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Figure 1: Methods for hair follicle regeneration.
We first investigated whether bioengineered hair follicle germs, reconstituted by our previously developed organ germ method, could orthotopically erupt from a hair shaft with the proper tissue structure and skin connections in the cutaneous environment of an adult mouse (Fig. 1a). To regenerate a bioengineered pelage follicle germ, the bioengineered pelage follicle germ was reconstituted with E18 mouse embryonic back skin-derived epithelial and mesenchymal cells (Fig. 1b). The bioengineered vibrissa follicle germ was regenerated using dissociated epithelial cells (1×10[SUP]4[/SUP] cells) isolated from the adult vibrissa-derived bulge region, which expressed CD49f and CD34 antigens (Fig. 1c), and primary cultured DP cells (3×10[SUP]3[/SUP] cells; Fig. 1d).
Figure 2: Hair follicle regeneration via the intracutaneous transplantation of a bioengineered hair follicle germ.
Both the bioengineered pelage follicle and the vibrissa follicle formed correct structures comprising an infundibulum and sebaceous gland in the proximal region as well as a hair matrix, hair shaft, inner root sheath, outer root sheath (ORS), DS and DP (Fig. 2a). Each natural and bioengineered vibrissa follicle contained 500-1,000 DP cells (Fig. 2a). The bioengineered vibrissa follicle germs generated not only hair follicles in the variable region but also infundibulum and sebaceous gland structures in the permanent region, and in contrast to the bioengineered pelage follicle, the follicle-derived cells did not distribute among the surrounding cutaneous tissues (Fig. 2a). DP cells in the bioengineered vibrissa follicle were also found to express alkaline phosphatase (ALP) and versican, and the thin outermost dermal layer of the hair bulb was observed to express α-smooth muscle actin (α-SMA; Fig. 2a). These results indicate that the bioengineered pelage and vibrissa follicle germs can regenerate structurally correct follicles and hair shafts following intracutaneous transplantation.
Figure 3: Control of the number and features of the bioengineered hairs.
We next analysed the formation of pelage follicles from the bioengineered follicle germ (Fig. 3a). These analyses demonstrated that the bioengineered pelage follicles, which contain EGFP-labelled mouse-derived mesenchymal cells, could be induced to form autonomously assembled cell aggregates with high-intensity green fluorescence and alkaline phosphatase expression at the boundary between epidermal and mesenchymal cell layers after 2 days in an organ culture (Fig. 3a). After 3 days in an SRC, the bioengineered pelage follicle germ faithfully reproduced the expression of required signalling network genes, such as Shh, Wnt10b, Msx2, β-catenin, Versican, Lef1, Bmp4 and Notch1, which have essential roles in early hair follicle development (Fig. 3a,b). After the isolation of the bioengineered pelage follicle germ with different numbers of condensed dermal cell aggregates, we found that the number of bioengineered pelage follicles was linearly correlated with that of the condensed dermal cell aggregates (Fig. 3a). These results also indicated that the condensed dermal cells and surrounding epithelial cells recapitulated hair follicle morphogenesis during embryonic organogenesis. The outermost layer of the natural and bioengineered pelage-type hair shafts were surrounded by a thin cuticle comprising two to four layers (Fig. 3e). The bioengineered vibrissae, which had a six-layer-thick cuticle, were similar to natural vibrissae (Fig. 3e). Melanin granules were found in the pigmented natural hairs. However, the unpigmented bioengineered vibrissa hair had no detectable melanin granules (Fig. 3e). These results indicate that our bioengineering method for hair follicle regeneration can reproduce all hair types with correct hair structures in accordance with the fate of their follicle, which is determined during embryonic development.
Figure 4: Rearrangement of follicular stem cells and their niches in the bioengineered pelage and vibrissa follicles. (a) Immunohistochemical analysis of the bulge region in a bioengineered pelage (upper) and vibrissa (lower) follicle.
Hair shaft pigmentation is provided by neural crest-derived melanocyte progenitor cells associated with the hair follicle pigmentary unit in the sub-bulge region of vibrissa hair follicles at anagen phases. The white hair shafts of the bioengineered vibrissae could be caused by the loss of neural crest-derived melanocyte stem/progenitor cells (Figs 1c, d, f and 4c). The addition of cells from the sub-bulge (SB) region (Fig. 1d), which contains melanoblasts in the ORS, caused the bioengineered vibrissae to be pigmented with a black colour at a frequency of 68.3% (n=26; Fig. 4c,d) at 3 weeks after transplantation. The shape, however, was normal with a frequency of 27.0% (n=17; Fig. 4c,d). In contrast, the bioengineered vibrissae derived from regenerated follicles with proximal hair matrix (PHM) region-derived cells (Fig. 2c), which contain immature melanocytes that are the progeny of melanoblasts, were pigmented black with a frequency of 40.1% (n=19; Fig. 4c,d) and had the structural properties of natural vibrissae, as assessed by electron microscopy (83.3%, n=6; Fig. 4c,d). The fine structure of the bioengineered vibrissa harbouring PHM region cells was similar to that of natural vibrissae, but the hair that was bioengineered with SB region cells had an abnormal shape with regard to the structure of the cuticle layer and the cortex region (Fig. 4c). Dopachrome tautomerase (Dct) messenger RNA-positive cells, which are neural crest-derived melanocyte stem/progenitor cells, were also detected in the SB region of pigmented bioengineered pelage and vibrissa follicles, but not the unpigmented bioengineered vibrissa follicle, by in situ hybridization (n=10, Fig. 4e). These results indicated that various follicle stem cells and their niches were successfully rearranged in the bioengineered pelage and vibrissa follicles using appropriate epithelial and mesenchymal cell populations.
