Dedifferentiation maintains melanocyte stem cells in a dynamic niche.

For unknow reasons, the melanocyte stem cell (McSC) system fails earlier than other adult stem cell populations1, which leads to hair greying in most humans and mice2,3. Current dogma states that McSCs are reserved in an undifferentiated state in the hair follicle niche, physically segregated from differentiated progeny that migrate away following cues of regenerative stimuli4-8. Here we show that most McSCs toggle between transit-amplifying and stem cell states for both self-renewal and generation of mature progeny, a mechanism fundamentally distinct from those of other self-renewing systems. Live imaging and single-cell RNA sequencing revealed that McSCs are mobile, translocating between hair follicle stem cell and transit-amplifying compartments where they reversibly enter distinct differentiation states governed by local microenvironmental cues (for example, WNT). Long-term lineage tracing demonstrated that the McSC system is maintained by reverted McSCs rather than by reserved stem cells inherently exempt from reversible changes. During ageing, there is accumulation of stranded McSCs that do not contribute to the regeneration of melanocyte progeny. These results identify a new model whereby dedifferentiation is integral to homeostatic stem cell maintenance and suggest that modulating McSC mobility may represent a new approach for the prevention of hair greying.

Coordinated activation of Wnt in epithelial and melanocyte stem cells initiates pigmented hair regeneration.

Melanocyte stem cells (McSCs) intimately interact with epithelial stem cells (EpSCs) in the hair follicle bulge and secondary hair germ (sHG). Together, they undergo activation and differentiation to regenerate pigmented hair. However, the mechanisms behind this coordinated stem cell behavior have not been elucidated. Here, we identified Wnt signaling as a key pathway that couples the behavior of the two stem cells. EpSCs and McSCs coordinately activate Wnt signaling at the onset of hair follicle regeneration within the sHG. Using genetic mouse models that specifically target either EpSCs or McSCs, we show that Wnt activation in McSCs drives their differentiation into pigment-producing melanocytes, while EpSC Wnt signaling not only dictates hair follicle formation but also regulates McSC proliferation during hair regeneration. Our data define a role for Wnt signaling in the regulation of McSCs and also illustrate a mechanism for regeneration of complex organs through collaboration between heterotypic stem cell populations.

OPN4 belongs to the photosensitive system of the human skin.

The human skin has previously been described to be affected by light; however, the underlying mechanism remains unknown. OPN4 (melanopsin) expression was first identified in the skin of amphibians; however, whether it is also expressed and functioned in the human skin has not yet been identified. Here, we show that OPN4 was expressed in the human skin tissue and cultures of isolated keratinocytes, melanocytes and fibroblasts. Additionally, Ca2+ influx in vitro and ex vivo and phosphorylation of extracellular signal-regulated kinases 1/2 in human fibroblasts were observed by stimulation of blue light irradiation. Notably, our findings showed that this Ca2+ influx and phosphorylation of extracellular signal-regulated kinases 1/2 are promoted in an intensity-dependent manner, indicating that the light signal is converted to an intracellular signal via OPN4 in the human skin. Overall, in this study we showed that the human skin functions as a photoreceptor by demonstrating that in human skin, the photoreceptive protein was expressed, and photoreception was conducted via photoreceptive protein.

Wnt activation in nail epithelium couples nail growth to digit regeneration.

The tips of mammalian digits can regenerate after amputation, like those of amphibians. It is unknown why this capacity is limited to the area associated with the nail. Here we show that nail stem cells (NSCs) reside in the proximal nail matrix and that the mechanisms governing NSC differentiation are coupled directly with their ability to orchestrate digit regeneration. Early nail progenitors undergo Wnt-dependent differentiation into the nail. After amputation, this Wnt activation is required for nail regeneration and also for attracting nerves that promote mesenchymal blastema growth, leading to the regeneration of the digit. Amputations proximal to the Wnt-active nail progenitors result in failure to regenerate the nail or digit. Nevertheless, ß-catenin stabilization in the NSC region induced their regeneration. These results establish a link between NSC differentiation and digit regeneration, and suggest that NSCs may have the potential to contribute to the development of novel treatments for amputees.

Comparative studies of hair shaft components between healthy and diseased donors.

