Reduced Elastin Fibers and Melanocyte Loss in Vitiliginous Skin Are Restored after Repigmentation by Phototherapy and/or Autologous Minigraft Transplantation.

Vitiligo is a hypopigmentation disease characterized by melanocyte death in the human epidermis. However, the mechanism of vitiligo development and repigmentation is largely unknown. Dermal fiber components might play an important role in vitiligo development and repigmentation. Indeed, our preliminary study demonstrated that elastin fibers were decreased in vitiliginous skin, suggesting that the elastin fiber is one of the factors involved in vitiligo development and repigmentation. To confirm our hypothesis, we investigated whether elastin fibers can be restored after treatment using phototherapy and/or autologous skin transplantation. Punch biopsies from 14 patients of stable nonsegmental vitiligo vulgaris were collected from nonlesional, lesional, and repigmented skin, and processed to dopa and combined dopa-premelanin reactions. Melanocytes positive to the dopa reaction and melanoblasts/melanocytes positive to the combined dopa-premelanin reaction were surveyed. Moreover, elastin fibers were detected by Victoria blue staining. Numerous melanocytes and melanoblasts were observed in the epidermis of repigmented skin after the treatment. Moreover, in the dermis of repigmented skin, elastin fibers were completely recovered or even upregulated. These results suggest that melanocyte loss in the vitiliginous skin, as well as melanocyte differentiation in repigmented skin, may be at least in part regulated by elastin fibers in the dermis.

The human melanocyte and melanoblast populations per unit area of epidermis in the rete ridge are greater than in the inter-rete ridge.

OBJECTIVE: In human skin, melanocytes and melanoblasts are mostly located in the epidermis in addition to hair follicles. In hairy skin such as mouse skin, the inter-follicular epidermis is generally flat. In contrast, in human skin, the epidermis is wavy and possesses well-developed rete ridges. It is not well understood what difference exists in cell function between melanocytes present in the inter-rete ridge and those in the rete ridge. To clarify this problem, we first tried to determine the melanocyte and melanoblast populations per unit area of epidermis both in the rete ridge and inter-rete ridge epidermis. METHODS: Thirty-eight samples from normal skin sites of 28 patients (14 males and 14 females) aged from 5 to 76 years were fixed and processed to dopa and combined dopa-premelanin reactions. The numbers of cells positive to the dopa (melanocytes) and combined dopa-premelanin (melanoblasts and melanocytes) reactions were scored. RESULTS: The average melanocyte population/0.1 mm2 of the inter-rete ridge was 74 cells, whereas that of the rete ridge was 147 cells. Moreover, the average melanoblast population/0.1 mm2 in the inter-rete ridge was 43 cells, whereas that of the rete ridge was 131 cells. The melanoblast population in the rete ridge differed between female and male, but the melanocyte populations in the rete ridge and inter-rete ridge and the melanoblast population in the inter-rete ridge did not differ between female and male. However, no age difference in the melanocyte and melanoblast populations was observed between the rete ridge and inter-rete ridge. CONCLUSION: Human epidermal melanocytes and melanoblasts in the rete ridge exist more numerously than in the inter-rete ridge.