Figure 5: Hair cycle analyses of bioengineered hair follicles.
We further analysed the hair cycles, that is, the alternative growth (hair-growing) and regression (non-growing) phases (Fig. 5a), of the bioengineered pelage and vibrissa shafts that had erupted from bioengineered follicles for 80 days (Fig. 5b,c). The bioengineered pelage and vibrissa follicles repeated the hair cycle at least 3 times during the 80-day period (Fig. 5b,c). For natural pelage follicles transplanted into the back skin of nude mice, the average growth and regression periods were 10.7±2.0 and 10.5±1.6 days, respectively (Fig. 5c,d). For the bioengineered pelage follicles, these periods were 9.3±2.5 and 12.4±2.8 days, as calculated from 3 hair cycle periods (Fig. 5c,d). The growth and regression phases of the natural vibrissa follicles were 10.6±3.4 and 5.5±3.9 days, respectively, and those of the bioengineered vibrissa follicles were 10.7±4.3 and 5.4±3.3 days, respectively (Fig. 5c,d). No significant differences in the hair cycle periods were found between the natural and bioengineered follicles (both pelage and vibrissae; Fig. 5d).
Figure 6: Analyses of the connections to surrounding tissues and the piloerection capability of bioengineered hair follicles.
To achieve functional hair follicle regeneration, it is essential that the engrafted follicle be able to connect to the arrector pili muscle and nerve, and that the follicle has a piloerection capability. The possibility of reconstituting the niche for arrector pili muscle development, in which the cells express NPNT, was demonstrated in the bulge region of the bioengineered pelage (Fig. 4a). The bulge region of a natural pelage follicle is connected to the arrector pili muscle, which is composed of calponin-positive smooth muscle, but not troponin-positive striated muscle, and is innervated through the development of neuromuscular junctions, which surround the arrector pili muscle with sympathetic nerve fibres that extend from a deep dermal nerve plexus (Fig. 6a). All bioengineered pelage follicles were observed to connect to the calponin-positive arrector pili muscle, but not the troponin-positive striated muscle, and to connect to the nerve fibres at the bulge region. These connections were also observed in the natural pelage follicle (n=10; Fig. 6a). The bioengineered vibrissa follicles were connected to the troponin-positive striated muscle cells at the hair bulb regions but not to calponin-positive smooth muscle, in any other areas, which is similar to the structure of the natural vibrissa follicle (Fig. 6a). The bioengineered vibrissa follicles were also connected to nerve fibres, and they formed neuron-follicular junctions in an ORS layer of the bulge region (Fig. 6a).These results indicate that the bioengineered hair follicles have a piloerection ability that is comparable to that of natural follicles. These findings indicated that our bioengineered follicles could induce selective connections with the appropriate types of muscles and nerve fibres, and they showed that these follicles were able to achieve piloerection through the rearrangement of stem cells and their niches
Figure 7: Model of the autonomous connections of the bioengineered hair follicles with surrounding tissues in adult skin.
The bioengineered pelage and vibrissa follicles were connected with other tissues such as nerve fibres, arrector pili muscles and striated muscle, which were derived from host and/or donor cells. The bioengineered pelage follicle autonomously connected with smooth muscle as a result of the reproduction of the NPNT-expressing bulge region in a manner similar to that of the natural pelage. Neither NPNT expression nor smooth muscle connection was detected in bulge regions of the natural and bioengineered vibrissa follicles.
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In the developing embryo, hair follicle morphogenesis is regulated by reciprocal epithelial and mesenchymal interactions that occur in almost all organs. The hair follicle is divided into a permanent upper region, which consists of the infundibulum and isthmus, and a variable lower region, which is the actual hair-shaft factory that contains the hair matrix, differentiated epithelial cells and dermal papilla (DP) cells. DP cells are responsible for the production of dermal-cell populations such as dermal sheath (DS) cells, and they generate dermal fibroblasts and adipocytes. After morphogenesis, various stem cell types are maintained in certain regions of the follicle. For instance, follicle epithelial cells are found in the follicle stem cell niche of the bulge region; multipotent mesenchymal precursors are found in DP cells; neural crest-derived melanocyte progenitors are located in the sub-bulge region, and follicle epithelial stem cells in the bulge region that is connected to the arrector pili muscle. The follicle variable region mediates the hair cycle, which depends on the activation of follicle epithelial stem cells in the bulge stem cell niche during the telogen-to-anagen transition. This transition includes phases of growth (anagen), apoptosis-driven regression (catagen) and relative quiescence (telogen), whereas the organogenesis of most organs is induced only once during embryogenesis.
In this study, we successfully demonstrate fully functional bioengineered hair follicle regeneration that produces follicles that can repeat the hair cycle, connect properly with surrounding skin tissues and achieve piloerection. This regeneration occurs through the rearrangement of various follicular stem cells and their niches. These findings significantly advance the technological development of bioengineered hair follicle regenerative therapy.
In conclusion, this study provides novel evidence of fully functional hair follicle regeneration through the rearrangement of various stem cells and their niches in bioengineered hair follicles. Our study provides a substantial contribution to the development of bioengineering technologies that will enable future regenerative therapy for hair loss caused by injury or by diseases such as alopecia and androgenic alopecia. Further studies on the optimization of human hair follicle-derived stem cell sources for clinical applications and further investigations of stem cell niches will contribute to the development of hair regenerative therapy as a prominent class of organ replacement regenerative therapy in the future
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