Requirement of zinc transporter ZIP10 for epidermal development: Implication of the ZIP10-p63 axis in epithelial homeostasis.

Skin tissues, in particular the epidermis, are severely affected by zinc deficiency. However, the zinc-mediated mechanisms that maintain the cells that form the epidermis have not been established. Here, we report that the zinc transporter ZIP10 is highly expressed in the outer root sheath of hair follicles and plays critical roles in epidermal development. We found that ZIP10 marked epidermal progenitor cell subsets and that ablating Zip10 caused significant epidermal hypoplasia accompanied by down-regulation of the transactivation of p63, a master regulator of epidermal progenitor cell proliferation and differentiation. Both ZIP10 and p63 are significantly increased during epidermal development, in which ZIP10-mediated zinc influx promotes p63 transactivation. Collectively, these results indicate that ZIP10 plays important roles in epidermal development via, at least in part, the ZIP10-zinc-p63 signaling axis, thereby highlighting the physiological significance of zinc regulation in the maintenance of skin epidermis.

Cyclical dermal micro-niche switching governs the morphological infradian rhythm of mouse zigzag hair.

Biological rhythms are involved in almost all types of biological processes, not only physiological processes but also morphogenesis. Currently, how periodic morphological patterns of tissues/organs in multicellular organisms form is not fully understood. Here, using mouse zigzag hair, which has 3 bends, we found that a change in the combination of hair progenitors and their micro-niche and subsequent bend formation occur every three days. Chimeric loss-of-function and gain-of-function of Ptn and Aff3, which are upregulated immediately before bend formation, resulted in defects in the downward movement of the micro-niche and the rhythm of bend formation in an in vivo hair reconstitution assay. Our study demonstrates the periodic change in the combination between progenitors and micro-niche, which is vital for the unique infradian rhythm.

Expansion and characterization of epithelial stem cells with potential for cyclical hair regeneration.

In mammals, organ induction occurs only during embryonic development except for hair follicles (HFs). However, HF-resident epithelial stem cells (HFSCs), which are responsible for repetitive HF regeneration, are not fully characterized. Here, we establish in vitro culture systems that are capable of controlling the ability of HFSCs to regenerate HFs. Based on a method that precisely controlled the number of HFs for regeneration, functional analysis revealed that CD34/CD49f/integrin ß5 (Itgß5)-triple-positive (CD34+/CD49f+/Itgß5+) cells have multipotency and functional significance for continual hair regeneration. In native HFs, these cells reside in the uppermost area of the bulge region, which is surrounded by tenascin in mice and humans. This study unveils the subpopulation of HFSCs responsible for long-term hair cycling of HFs regenerated from bioengineered HF germ, suggesting the presence of functional heterogeneity among bulge HFSCs and the utility of our culture system to achieve HF regenerative therapy.

Regeneration of a bioengineered 3D integumentary organ system from iPS cells.

Organ systems play essential roles in the physiological functions required for homeostasis. A 3D integumentary organ system (3D-IOS) comprises the skin and skin appendages such as hair follicles and sebaceous glands. This protocol describes how to induce the differentiation of murine induced pluripotent stem (iPS) cells into a 3D-IOS. First, iPS cells are grown for 7 d under conditions that encourage the formation of embryoid bodies (EBs). The iPS cell-derived EBs are stimulated by Wnt10b one day before transplantation of multiple EBs in vivo (a method we describe as the clustering-dependent embryoid body (CDB) transplantation method). After a further 30 d, the transplanted EBs will have differentiated into a 3D-IOS containing mature hair follicles and sebaceous glands. These can be removed and transplanted into wounds in the skin of other mice. After transplantation of a 3D-IOS, the organ system shows full physiological function in vivo starting 14 d following transplant. Thus, this protocol enables a whole functional organ system to be generated from pluripotent stem cells.

Functional Hair Follicle Regeneration by the Rearrangement of Stem Cells.

Hair follicles develop from the ectoderm in embryos and cyclically regenerate using proper spatiotemporal signaling molecules, which are conserved in organogenesis during adulthood. Previously, we demonstrated that bioengineered hair follicle germs could regenerate functional hair follicles via a three-dimensional cell manipulation technique, which we named the "organ germ method ." We could also regulate the type of hair follicle and pigmentation with correct structures by rearranging the source of the cells. In this article, we describe a detailed protocol for the regeneration of functional hair follicles and their stem cell niches by the rearrangement of embryonic or adult hair follicle-derived epithelial and mesenchymal cells.

