Murine Vibrissae Cultured in Serum-Free Medium Reinitiate Anagen Jonghyeob Lee, Wei Wu, Raphael Kopan  Journal of Investigative Dermatology  Volume 128, Issue 2, Pages 482-485 (February 2008) DOI: 10.1038/sj.jid.5701024 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

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 2mM L-glutamine, insulin (10g/ml, Invitrogen), hydrocortisone (10ng/ml, Sigma-Aldrich, St Louis, MO), penicillin (100U/ml), and streptomycin (100ug/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=1mm. (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=300μm. Journal of Investigative Dermatology 2008 128, 482-485DOI: (10.1038/sj.jid.5701024) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

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=1mm (a); 300μm (b and i–n); 50μm (e–h). See serial sections of follicle (c) in Figure S1. Journal of Investigative Dermatology 2008 128, 482-485DOI: (10.1038/sj.jid.5701024) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions