been implicated in human boldness (Reviewed in (Nakamura et al, 2001; Paus and Cotsarelis, 1999)). Although several modulators of mammalian hair follicle cycle have been recently described, discovery of effective treatment for boldness has suffered from the absence of a reliable in vitro culture systems in which the hair cycle can be assessed easily and inexpensively (Stenn and Paus, 2001). Several laboratories have established serum-free culture system where vibrissa can grow and differentiate in vitro (Supplemental Table 1, (Jindo et al, 1993; Robinson et al, 1997; Yano et al, 2001). Although rat vibrissae cultured for 23 days were reported to share histological similarities with catagen or pro-anagen (telogen) stage follicles (Philpott and Kealey, 2000), these follicles did not show any progress beyond pro-anagen phase nor did they produce a new hair shaft (Philpott and Kealey, 2000). In this report, we demonstrate that a simple modification permits murine vibrissae in the current in vitro culture system to reinitiate anagen. This will accelerate the development of screening systems aimed at modifying the hair cycle. To establish a modified in vitro vibrissa culture system, anagen-stage vibrissae were carefully isolated from 14-day-old mice and the tip of the vibrissa shafts were anchored in a stripe of sterilized silicone grease placed on the culture dish through a 3 ml syringe. The plate was then filled with serum-free medium (Figure 1a, Supplemental Material and Method). Out of 86 cultured vibrissae collected from three pups, 81 vibrissae (94%) showed measurable shaft growth (Table 1, Figure 1b). Two of 81 vibrissae were lost, and 17 developed abnormal (kinked) fiber and were omitted from growth measurement. Of the remaining 62 vibrissae, straight shafts were produced by all at a rate of 0.3–0.5mm/day for the first three days. While some follicles maintained this growth rate through the fifth day of culture (Figure 1b and 1c and data not shown), others began a gradual decline in growth rate (Figure 1b and 1c, ​,2a2a and data not shown). As previously reported (Jindo et al, 1993; Robinson et al, 1997), growth rates of all follicles slowed down considerably after 5 days in culture, indicating independence from culture conditions (Supplemental Table 1). Figure 1 Vibrissae produce hair shafts and reinitiate anagen in serum-free culture medium Figure 2 Histological and immunohistochemical characteristics of new anagen follicles Table 1 Summary of the in vitro vibrissa culture Hair shaft elongation rate decreases and eventually stops when follicles enter catagen/telogen phase in vivo (Alonso and Fuchs, 2006). Several days after cessation of hair shaft growth, cultured vibrissae follicles continuously changed their morphology, at times visible through their collagen capsules (Figure 1d). Strikingly, dissection and removal of collagen capsules from 21-day-cultured vibrissae revealed that all vibrissae produced a second shaft (Figures 1d and 2a–h, arrows; Figure S1), readily distinguishable from the original shaft whose proximal end formed club-end indicative of a completed catagen (Figures 1 and ​and22 closed arrowheads). Histological and immunohistochemical analyses using AE13, a marker for cortex/cuticle specific keratins, confirmed the formation of new hair shafts (Figures 2c–f and S1). The new shafts were produced in proximal bulbs that appeared smaller in size than the original ones before culture. Since the original hair growth stopped within the first 5 days of culture, and since a new shaft emerges from a secondary bulb, we concluded that vibrissa follicles from CD1 mice reinitiated anagen phase by regenerating hair bulbs (Figure 2). Staining for alkaline phosphatase activity identified an intact (but small) dermal papilla within the new bulb (Figure 1d, open arrowhead; Figure 2g, alkaline phosphatase), as seen in the early anagen phase of vibrissae in vivo (Oshima et al, 2001; Young and Oliver, 1976). Moreover, the hair matrix cells were positive for Ki-67, a proliferation marker, even after 13 days in culture (Figure 2h), indicating that the formation of new shaft was the result of active proliferation and differentiation of matrix cells in cultured vibrissae. Similar cycling patterns were also observed with vibrissae from 14-day-old C57BL/6 mice (Figure S2), indicating that hair cycle in vitro is independent of mouse strains. To better visualize the hair cycle process, we next performed vibrissae culture after careful removal of their collagen capsules (Figure 2i, Supplemental Material and Method). Within 2 days, the proximal end of the vibrissa keratinized, forming a club hair, at which time dermal papilla structures were no longer discernable (Figure 2i and data not shown). A day later, however, a bud-like structure emerged from the proximal part of the bulb in eight of ten cultured vibrissae. Of these, one vibrissa continued growing outwards, eventually forming a new bulb producing a vibrissa thinner than the club hair (Figure 2i). Strikingly, we observed that this vibrissa appeared to enter a third anagen, producing a new bud from the second bulb (Figure 2i, open arrow). Collectively, the data presented here indicate that in our in vitro culture system, vibrissa follicles enter a second anagen phase and it may be possible for a few to enter a third cycle in culture. We are not certain which modification in our culture system permits vibrissae to cycle. We do not think this is caused by differences in media composition, mouse strain, or frequency of media change. Our attempt to grow vibrissae at the air-fluid interface as in (Jindo et al, 1993) showed that one of five vibrissae formed a new hair shaft (Figure S3, Arrows), indicating that hair cycle is not strictly dependent on culture media or on the frequency of media change. However, we observed that on a solid substrate most vibrissae gradually lost their hair matrix possibly due to a pressure caused by friction generated while extending a hair shaft on a solid surface or in the absence of sebum (Sundberg et al, 2000) (Figure S3, Arrowheads). Although not proven, we speculate that the critical modification was our method of immobilizing the hair shaft in silicon grease that provided friction-free environment; anchoring the tip of shafts may have allowed gravity to facilitate the separation of club hairs from the hair matrix and that supports new hair shaft formation. In addition, the smaller size of mouse vibrissae, in comparison with human or rat vibrissae, could also be a contributing factor in the anagen reinitiation in vitro As this culture system is the first to support reinitiation of anagen in culture dish, we hope this improvement will expedite the testing and development of hair cycle modifying pharmaceuticals.