Hair Follicle Regeneration by Transplantation of a Bioengineered Hair Follicle Germ.

Hair follicle morphogenesis is first induced by epithelial-mesenchymal interactions in the developing embryo. In the hair follicle, various stem-cell populations are maintained in specialized niches to promote repetitive hair follicle-morphogenesis, which is observed in the variable lower region of the hair follicle as a postnatal hair cycle. In contrast, the genesis of most organs is induced only once during embryogenesis. We developed a novel bioengineering technique, the Organ Germ Method, that employs three-dimensional stem cell culture for regenerating various organs and reproducing embryonic organogenesis. In this chapter, we describe a protocol for hair follicle germ reconstitution using adult follicle-derived epithelial stem cells and dermal papilla cells with intracutaneous transplantation of the bioengineered hair-follicle organ germ. This protocol can be useful not only for the clinical study of hair regeneration but also for studies of stem cell biology and organogenesis.

Bioengineering a 3D integumentary organ system from iPS cells using an in vivo transplantation model.

The integumentary organ system is a complex system that plays important roles in waterproofing, cushioning, protecting deeper tissues, excreting waste, and thermoregulation. We developed a novel in vivo transplantation model designated as a clustering-dependent embryoid body transplantation method and generated a bioengineered three-dimensional (3D) integumentary organ system, including appendage organs such as hair follicles and sebaceous glands, from induced pluripotent stem cells. This bioengineered 3D integumentary organ system was fully functional following transplantation into nude mice and could be properly connected to surrounding host tissues, such as the epidermis, arrector pili muscles, and nerve fibers, without tumorigenesis. The bioengineered hair follicles in the 3D integumentary organ system also showed proper hair eruption and hair cycles, including the rearrangement of follicular stem cells and their niches. Potential applications of the 3D integumentary organ system include an in vitro assay system, an animal model alternative, and a bioengineered organ replacement therapy.

Saliva secretion in engrafted mouse bioengineered salivary glands using taste stimulation.

PURPOSE: The aim of this study was to compare saliva flow and protein composition induced using five basic taste stimulations between natural and bioengineered salivary glands. MATERIALS AND METHODS: We developed a mouse saliva secretion model using taste stimulation and analyzed the saliva secretion from natural and bioengineered salivary glands using an assay. The protein components and alpha-amylase in the natural and bioengineered saliva were analyzed by gel electrophoresis and Western blotting. RESULTS: The salivary flow responses induced by sour (citric acid) and bitter (quinine-HCl) stimuli were significantly high in the natural and bioengineered salivary glands. Although the protein concentrations in the natural and bioengineered saliva induced using five basic taste stimulations were similar, the protein composition and the amylase concentration in the natural saliva after taste stimulation had different profiles. Sympathetic and non-sympathetic nerves were observed around the acini and ducts in the natural and bioengineered salivary glands. However, the frequency of neuropeptide Y-positive sympathetic nerves in the bioengineered gland was relatively high compared to that in the natural gland. CONCLUSIONS: These results suggest that the signal balance between the sympathetic and parasympathetic components of the efferent nerves in an engrafted bioengineered salivary gland may differ from that in a natural salivary gland.

Single follicular unit transplantation reconstructs arrector pili muscle and nerve connections and restores functional hair follicle piloerection.

The autologous transplantation of hair follicles that have been separated into single follicular units is an accepted treatment for androgenetic alopecia. Recent studies demonstrate that the multiple stem cell populations and surrounding cutaneous tissues coordinately regulate the hair follicle functions and skin homeostasis. Therefore, the critical issues for consideration regarding functional hair restoration therapy are reproduction the correct connectivity and cooperation with host cutaneous tissues, including the arrector pili muscle (APM) and nerve system. We report successful establishment of mouse single follicular transplantation model and autonomous restoration of transplanted hair follicle piloerection in mouse skin. Transplanted hair follicles were responsive to the neurotransmitter acetylcholine and formed proper connections with surrounding host tissues such as APM and nerve fibers, which in turn connect with not only the hair follicle bulge region but also the APM. These results demonstrate that the piloerection ability of transplanted hair follicles can be estimated quantitatively. This study makes a substantial contribution towards the development of transplantation therapy that will facilitate future functional regeneration therapy for skin and skin appendages.