OBJECTIF: dans la peau humaine, les mélanocytes et les mélanoblastes sont principalement situés dans l'épiderme ainsi que dans les follicules pileux. Dans la peau poilue, comme la peau de souris, l'épiderme interfolliculaire est généralement plat. En revanche, dans la peau humaine, l'épiderme a un aspect ondulé et possède des crêtes épidermiques bien développées. On ne comprend pas bien la différence entre la fonction cellulaire des mélanocytes présents dans la crête inter-épidermique et celle des mélanocytes présents dans la crête épidermique. Afin de clarifier ce problème, nous avons d'abord essayé de déterminer les populations mélanocytaires et mélanoblastiques par unité de surface de l'épiderme, à la fois dans l'épiderme de la crête épidermique et dans celui de la crête inter-épidermique. MÉTHODES: trente-huit échantillons provenant de sites cutanés normaux de 28 patients (14 hommes et 14 femmes), âgés de 5 à 76 ans, ont été fixés et traités pour des réactions à la dopa et à l'association dopa-prémélanine. Le nombre de cellules positives aux réactions à la dopa (mélanocytes) et à l'association dopa-prémélanine (mélanoblastes et mélanocytes) a été évalué. RÉSULTATS: la population mélanocytaire moyenne/0,1 mm2 de la crête inter-épidermique était de 74 cellules, alors que celle de la crête épidermique était de 147 cellules. De plus, la population mélanoblastique moyenne/0,1 mm2 de la crête inter-épidermique était de 43 cellules, alors que celle de la crête épidermique était de 131 cellules. La population mélanoblastique dans la crête épidermique était différente entre les femmes et les hommes, mais les populations mélanocytaires dans la crête épidermique et la crête inter-épidermique, ainsi que la population mélanoblastique dans la crête inter-épidermique, n'étaient pas différentes entre les femmes et les hommes. Cependant, aucune différence liée à l'âge n'a été observée entre la crête épidermique et la crête inter-épidermique dans les populations mélanocytaires et mélanoblastiques. CONCLUSION: chez l'homme, les mélanocytes et les mélanoblastes épidermiques sont plus nombreux dans la crête épidermique que dans la crête inter-épidermique.

Improved HPLC Conditions to Determine Eumelanin and Pheomelanin Contents in Biological Samples Using an Ion Pair Reagent.

Alkaline hydrogen peroxide oxidation (AHPO) of eumelanin and pheomelanin, two major classes of melanin pigments, affords pyrrole-2,3,5-tricarboxylic acid (PTCA), pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) from eumelanin and thiazole-2,4,5-tricarboxylic acid (TTCA) and thiazole-4,5-dicarboxylic acid (TDCA) from pheomelanin. Quantification of these five markers by HPLC provides useful information on the quantity and structural diversity of melanins in various biological samples. HPLC analysis of these markers using the original method of 0.1 M potassium phosphate buffer (pH 2.1):methanol = 99:1 (85:15 for PTeCA) on a reversed-phase column had some problems, including the short lifetime of the column and, except for the major eumelanin marker PTCA, other markers were occasionally overlapped by interfering peaks in samples containing only trace levels of these markers. These problems can be overcome by the addition of an ion pair reagent for anions, such as tetra-n-butylammonium bromide (1 mM), to retard the elution of di-, tri- and tetra-carboxylic acids. The methanol concentration was increased to 17% (30% for PTeCA) and the linearity, reproducibility, and recovery of the markers with this improved method is good to excellent. This improved HPLC method was compared to the original method using synthetic melanins, mouse hair, human hair, and human epidermal samples. In addition to PTCA, TTCA, a major marker for pheomelanin, showed excellent correlations between both HPLC methods. The other markers showed an attenuation of the interfering peaks with the improved method. We recommend this improved HPLC method for the quantitative analysis of melanin markers following AHPO because of its simplicity, accuracy, and reproducibility.

Activation of melanoblasts and melanocytes after treatment with monochromatic excimer light and narrowband-ultraviolet B of skin of vitiligo patients.

BACKGROUND: Vitiligo is a hypopigmentation disease characterized by achromatic or hypochromatic macules in several sites of human skin. Although monochromatic excimer light (MEL) and narrowband-ultraviolet B (NB-UVB) have recently been used for efficacious treatment of vitiligo, the repigmentation process and role of melanocytes/melanoblasts are not well known. METHODS: White macules of 28 vitiligo patients were repeatedly exposed with MEL/NB-UVB. After the exposures, punch biopsies of the center and edge of the lesional skin in addition to normal skin were fixed and processed to dopa and combined dopa-premelanin reactions. Melanocytes positive to the dopa reaction and melanoblasts/melanocytes positive to the combined dopa-premelanin reaction were surveyed. RESULTS: Two repigmentation patterns were observed; one was "marginal" and the other was "perifollicular." Although the frequency of the former pattern was much lower than that of the latter pattern, no differences were observed in average percentage of repigmentation and the number of melanocytes and melanoblasts observed at the center and edge of the lesional epidermis. CONCLUSIONS: These results suggest that exposures with MEL/NB-UVB induce the marginal or perifollicular repigmentation pattern in the white macules, though the two patterns produce no differences in the distribution and density of melanoblasts/melanocytes in the lesional epidermis.