Globally, the rapid aging of the population is predicted to become even more severe in the second half of the 21st century. Thus, it is expected to establish a growing expectation for innovative, non-invasive health indicators and diagnostic methods to support disease prevention, care, and health promotion efforts. In this study, we aimed to establish a new health index and disease diagnosis method by analyzing the minerals and free amino acid components contained in hair shaft. We first evaluated the range of these components in healthy humans and then conducted a comparative analysis of these components in subjects with diabetes, hypertension, androgenetic alopecia, major depressive disorder, Alzheimer's disease, and stroke. In the statistical analysis, we first used a student's t test to compare the hair components of healthy people and those of patients with various diseases. However, many minerals and free amino acids showed significant differences in all diseases, because the sample size of the healthy group was very large compared to the sample size of the disease group. Therefore, we attempted a comparative analysis based on effect size, which is not affected by differences in sample size. As a result, we were able to narrow down the minerals and free amino acids for all diseases compared to t test analysis. For diabetes, the t test narrowed down the minerals to 15, whereas the effect size measurement narrowed it down to 3 (Cr, Mn, and Hg). For free amino acids, the t test narrowed it down to 15 minerals. By measuring the effect size, we were able to narrow it down to 7 (Gly, His, Lys, Pro, Ser, Thr, and Val). It is also possible to narrow down the minerals and free amino acids in other diseases, and to identify potential health indicators and disease-related components by using effect size.

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.

Mechanical homeostasis of liver sinusoid is involved in the initiation and termination of liver regeneration.

Organogenesis and regeneration are fundamental for developmental progress and are associated with morphogenesis, size control and functional properties for whole-body homeostasis. The liver plays an essential role in maintaining homeostasis of the entire body through various functions, including metabolic functions, detoxification, and production of bile, via the three-dimensional spatial arrangement of hepatic lobules and has high regenerative capacity. The regeneration occurs as hypertrophy, which strictly controls the size and lobule structure. In this study, we established a three-dimensional sinusoidal network analysis method and determined valuable parameters after partial hepatectomy by comparison to the static phase of the liver. We found that mechanical homeostasis, which is crucial for organ morphogenesis and functions in various phenomena, plays essential roles in liver regeneration for both initiation and termination of liver regeneration, which is regulated by cytokine networks. Mechanical homeostasis plays critical roles in the initiation and termination of organogenesis, tissue repair and organ regeneration in coordination with cytokine networks.

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.

Tissue-scale tensional homeostasis in skin regulates structure and physiological function.

Tensional homeostasis is crucial for organ and tissue development, including the establishment of morphological and functional properties. Skin plays essential roles in waterproofing, cushioning and protecting deeper tissues by forming internal tension-distribution patterns, which involves aligning various cells, appendages and extracellular matrices (ECMs). The balance of traction force is thought to contribute to the formation of strong and pliable physical structures that maintain their integrity and flexibility. Here, by using a human skin equivalent (HSE), the horizontal tension-force balance of the dermal layer was found to clearly improve HSE characteristics, such as the physical relationship between cells and the ECM. The tension also promoted skin homeostasis through the activation of mechano-sensitive molecules such as ROCK and MRTF-A, and these results compared favourably to what was observed in tension-released models. Tension-induced HSE will contribute to analyze skin physiological functions regulated by tensional homeostasis as an alternative animal model.

Functional ectodermal organ regeneration as the next generation of organ replacement therapy.

In this decade, substantial progress in the fields of developmental biology and stem cell biology has ushered in a new era for three-dimensional organ regenerative therapy. The emergence of novel three-dimensional cell manipulation technologies enables the effective mimicking of embryonic organ germ formation using the fate-determined organ-inductive potential of epithelial and mesenchymal stem cells. This advance shows great potential for the regeneration of functional organs with substitution of complete original function in situ. Organoids generated from multipotent stem cells or tissue stem cells via establishment of an organ-forming field can only partially recover original organ function owing to the size limitation; they are considered 'mini-organs'. Nevertheless, they hold great promise to realize regenerative medicine. In particular, regeneration of a functional salivary gland and an integumentary organ system by orthotopic and heterotopic implantation of organoids clearly points to the future direction of organ regeneration research. In this review, we describe multiple strategies and recent progress in regenerating functional three-dimensional organs, focusing on ectodermal organs, and discuss their potential and future directions to achieve organ replacement therapy as a next-generation regenerative medicine.

A novel mouse model demonstrates that oncogenic melanocyte stem cells engender melanoma resembling human disease.

Melanoma, the deadliest skin cancer, remains largely incurable at advanced stages. Currently, there is a lack of animal models that resemble human melanoma initiation and progression. Recent studies using a Tyr-CreER driven mouse model have drawn contradictory conclusions about the potential of melanocyte stem cells (McSCs) to form melanoma. Here, we employ a c-Kit-CreER-driven model that specifically targets McSCs to show that oncogenic McSCs are a bona fide source of melanoma that expand in the niche, and then establish epidermal melanomas that invade into the underlying dermis. Further, normal Wnt and Endothelin niche signals during hair anagen onset are hijacked to promote McSC malignant transformation during melanoma induction. Finally, molecular profiling reveals strong resemblance of murine McSC-derived melanoma to human melanoma in heterogeneity and gene signatures. These findings provide experimental validation of the human melanoma progression model and key insights into the transformation and heterogeneity of McSC-derived melanoma.

