Hair follicle germs containing vascular endothelial cells for hair regenerative medicine

Abstract

Hair regenerative medicine has emerged as a promising approach for the treatment of severe hair loss. Recent advances in three-dimensional tissue engineering, such as formation of hair follicle germs (HFGs), have considerably improved hair regeneration after transplantation in animal models. Here, we proposed an approach for fabricating HFGs containing vascular endothelial cells. Epithelial, dermal papilla, and vascular endothelial cells initially formed a single aggregate, which subsequently became a dumbbell-shaped HFG, wherein the vascular endothelial cells localized in the region of dermal papilla cells. The HFGs containing vascular endothelial cells exhibited higher expression of hair morphogenesis-related genes in vitro, along with higher levels of hair shaft regeneration upon transplantation to the dorsal side of nude mice, than those without vascular endothelial cells. The generated hair follicles represented functional characteristics, such as piloerection, as well as morphological characteristics comparable to those of natural hair shafts. This approach may provide a promising strategy for fabricating tissue grafts with higher hair inductivity for hair regenerative medicine.

Figure 1

Preparation of vHFGs through self-organization. Human DP cells, murine fetal skin-derived epithelial cells, and HUVECs were mixed and seeded in a non-cell-adhesive 96-well plate and lab-created HFG chip. In each culture vessel, the cells formed single aggregates in the microwells, wherein the three types of cells were randomly distributed after 1 day of culture and spontaneously formed vHFGs in the following 2 days of culture. vHFGs were then transplanted into the dorsal skin of nude mice to evaluate hair regeneration potential.

Figure 2

Self-organization of cells into vHFGs. (a) Changes in spatial distribution of DP and epithelial cells (1:1 in cell number) in individual aggregates after 1 and 3 days of culture. The DP cells were labeled with fluorescent Vybrant DiI dye before seeding. The fluorescence and phase-contrast images were overlaid. The broken lines indicate the boundary of aggregates. (b) Changes in spatial distribution of DP cells, epithelial cells, and HUVECs (4:4:1 in cell number) in individual aggregates at 1 and 3 days of culture. The RFP-HUVECs were used to distinguish them from others. The fluorescence and phase-contrast images are overlaid. The broken lines indicate the boundary of aggregates. (c) Confocal microscopic images of immunohistochemically stained vHFGs (9 × 10³ cells/HFG). (i) Green, GFP-HUVEC; red, vimentin. (ii) Green, GFP-HUVEC; red, cytokeratin 15 (K15). (d) Effects of Y-27632 treatment on self-organization of the three cell types. Red, DiI-labeled mouse epithelial cells; green, GFP-HUVECs. (e) Putative schematic of cell sorting behaviors in the presence/absence of Y-27632.

Figure 3

vHFG preparation in HFG chip. (a) Fabricated HFG chip. The inset shows a magnified view of vHFGs in microwells. (b) Representative appearance of vHFGs and HFGs after 3 days of culture in the HFG chip. The number of cells seeded in the HFG chip was 1.13 × 10⁶ total cells/chip for vHFG and 1 × 10⁶ total cells/chip for HFG. GFP-HUVECs were visualized, and fluorescence and phase-contrast images were overlaid for vHFG. (c) HE-staining (i) and fluorescence staining (ii, iii) of cross-sections of vHFGs after 3 days of culture in the HFG chip. GFP-HUVECs (green) and nuclei (blue) were visualized. (d) Relative expression of trichogenic genes. GAPDH was used as a reference gene to normalize expression. Error bars represent the standard error of mean calculated from three independent experiments for each condition. Variables were statistically evaluated using Student’s t-test. *p < 0.05 was considered significant.

Figure 4

Hair-patch assay with vHFGs. (a) Hair shafts generated from 30 vHFGs and 30 HFGs in the lateral dorsal skin. (b) Quantification of generated hair shafts. The values and error bars represent at least four experiments for each condition. *p < 0.05 was considered significant. The values and error bars were calculated based on at least five samples.

Figure 5

Intracutaneous transplantation of vHFGs and piloerection assay. (a) Hair shafts generated 3 weeks after transplantation of vHFGs and HFGs into the dorsal murine skin. (b) Scanning electron microscopic images of generated hair shafts. (c) Number of generated hair shafts. Values and error bars were calculated from at least 15 HFG transplantations for each condition in three independent experiments. Numerical variables were statistically evaluated using the Student's t-test. p < 0.1 is represented by †. (d) Piloerection of generated hair shafts after injection of (i) PBS, (ii) acetylcholine, and (iii) acetylcholine plus atropine sulfate.