Histochemical study of the distribution of epidermal melanoblasts and melanocytes in Asian human skin.

INTRODUCTION: The classical study revealed the melanocyte density in the epidermis of many skin sites of humans. However, these data were obtained by counting melanocytes using dihydroxyphenylalanine (dopa)-treated epidermal sheets. Since rete ridges are developed well in human epidermis, there is a concern about the accuracy of these data. The accurate counting of the melanocyte density in the epidermis including the rete ridges should be performed using histological sections after the dopa treatment. Moreover, it is not known well how many melanoblasts are present in Asian epidermis. The aim of this study was to count the accurate number of melanocytes and melanoblasts. METHODS: Normal skin sites of 9- to 77-year-old patients were fixed with buffered formalin and processed to the dopa and combined dopa-premelanin reactions. The numbers of cells positive to the dopa reaction (melanocytes) and to the combined dopa-premelanin reaction (melanoblasts and melanocytes) were scored. RESULTS: In the skin of arms, legs, back, and belly, similar density (approximately 110-120 cells/0.1 mm2 ) of melanocytes was observed, whereas in the skin of scalp, melanocyte density was much lower (approximately 70 cells/0.1 mm2 ). By contrast, the melanoblast density did not differ between skin sites (approximately 100 cells/0.1 mm2 ). CONCLUSIONS: These results suggest that the melanocyte density does not differ between skin sites except the scalp skin, and a certain number of melanoblasts are present in each skin site of Asian. Melanoblasts seem to be required for producing new melanocytes required to maintain epidermal homeostasis.

The Pro-Oxidant Activity of Pheomelanin is Significantly Enhanced by UVA Irradiation: Benzothiazole Moieties Are More Reactive than Benzothiazine Moieties.

It is generally considered that eumelanin (EM) is photoprotective while pheomelanin (PM) is phototoxic. A recent study using a mouse model demonstrated that PM produces reactive oxygen species (ROS) that cause DNA damage and eventually lead to melanomagenesis. A biochemical study showed that PM possesses a pro-oxidant activity. PM consists of benzothiazine (BT) and benzothiazole (BZ) moieties, BT moieties being transformed to BZ moieties by heat or light. In this study, we compared the effects of ultraviolet A (UVA) irradiation using synthetic PMs with different BT to BZ ratios and using various coat color mouse hairs. We found that UVA irradiation of BZ-PM increased glutathione (GSH) depletion and generated more H2O2 than UVA irradiation of BT-PM. Non-irradiated controls did not exhibit strong pro-oxidant activities. Upon UVA irradiation, yellow mouse hairs oxidized GSH and produced H2O2 faster than black or albino mouse hairs. Next, to examine the mechanism of the pro-oxidant activity of BT-PM and BZ-PM, we examined the pro-oxidant activities of 7-(2-amino-2-carboxyethyl)-dihydro-1,4-benzothiazine-3-carboxylic acid (DHBTCA) and 6-(2-amino-2-carboxyethyl)-4-hydroxybenzothiazole (BZ-AA) as BT and BZ monomers, respectively. Their pro-oxidant activities were similar, but a large difference was seen in the effects of ROS scavengers, which suggests that the redox reactions may proceed via singlet oxygen in BZ-AA and via superoxide anions in DHBTCA. These results show that UVA enhances the pro-oxidant activity of PM, in particular BZ-PM.

Approach for the Derivation of Melanocytes from Induced Pluripotent Stem Cells.

