We previously demonstrated that hair follicle germ (HFG)-like aggregates consisting of epithelial and mesenchymal cells efficiently generated de novo hair follicles when transplanted into the skin [1]. However, a challenge of this approach was to control the density and orientation of de novo hair shafts. Our recent study showed that HFGs sprouted hair shafts in vitro in long term culture[2]. This study proposes an approach to further grow the hair shafts to several millimeters in length that can be transplanted into the skin similar to surgical hair transplantation. Using a mouse model, we demonstrated this approach is useful for controlling the density and orientation of regenerated hair shafts.

　Epithelial and mesenchymal cells were isolated from the skin of mice. These cells were suspended in culture medium supplemented with 2% Matrigel, and seeded into U-shaped 96 microwells. Interestingly, they formed a core-shell shape, consisting of an epithelial core surrounded by a mesenchymal shell. After 2 days of culture, the aggregates were encapsulated in 100% Matrigel to induce longer and straight hair shafts. The length of the hair shafts reached over 1.0 mm at 14 days. The immunostaining analysis of the hair follicle at 14 days revealed the formation of the hair follicle-like morphology with tissue-specific marker expression. Single hair follicles were isolated from the organoids at day 14, which were then embedded in 100% Matrigel. In following culture, the hair shafts further elongated to be 2.5 mm in length at day 20. The hair follicles were transplanted into the skin of nude mice. The transplanted hair follicles stably remained in the skin and repeated hair cycles for at least 5 months. Notably, hair shaft orientation was controllable by the direction at transplantation. These results demonstrated our approach is promising for highly oriented hair regeneration.