Organ regeneration based on developmental biology: past and future.

In this decade, great progress has been made in the field of organ regeneration by incorporating emerging concepts from the fields of stem cell biology and developmental biology, and this progress has pioneered a new frontier in regenerative medicine. The generation of bioengineered organ germ-utilizing, fate-determined, organ-inductive epithelial and mesenchymal cells has provided evidence for the concept of functional organ regeneration in vivo. Organoid studies have verified that nearly all organs can be generated in the form of a mini-organ by recapitulating embryonic body patterning and establishing an organ-forming field among self-organizing pluripotent stem cells by utilizing cytokines that mimic the patterning and positional signals of organogenesis. More recently, the regeneration of an integumentary organ system composed of multiple organs, including hair follicles, has been achieved, demonstrating that regenerative medicine is forthcoming. In this review, we will introduce current research trends aimed at regenerating a functional three-dimensional (3D) organ, and we will discuss the potential use of these recent achievements and future directions needed to realize the next-generation of regenerative therapy for organ replacement.

Dissecting Wnt Signaling for Melanocyte Regulation during Wound Healing.

Abnormal pigmentation is commonly seen in the wound scar. Despite advancements in the research of wound healing, little is known about the repopulation of melanocytes in the healed skin. Previous studies have shown the capacity of melanocyte stem cells in the hair follicle to contribute skin epidermal melanocytes after injury in mice and humans. Here, we focused on the Wnt pathway, known to be a vital regulator of melanocyte stem cells in efforts to better understand the regulation of follicle-derived epidermal melanocytes during wound healing. We showed that transgenic expression of Wnt inhibitor Dkk1 in melanocytes reduced epidermal melanocytes in the wound scar. Conversely, forced activation of Wnt signaling by genetically stabilizing ß-catenin in melanocytes increases epidermal melanocytes. Furthermore, we show that deletion of Wntless (Wls), a gene required for Wnt ligand secretion, within epithelial cells results in failure in activating Wnt signaling in adjacent epidermal melanocytes. These results show the essential function of extrinsic Wnt ligands in initiating Wnt signaling in follicle-derived epidermal melanocytes during wound healing. Collectively, our results suggest the potential for Wnt signal regulation to promote melanocyte regeneration and provide a potential molecular window to promote proper melanocyte regeneration after wounding and in conditions such as vitiligo.

Hedgehog stimulates hair follicle neogenesis by creating inductive dermis during murine skin wound healing.

Mammalian wounds typically heal by fibrotic repair without hair follicle (HF) regeneration. Fibrosis and regeneration are currently considered the opposite end of wound healing. This study sought to determine if scar could be remodeled to promote healing with HF regeneration. Here, we identify that activation of the Sonic hedgehog (Shh) pathway reinstalls a regenerative dermal niche, called dermal papilla, which is required and sufficient for HF neogenesis (HFN). Epidermal Shh overexpression or constitutive Smoothened dermal activation results in extensive HFN in wounds that otherwise end in scarring. While long-term Wnt activation is associated with fibrosis, Shh signal activation in Wnt active cells promotes the dermal papilla fate in scarring wounds. These studies demonstrate that mechanisms of scarring and regeneration are not distant from one another and that wound repair can be redirected to promote regeneration following injury by modifying a key dermal signal.

Epithelium-Derived Wnt Ligands Are Essential for Maintenance of Underlying Digit Bone.

Clinically, many nail disorders accompany bone deformities, but whether the two defects are causally related is under debate. To investigate the potential interactions between the two tissue types, we analyzed epithelial-specific ß-catenin-deficient mice, in which nail differentiation is abrogated. These mice showed regression of not only the nail plate but also of the underlying digit bone. Characterization of these bone defects revealed active bone resorption, which is suppressed by Wnt activation in osteoblast and osteoclast precursors. Furthermore, we found that Wntless expression, essential for Wnt ligand secretion, was lacking in the ß-catenin-deficient nail epithelium and that genetic deletion of Wntless (Wls) in the nail epithelium led to the lack of Wnt activation in osteoblast and osteoclast precursors and subsequently led to defective regression of the underlying digit bone. Together, these data show that epithelial Wnt ligands can ultimately regulate Wnt signaling in osteoblast and osteoclast precursors, known to regulate bone homeostasis. These results reveal a critical role for the nail epithelium on the digit bone during homeostatic regeneration and show that Wnt/ß-catenin signaling is critical for this interaction.

EdnrB Governs Regenerative Response of Melanocyte Stem Cells by Crosstalk with Wnt Signaling.