Induced pluripotent stem (iPS) cells have the ability to differentiate into multiple cell types in the body and have an unlimited growth potential. However, iPS cell-derived melanocytes produced by existing protocols have significant limitations in developing novel strategies for regenerative medicine and cell therapies of pigmentation disorders in humans because they involve culture in media containing fetal bovine serum and nonphysiological agents. In this study, we established an in vitro approach to generate iPS cell-derived human melanocytes that have higher proliferation rates and increased melanin production compared with melanocytes prepared by previously reported approaches. Importantly, our iPS cell-derived human melanocytes are prepared in fetal bovine serum-free culture conditions that do not contain any nonphysiological agents. We designed two original methods, transferring black colonies by pipette and recovering black cell pellets from centrifuged medium, and numerous human iPS cell-derived melanocytes proliferated in gelatinous dishes coated with Matrigel after 12 days. We also succeeded in inducing melanin pigmentation in the nude mouse skin in vivo using those human iPS cell-derived melanocytes. We propose that this method using iPS cells established from T cells in the blood of normal human volunteers could be applied clinically to develop regenerative medicine and cell therapies for various forms of human pigmentation disorders.

Regeneration of Murine Hair Follicles is Inhibited by Low-Dose-Rate Gamma Irradiation.

To determine whether the effects of low-dose-rate gamma (γ) irradiation are identifiable in the regeneration of murine hair follicles, we irradiated whole bodies of C57BL/10JHir mice in the first telogen phase of the hair cycle with 137Cs γ-rays. The mice were examined for effects on hair follicles, including number, morphology, and pigmentation in the second anagen phase. Effects of γ-radiation on melanocyte stem cells were also investigated by the indirect immunolabeling of tyrosinase-related protein 2 (TRP2). Irradiated skin showed a decrease in hair follicle density and the induction of curved hair follicles along with the presence of white hairs and hypopigmented hair bulbs. There was a small, but not significant, change in the number of TRP2-positive melanocyte stem cells in the hair bulge region of the irradiated skin. These results suggest that low-dose rate γ-irradiation does not deplete melanocyte stem cells, but can damage stem cells and progenitors for both keratinocytes and melanocytes, thereby affecting the structure and pigmentation of regenerated hair follicles in the 2nd anagen phase.

Human fibroblasts treated with hydrogen peroxide stimulate human melanoblast proliferation and melanocyte differentiation, but inhibit melanocyte proliferation in serum-free co-culture system.

BACKGROUND: Oxidative stress caused by hydrogen peroxide (H2O2) elicits harmful effects on human melanocytes such as DNA damage and cell death. On the contrary, H2O2 is known to possess beneficial effects on melanocytes. However, mechanisms of the beneficial effects of H2O2 on melanocytes have not been fully understood, especially the indirect effects on melanocyte proliferation and differentiation from cells constituting surrounding tissue environment such as fibroblasts. OBJECTIVE: The aim of this study was to clarify whether H2O2-treated human fibroblasts affect the proliferation and differentiation of human melanocytes using serum-free co-culture system. METHODS: Epidermal melanoblasts and melanocytes were co-cultured with H2O2-treated or control fibroblasts in serum-free culture media. The effects of H2O2-treated fibroblasts were detected by changes in the proliferation and differentiation of melanoblasts/melanocytes. RESULTS: H2O2-treated fibroblasts stimulated the proliferation of melanoblasts and the differentiation, melanogenesis, and dendritogenesis of melanocytes, but inhibited the proliferation of melanocytes. In the melanocytes co-cultured with H2O2-treated fibroblasts, the expression of tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and KIT was increased, whereas TYRP2 and microphthalmia-associated transcription factor showed no change. CONCLUSION: These results suggest that H2O2-treated fibroblasts can produce and release some mitogenic and melanogenic factors toward melanoblasts in addition to some proliferation-inhibiting factors toward melanocytes. The stimulation of melanocyte differentiation seems to be performed through the upregulation of TYR, TYRP1, and KIT.

Platelet-derived growth factor regulates the proliferation and differentiation of human melanocytes in a differentiation-stage-specific manner.