Fully functional hair follicle regeneration through the rearrangement of stem cells and their niches.

Organ replacement regenerative therapy is purported to enable the replacement of organs damaged by disease, injury or aging in the foreseeable future. Here we demonstrate fully functional hair organ regeneration via the intracutaneous transplantation of a bioengineered pelage and vibrissa follicle germ. The pelage and vibrissae are reconstituted with embryonic skin-derived cells and adult vibrissa stem cell region-derived cells, respectively. The bioengineered hair follicle develops the correct structures and forms proper connections with surrounding host tissues such as the epidermis, arrector pili muscle and nerve fibres. The bioengineered follicles also show restored hair cycles and piloerection through the rearrangement of follicular stem cells and their niches. This study thus reveals the potential applications of adult tissue-derived follicular stem cells as a bioengineered organ replacement therapy.

Hair organ regeneration via the bioengineered hair follicular unit transplantation.

Organ regenerative therapy aims to reproduce fully functional organs to replace organs that have been lost or damaged as a result of disease, injury, or aging. For the fully functional regeneration of ectodermal organs, a concept has been proposed in which a bioengineered organ is developed by reproducing the embryonic processes of organogenesis. Here, we show that a bioengineered hair follicle germ, which was reconstituted with embryonic skin-derived epithelial and mesenchymal cells and ectopically transplanted, was able to develop histologically correct hair follicles. The bioengineered hair follicles properly connected to the host skin epithelium by intracutaneous transplantation and reproduced the stem cell niche and hair cycles. The bioengineered hair follicles also autonomously connected with nerves and the arrector pili muscle at the permanent region and exhibited piloerection ability. Our findings indicate that the bioengineered hair follicles could restore physiological hair functions and could be applicable to surgical treatments for alopecia.

Polymorphic CAG repeat numbers in the androgen receptor gene of female pattern hair loss patients.

Female pattern hair loss (FPHL) is frequently referred to as female androgenetic alopecia (FAGA). However, the role of androgen in this type of hair loss remains uncertain. We previously reported greater therapeutic efficacy of finasteride in Japanese male androgenetic alopecia (MAGA) patients in cases where the CAG repeats of the androgen receptor (AR) gene were short. To examine the correlation between CAG repeat numbers and the therapeutic efficacy of finasteride in FPHL patients, the efficacy of finasteride (1 mg/day) was evaluated macroscopically. Because women have two X-chromosomes, the shorter and longer CAG repeat numbers were analyzed in 37 Japanese FPHL patients, then the correlation of these factors was statistically analyzed by anova. No statistical significance in terms of the differences in CAG repeat numbers was detected among the four groups classified on the basis of the efficacy of finasteride. From these results, it may be concluded that the efficacy of this medicine in each FPHL patient cannot be predicted by the CAG repeat numbers in the AR gene.

Contact between dermal papilla cells and dermal sheath cells enhances the ability of DPCs to induce hair growth.

We previously showed that cultured rat dermal papilla cells (DPCs) retain their hair-inducing capacity on afollicular epidermal cell (EPCs). Here, we examined the hair growth-inducing capacity of differently subcultured DPCs by transplanting them, along with rat EPCs, onto the backs of nude mice (graft chamber assay). DPCs at passage (p) 6 (DPCs(p6) or, more generally, low-passage DPCs) induced hair formation. However, DPCs(p>30) (high-passage DPCs) had no such activity and induced only subepidermal hair follicles (HFs) that were not encapsulated by the dermal sheath (DS). Thus, we examined the effect of DS cells (DSCs(p=1)) on the ability of DPCs(p=60) to induce hair growth by testing a mixture of these two cell types (cotransplant) in the graft chamber assay, in which DSCs(p=1) and DPCs(p=60) were labeled with enhanced green fluorescent protein (EGFP) and 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI), respectively. These cotransplants generated hairs as actively as did DPCs(p=6) transplants. Their HFs were encapsulated with EGFP(+)-DS and had DPs consisting largely of EGFP(+)-DPCs (47%) and DiI(+)-DPCs (43%), indicating a major contribution of DSC(p=1)-derived DPCs to HF induction. In addition, the results of in vitro coculture of DPCs(p=60) and DSCs(p=1) suggest that high-passage DPCs stimulate the expression of certain trichogenic genes in DSCs.