Isolation of Mouse Hair Follicle Bulge Stem Cells and Their Functional Analysis in a Reconstitution Assay

Abstract

The hair follicle (HF) is a dynamic structure readily accessible within the skin, and contains various pools of stem cells that have a broad regenerative potential during normal homeostasis and in response to injury. Recent discoveries demonstrating the multipotent capabilities of hair follicle stem cells and the easy access to skin tissue make the HF an attractive source for isolating stem cells and their subsequent application in tissue engineering and regenerative medicine. Here, we describe the isolation and purification of hair follicle bulge stem cells from mouse skin, and hair reconstitution assays that allows the functional analysis of multipotent stem cells.

Keywords: Bulge; Hair follicle stem cell; Hair reconstitution assay; Regeneration.

Fig. 1

FACS analysis and reconstitution assays for hair-follicle stem cells. (a) FACS analysis revealing the presence of bulge stem cells (CD34⁺CD49f⁺, approximately 8 % of the total input cells) among the keratinocytes isolated from the back skin of a 2-month-old mouse. (b) Patch-assay skin sample showing dermal view of skin harvested from a nude mouse which received multiple injections of a mixed suspension of dermal and epidermal cells. The boxed area shows pigmented de novo hair follicles reconstituted from injected cells. (c) An enlarged view of a representative injected site similar to the one in the boxed area in (b). As many as several hundred hair follicles with hair shafts can be seen at one injection site. (d–g) The dimensions of the silicone chamber used in the chamber assay. The chamber assembly consists of two parts, a dome and a base onto which the dome snaps. The rulers have metric units, with the smallest division representing 1 mm. (d) Top view of the dome (left) and the base (right). The arrowhead indicates the small hole on top of the dome, through which cell slurry is pipetted into the chamber. (e) Side view of the dome. (f) Side view of the base. (g) Schematic representations of the chamber, with the dimensions indicated in cm. The right illustrates the assembled chamber in use. The thickness of the chamber is uniformly 0.8 mm. (h–j) Chamber assays in nude mice. (h) Excision of the back skin on a nude mouse to prepare for the insertion of the chamber. (i) The inserted chamber is securely anchored to the skin with clips. (j) Hairs growing out of the site where the chamber was implanted

Fig. 2

Flow chart of mouse neonatal dermal-cell isolation. Details are described in the procedures for neonatal dermal cell isolation. S (A): supernatant A; P (A): pellet A; S (B): supernatant B; P (B): pellet B; S (C): supernatant C; P (C): pellet C; S (D): supernatant D; P (D): pellet D; S (E): supernatant E; P (E): pellet E. Asterisk denotes fractions to be discarded