BACKGROUND: Although many kinds of keratinocyte-derived factors are known to regulate the proliferation and differentiation of human melanocytes, it is not well defined whether dermis-derived factors work in a similar way. OBJECTIVE: The aim of this study is to clarify whether dermal factors are involved in regulating the proliferation and differentiation of human melanocytes. METHODS: Human epidermal melanoblasts were cultured serially in a serum-free growth medium. Platelet-derived growth factor-BB (PDGF-BB) was supplemented to the medium, and the effects on the proliferation of melanoblasts/melanocytes and the differentiation of melanocytes were studied. RESULTS: PDGF-BB stimulated the proliferation of melanoblasts cultured in melanoblast-proliferation medium, but inhibited the proliferation of melanocytes cultured in melanocyte-proliferation medium. By contrast, PDGF-BB stimulated the differentiation, dendritogenesis, and melanogenesis of melanocytes through the stimulation of tyrosinase activity and the expressions of tyrosinase and tyrosinase-related protein-1. CONCLUSION: These results suggest that PDGF-BB regulates the proliferation and differentiation of human melanocytes in a differentiation-stage-specific manner. PDGF-BB seems to be one of the dermal factors that regulate the proliferation and differentiation of human melanocytes.

Multipotency of melanoblasts isolated from murine skin depends on the Notch signal.

BACKGROUND: Melanoblasts (MBs), derived from neural crest cells, only differentiate into melanocytes (Ms) in vivo. We previously showed that MBs isolated from mouse skin were multipotent, generating neurons (Ns) and glial cells (Gs) together with Ms. Using Sox10-IRES-Venus mice and mouse embryonic stem cells, we investigated how MBs expressed their multipotency. RESULTS: MBs generated colonies containing Ns, Gs, and Ms in the presence of ST2 stromal cells, but they generated only M colonies when incubated with keratinocytes or ST2 culture supernatant, thus showing that MBs required contact with ST2 stromal cells for expression of their multipotency. Notch signaling was shown to be one of the important cues for the maintenance and differentiation of MBs through cell-cell contact. When Notch signaling was inhibited, MBs mainly generated colonies that contained just one type of cells, Ns, Gs, or Ms; the number of colonies containing two or three types of cells markedly decreased even on ST2 stromal cells, showing restriction of their differentiation potency. Whereas when Notch signaling was activated, the number of colonies containing two or three types of cells increased, indicating that their multipotency had been maintained. CONCLUSIONS: Our results demonstrate that Notch signaling played novel roles in MB multipotency.

The effects of gamma rays on the regeneration of hair follicles are carried over to later hair cycles.

PURPOSE: The purpose of this study was to determine whether the effects of gamma rays on the regeneration of hair follicles are carried over to later hair cycles. MATERIALS AND METHODS: The whole bodies of C57BL/10JHir mice in the 1st telogen phase were irradiated with (60)Co γ-rays. Mice were examined for the effects on hair follicles, including their number, morphology and pigmentation in the 3rd anagen phase. Effects of γ-rays on hair follicle stem cells were investigated by the indirect immunolabeling of keratin 15 (K15). RESULTS: Decreased hair follicle density and induction of curved hair follicles were observed in the dermis of irradiated skin. In addition, white hair and hypopigmented hair bulbs were found. The number of K15-positive hair follicle stem cells in the hair bulge region of irradiated skin appeared to decrease slightly but not significantly. CONCLUSIONS: These results suggest that the effects of γ-rays are carried over to a later hair cycle to affect the number, structure and pigmentation of hair follicles in the 3rd anagen phase when stem cells and committed progenitors for keratinocytes and melanocytes are irradiated in the 1st telogen phase.

l-tyrosine induces melanocyte differentiation in novel pink-eyed dilution castaneus mouse mutant showing age-related pigmentation.

