1. ¹Department of Molecular Biology and Pharmacology, Washington University School of Medicine, Saint Louis, Missouri, USA
2. ²Division of Dermatology, Department of Medicine, Washington University School of Medicine, Saint Louis, Missouri, USA
3. ³Synaptic Transmission Unit, National Institute of Neurological Disorders and Stroke, NINDS, National Institutes of Health, Bethesda, Maryland, USA

Correspondence: Raphael Kopan, E-mail: kopan@wustl.edu

⁴Current Address: Department of Developmental Biology, Stanford University School of Medicine, 279 Campus Drive, Stanford, California 94305-5329, USA

TO THE EDITOR

Aberrant regulation of the hair cycle has been implicated in human baldness (reviewed in Paus and Cotsarelis, 1999; Nakamura et al., 2001). Although several modulators of mammalian hair follicle cycle have been recently described, discovery of effective treatment for baldness has suffered from the absence of a reliable in vitro culture system in which the hair cycle can be assessed easily and inexpensively (Stenn and Paus, 2001). Several laboratories have established serum-free culture systems where vibrissa can grow and differentiate in vitro (Table S1; Jindo et al., 1993; Robinson et al., 1997; Yano et al., 2001). Although rat vibrissae cultured for 23 days were reported to share histologic similarities with catagen or pro-anagen (telogen) stage follicles (Philpott and Kealey, 2000), these follicles did not show any progress beyond the pro-anagen phase nor did they produce a new hair shaft (Philpott and Kealey, 2000). In this study, 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 tips 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 and Supplementary Materials and Methods). Out of the 86 cultured vibrissae collected from three pups, 81 vibrissae (94%) showed measurable shaft growth (Table 1 and Figure 1b). Two of the 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 0.3–0.5 mm/day for the first 3 days. While some follicles maintained this growth rate through the fifth day of culture (Figure 1b and c and data not shown), others began a gradual decline in growth rate (Figure 1b and c and 2a and data not shown). As reported previously (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 (Table S1).

Figure 1.

Vibrissae produce hair shafts and reinitiate anagen in serum-free culture medium. (a) Schematic diagram of the in vitro vibrissa culture procedure. The culture media; Williams E medium (Invitrogen, Carlsbad, CA) supplemented with 2 mM L-glutamine, insulin (10 g/ml, Invitrogen), hydrocortisone (10 ng/ml, Sigma-Aldrich, St Louis, MO), penicillin (100 U/ml), and streptomycin (100 ug/ml, Invitrogen). In addition, tips of the harvested vibrissae were immobilized in silicone grease on culture dish. (b) Growth curve of the vibrissal shafts over time. Error bars represent SD. (c) Representative pictures of a vibrissal shaft growth cultured for 15 days. Pictures were taken on 1, 2, 3, 5, 8, 13, and 15 days after culture. Note that some vibrissae show growth retardation 3 days after culture, as shown in Figure 2a. Bar=1 mm. (d) Representative vibrissa grown in culture exhibit an emergence of new shaft (arrows) and bulb (open arrowhead). New shafts were clearly distinguished from the original shafts which become club hairs (arrowheads). Bar=300 m.

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Figure 2.

Histological and immunohistochemical characteristics of new anagen follicles. (a) Representative pictures of a vibrissa cultured for 13 days. Pictures were taken on 0, 1, 2, 3, 5, 7, 9, 11, and 13 days after culture. (b) Magnified view of the 13-day cultured vibrissa shown in (a). Note the shape of bulb and dermal papilla. (c) Hematoxylin and eosin staining of the sections from (b). Note that both new hair shaft (arrow) and the club hair (arrowhead) are shown. (d) AE13 staining (green) on the adjacent section of (c). (e and f) Magnified views of the area marked by green and red box shown in (d). Note both club hair (e) and new hair shaft (f) are AE13-positive. (g) The presence of dermal papilla was confirmed by alkaline phosphatase substrate staining (purple) in adjacent section of the same vibrissa. (h) Ki-67 staining (green) on the adjacent section revealed the presence of proliferating matrix cells in the vibrissal bulb. (i–n) Pictures were taken on 1 (i), 2 (j), 3 (k), 7 (l), 14 (m), and 18 (n) days after culture of a vibrissa without collagen capsule. Inset in (n) is a magnified view. Note that a bud grew from the proximal part of the follicle to form a hair bulb that produces a new hair shaft (arrow in (m)) distinguished from a club hair (arrowheads in (l) and (m)). This follicle also shows a possible second bud formation (open arrowhead in (n)). Bars=1 mm (a); 300 m (b and i–n); 50 m (e–h). See serial sections of follicle (c) in Figure S1.

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Table 1 - Summary of the in vitro vibrissa culture.

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Hair shaft elongation rate decreases and eventually stops when follicles enter the 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-hair, indicative of a completed catagen (Figures 1 and 2 closed arrowheads). Histological and immunohistochemical analyses using AE13, a marker for cortex/cuticle-specific keratins, confirmed the formation of new hair shafts (Figure 2c–f and Figure 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 emerged from a secondary bulb, we concluded that vibrissa follicles from CD1 mice reinitiated the 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 (Young and Oliver, 1976; Oshima et al., 2001). 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 a 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 visualize better the hair cycle process, we next performed vibrissae culture after careful removal of their collagen capsules (Figure 2i–n, Supplementary Materials and Methods). Within 2 days, the proximal end of the vibrissa keratinized, forming a club hair, at that time when dermal papilla structures were no longer discernible (Figure 2j and data not shown). A day later, however, a bud-like structure emerged from the proximal part of the bulb in eight of the 10 cultured vibrissae. Of these, one vibrissa continued growing outwards, eventually forming a new bulb producing a vibrissa thinner than the club hair (Figure 2m and n). Strikingly, we observed that this vibrissa appeared to enter a third anagen, producing a new bud from the second bulb (Figure 2n, open arrowhead). 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 that 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 the 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 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 a 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 a culture dish, we hope this improvement will expedite the testing and development of hair cycle modifying pharmaceuticals.