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.
Hair regeneration methods that rely on the reproduction of epithelial–mesenchymal interactions have been attempted in several previous studies31
. It was also reported that the aggregation of dissociated epithelial and mesenchymal cells induced to form hair follicle self-assemblies during embryonic hair follicle development could be reproduced34
. The replacement of hair-inductive mesenchymal cells in an amputated hair follicle19
or in glabrous skin33
has also been reported as an analogy to the regeneration of the anagen phase in the adult hair cycle. In hair growth after birth, the position of the infundibular opening and hair shaft eruption are determined by the connection between the hair follicle and the interfollicular epidermis, and this position is maintained as a follicle-invariable region, during hair cycling16
. Our bioengineered vibrissa follicle germ that was reconstituted using adult follicle-derived stem cells successfully regenerated the hair follicle following intracutaneous transplantation. Hair shafts also erupted from this bioengineered germ with the proper connections to the surrounding host tissues. Hence, our findings indicate that it is possible to not only restore a hair follicle but also to re-establish successful connections with the recipient skin by intracutaneous transplantation of the bioengineered follicle germ.
Stem cells with organ-inductive potential exist only during embryonic organogenesis and are maintained as tissue stem cells in the stem cell niche of each organ to enable tissue repair after birth17
. It is well known that the hair follicle organ-inductive epithelial and mesenchymal stem cells provide a source of differentiated hair follicle cells that enable hair cycling to occur over the lifetime of a mammal16
. Thus, it is logical for hair follicle regenerative therapy to utilize organ-inductive stem cells of epithelial and mesenchymal origin that are isolated from adult tissues34
. It is also essential to rearrange these various stem cells and their niches in the bioengineered follicle to reproduce enduring hair cycles15
. The sub-bulge, bulge and the bulge-to-sebaceous gland regions provide niches for epithelial stem cells and melanoblast stem cells21
. In contrast, stem/progenitor cell populations that can differentiate into DS cells, adipocytes, cartilage cells and fibroblasts are found among the DP cells18
. Our bioengineered hair follicle can regenerate and sustain hair cycles according to the origin of its cells (that is, pelage or vibrissa), which suggests that bioengineered follicles have the potential to maintain stem cells through the reconstitution of niches for epithelial stem cells and DP cells. These observations also provide insight into the formation and maintenance of stem cell niches in their microenvironments9
For hair regenerative therapy, it is critical to consider whether bioengineered hair follicles can regenerate normal inherent traits41
, physiological functions such as hair shaft types43
, and cooperation with host cutaneous tissues including the arrector pili muscle and nerve system26
. Properties of the hair shaft, which reflect the function of the hair in the body region14
, are regulated by hair follicle mesenchymal DP and DS cells and are also modulated by the expression of various genes in the epidermis15
. It is also thought that hair pigmentation is controlled by melanocyte differentiation and the proliferation of melanocyte-lineage stem cells below the bulge region23
. Thus, hair properties can be controlled by the arrangement of cell types during the regeneration of the bioengineered hair follicle germ34
. We provide evidence for this arrangement by showing that bioengineered hair with a proper shape and colour can be generated through the appropriate cell populations, such as bulge-derived epithelial cells, DP cells, and the PHM region-derived cells, but not sebaceous gland region-derived cells. Our findings thus provide new insights into the regulation of hair properties and strongly suggest that these characteristics could be properly restored by cell processing for organ regeneration and by the transplantation of bioengineered hair follicle germ.
The peripheral nervous system has essential roles in organ function and the perception of noxious stimuli, such as pain and mechanical stress52
. The restoration of the nervous system is thus a critical issue to be addressed by organ replacement regenerative therapy13
. Previously, we demonstrated, in the successful regeneration of a tooth, the proper perception of noxious stimuli mediated by trigeminal innervation13
. In the hair follicle, the follicles, pelage and vibrissae achieve piloerection using the surrounding arrector pili muscle through the activation of the sympathetic nerves26
. The rodent vibrissa also functions as a sensory organ26
. We also demonstrate that the nerve fibres and muscles can connect autonomously to the pelage and vibrissa follicle, and that the bioengineered follicles exhibit ACh-induced piloerection. Our findings suggest that the transplantation of a bioengineered hair follicle germ can restore natural hair function and re-establish the cooperation between the follicle and the surrounding recipient muscles and nerve fibres. Thus, the transplantation of bioengineered hair follicle germ is potentially applicable to the future surgical treatment of alopecia.
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.