BACKGROUND: The mouse pink-eyed dilution (oculocutaneous albinism II; p/Oca2(p)) locus is known to control tyrosinase activity, melanin content, and melanosome development in melanocytes. Pink-eyed dilution castaneus (p(cas)/Oca2(p-cas)) is a novel mutant allele on mouse chromosome 7 that arose spontaneously in Indonesian wild mice, Mus musculus castaneus. Mice homozygous for Oca2(p-cas) usually exhibit pink eyes and beige-colored coat on nonagouti C57BL/6 (B6) background. Recently, a novel spontaneous mutation occurred in the progeny between this mutant and B6 mice. The eyes of this novel mutant progressively become black from pink and the coat becomes dark gray from beige with aging. OBJECTIVE: The aim of this study is to clarify whatever differences exist in melanocyte proliferation and differentiation between the ordinary (pink-eyed) and novel (black-eyed) mutant using serum-free primary culture system. METHODS: The characteristics of melanocyte proliferation and differentiation were investigated by serum-free primary culture system using melanocyte-proliferation medium (MDMD). RESULTS: The proliferation of melanoblasts in MDMD did not differ between the two mice. However, when the epidermal cell suspensions were cultured with MDMD supplemented with l-tyrosine (Tyr), the differentiation of black-eyed melanocytes was greatly induced in a concentration-dependent manner compared with pink-eyed melanocytes. Immunocytochemistry demonstrated that the expression of tyrosinase and tyrosinase-related protein-1 (Tyrp1) was greatly induced or stimulated both in pink-eyed and black-eyed melanocytes, whereas the expression of microphthalmia-associated transcription factor (Mitf) was stimulated only in black-eyed melanocytes. CONCLUSION: These results suggest that the age-related coat darkening in black-eyed mutant may be caused by the increased ability of melanocyte differentiation dependent on l-Tyr through the upregulation of tyrosinase, Tyrp1, and Mitf. This mutant mouse may be useful for animal model to clarify the mechanisms of age-related pigmentation in human skin, such as melasma and solar lentigines.

The roles of Frizzled-3 and Wnt3a on melanocyte development: in vitro studies on neural crest cells and melanocyte precursor cell lines.

BACKGROUND: Wnt3a and Frizzled-3 are both expressed in the dorsal neural tube that gives rise to the neural crest in Xenopus, zebrafish and mice. Melanocytes originate from the neural crest (NC) and postnatally, melanocyte stem cells reside in the hair follicle bulge and in the dermis. However, the roles of Wnt3a and Frizzled-3 in melanocyte development have not been clarified. OBJECTIVE: The aim of this study was to delineate the expression of Frizzled-3 in murine melanocyte lineage and human melanocytes, and to study the effects of Wnt3a on melanocyte development at various stages. METHODS: Murine NC explant cultures and three NC-derived melanocyte lineage cell lines, including NCCmelb4M5 (Kit(-) melanocyte precursors), NCCmelb4 (Kit(+) melanoblasts) and NCCmelan5 (differentiated melanocytes), and human epidermal melanocytes were treated with pure recombinant Wnt3a protein and their cell behaviors were analyzed including their proliferation, Kit expression, tyrosinase (Tyr) activity, melanin production, dendrite formation and migration. RESULTS: Frizzled-3 was expressed in Tyr-related protein (TRP)-1(+) cells in NC explant cultures, in all 3 melanocyte precursor cell lines and in human melanocytes. Wnt3a increased the population of TRP-1(+) cells, the number of L-3,4-dihydroxyphenylalanine (DOPA)(+) cells and dendrite formation in NC explant cultures. Wnt3a stimulated the proliferation of all 3 melanocyte precursor cell lines in a dose-dependent manner and also stimulated human melanocyte proliferation. Moreover, Wnt3a increased Tyr activity and melanin content of differentiated melanocytes, but did not activate Tyr activity in melanoblasts. Wnt3a stimulated dendrite formation in differentiated melanocytes, but not in melanoblasts. Wnt3a did not affect melanoblast or melanocyte migration. Wnt3a did not induce c-Kit expression in Kit(-) NCCmelb4M5 cells and did not affect c-Kit expression in any cell line tested. CONCLUSIONS: Frizzled-3 is constitutively expressed in murine melanocyte precursors, melanocytes and human melanocytes. Wnt3a and Frizzled-3 signalings play important roles in regulating the proliferation and differentiation of murine NCCs and various developmental stages of melanocyte precursors. The effect of Wnt3a on human melanocytes is similar to its effects on murine melanocytes. Therefore Wnt3a/Frizzled-3 signaling is a promising target for human melanocyte regeneration.