Delineating the crosstalk between distinct signaling pathways is key to understanding the diverse and dynamic responses of adult stem cells during tissue regeneration. Here, we demonstrate that the Edn/EdnrB signaling pathway can interact with other signaling pathways to elicit distinct stem cell functions during tissue regeneration. EdnrB signaling promotes proliferation and differentiation of melanocyte stem cells (McSCs), dramatically enhancing the regeneration of hair and epidermal melanocytes. This effect is dependent upon active Wnt signaling that is initiated by Wnt ligand secretion from the hair follicle epithelial niche. Further, this Wnt-dependent EdnrB signaling can rescue the defects in melanocyte regeneration caused by Mc1R loss. This suggests that targeting Edn/EdnrB signaling in McSCs can be a therapeutic approach to promote photoprotective-melanocyte regeneration, which may be useful for those with increased risk of skin cancers due to Mc1R variants.

Wound healing and skin regeneration.

The skin is a complex organ consisting of the epidermis, dermis, and skin appendages, including the hair follicle and sebaceous gland. Wound healing in adult mammals results in scar formation without any skin appendages. Studies have reported remarkable examples of scarless healing in fetal skin and appendage regeneration in adult skin following the infliction of large wounds. The models used in these studies have offered a new platform for investigations of the cellular and molecular mechanisms underlying wound healing and skin regeneration in mammals. In this article, we will focus on the contribution of skin appendages to wound healing and, conversely, skin appendage regeneration following injuries.

Epithelial Wnt ligand secretion is required for adult hair follicle growth and regeneration.

ß-Catenin, a key transducer molecule of Wnt signaling, is required for adult hair follicle growth and regeneration. However, the cellular source of Wnt ligands required for Wnt/ß-catenin activation during anagen induction is unknown. In this study, we genetically deleted Wntless (Wls), a gene required for Wnt ligand secretion by Wnt-producing cells, specifically in the hair follicle epithelium during telogen phase. We show that epithelial Wnt ligands are required for anagen, as loss of Wls in the follicular epithelium resulted in a profound hair cycle arrest. Both the follicular epithelium and dermal papilla showed markedly decreased Wnt/ß-catenin signaling during anagen induction compared with control hair follicles. Surprisingly, hair follicle stem cells that are responsible for hair regeneration maintained expression of stem cell markers but exhibited significantly reduced proliferation. Finally, we demonstrate that epidermal Wnt ligands are critical for adult wound-induced de novo hair formation. Collectively, these data show that Wnt ligands secreted by the hair follicle epithelium are required for adult hair follicle regeneration and provide new insight into potential cellular targets for the treatment of hair disorders such as alopecia.

Direct migration of follicular melanocyte stem cells to the epidermis after wounding or UVB irradiation is dependent on Mc1r signaling.

During wound healing, stem cells provide functional mature cells to meet acute demands for tissue regeneration. Simultaneously, the tissue must maintain a pool of stem cells to sustain its future regeneration capability. However, how these requirements are balanced in response to injury is unknown. Here we demonstrate that after wounding or ultraviolet type B irradiation, melanocyte stem cells (McSCs) in the hair follicle exit the stem cell niche before their initial cell division, potentially depleting the pool of these cells. We also found that McSCs migrate to the epidermis in a melanocortin 1 receptor (Mc1r)-dependent manner and differentiate into functional epidermal melanocytes, providing a pigmented protective barrier against ultraviolet irradiation over the damaged skin. These findings provide an example in which stem cell differentiation due to injury takes precedence over stem cell maintenance and show the potential for developing therapies for skin pigmentation disorders by manipulating McSCs.

Functional analysis of grimp, a novel gene required for mesodermal cell proliferation at an initial stage of regeneration in Enchytraeus japonensis (Enchytraeidae, Oligochaete).

Enchytraeus japonensis is a small oligochaete species, which has a remarkable regeneration capacity. It has been proposed as a new model animal for the study of regeneration, and some histological studies of this species have been carried out. On the other hand, the molecular biological mechanism of regeneration is almost unknown in this species. To clarify the molecular biological mechanism operating at an initial stage of regeneration in E. japonensis, we isolated by the cDNA subtraction method five genes whose expression levels changed in the regeneration process occurring between growing and early regenerating worms. One of the isolated genes (a novel gene named grimp) was expressed transiently from 3 to 12 h post amputation only in neoblasts and a population of mesodermal cells (the non-neoblast grimp-expressing cells) incorporating BrdU simultaneously showed mitotic activity. We succeeded in inhibiting grimp expression by RNA interference (RNAi), thus applying this technique for the first time in Oligochaeta. In knock-down worms, the number of BrdU-positive neoblasts and the non-neoblast grimp-expressing cells in the coelom drastically decreased. Moreover, the elongation and the segmentation of blastemas were inhibited, while no statistically significant inhibitory effect was observed in epidermal and intestinal cells. These results suggest that grimp is required for initial proliferation of neoblasts and some mesodermal cells for regeneration.