The patchwork mouse phenotype: implication for melanocyte replacement in the hair follicle.

Mice homozygous for the recessive patchwork (pwk) mutation are characterized by a variegated pigment pattern with a mixture of unpigmented and normally pigmented hairs. The pigmented hair bulbs contain functional melanocytes. By contrast, the unpigmented hair bulbs contain no melanocytes. This lack results from the death of melanoblasts in the hair follicle at the end of embryogenesis. Here, we report that melanoblasts and melanocytes are found in the epidermis of pwk/pwk mice. Furthermore, these epidermal pigment cells are able to colonize new hair follicles after skin wounding. Despite the presence of epidermal pigment cells with a colonization potential, a follicle that had produced an unpigmented hair produces a new unpigmented hair during the successive hair growth cycles. This hair color continuity is also true for the pigmented hair follicles. Thus, in normal conditions, the hair acts as an independent functional unit as regards its pigment cells population.     

The Patchwork Mouse Phenotype: Implication for Melanocyte Replacement in the Hair Follicle

Mice homozygous for the recessive patchwork (pwk) mutation are characterized by a variegated pigment pattern with a mixture of unpigmented and normally pigmented hairs. The pigmented hair bulbs contain functional melanocytes. By contrast, the unpigmented hair bulbs contain no melanocytes. This lack results from the death of melanoblasts in the hair follicle at the end of embryogenesis. Here, we report that melanoblasts and melanocytes are found in the epidermis of pwk/pwk mice. Furthermore, these epidermal pigment cells are able to colonize new hair follicles after skin wounding. Despite the presence of epidermal pigment cells with a colonization potential, a follicle that had produced an unpigmented hair produces a new unpigmented hair during the successive hair growth cycles. This hair color continuity is also true for the pigmented hair follicles. Thus, in normal conditions, the hair acts as an independent functional unit as regards its pigment cells population.     

Different Populations of Melanocytes Are Present in Hair Follicles and Epidermis

Melanocytes in human skin reside both in the epidermis and in the matrix and outer root sheath of anagen hair follicles. Comparative study of melanocytes in these different locations has been difficult as hair follicle melanocytes could not be cultured. In this study we used a recently described method of growing hair follicle melanocytes to characterize and compare hair follicle and epidermal melanocytes in the scalp of the same individual. Three morphologically and antigenically distinct types of melanocytes were observed in primary culture. These included (1) moderately pigmented and polydendritic melanocytes derived from epidermis; (2) small, bipolar, amelanotic melanocytes; and (3) large, intensely pigmented melanocytes; the latter two were derived from hair follicles. The three sub-populations of cells all reacted with melanocyte-specific monoclonal antibody. Epidermal and amelanotic hair follicle melanocytes proliferated well in culture, whereas the intensely pigmented hair follicle melanocytes did not. Amelanotic hair follicle melanocytes differed from epidermal melanocytes in being less differentiated, and they expressed less mature melanosome antigens. In addition, hair follicle melanocytes expressed some antigens associated with alopecia areata, but not antigens associated with vitiligo, whereas the reverse was true for epidermal melanocytes. Thus, antigenically different populations of melanocytes are present in epidermis and hair follicle. This could account for the preferential destruction of hair follicle melanocytes in alopecia areata and of epidermal melanocytes in vitiligo.     

Efficacy of hydroquinone in the treatment of cutaneous hyperpigmentation in hairless descendants of Mexican hairless dogs (Xoloitzcuintli)

The skin of adult hairless dogs is clinically nonpigmented, clinically lightly pigmented, or clinically hyperpigmented (spotty pigmented). The pigment noted clinically is attributable to melanin granules in the epidermis. Spotty pigmentation in the skin of adult hairless dogs was treated by administration of the depigmenting agent (3% hydroquinone, HQ) for 1 month. Depigmenting effects were examined by use of three methods: skin color, dihydroxyphenylalanine (DOPA)-positive melanocyte count, and histologic evaluation. The treated skin of hairless dogs began to become depigmented after application of HQ for 1 week. After 1 month of treatment with HQ, depigmentation spread over a quarter of the body. The number of DOPA-positive melanocytes in the HQ-treated sites decreased to less than approximately a fifth of that before treatment. In HQ-treated skin, histologic staining by use of Fontana-Masson's (FM) method revealed complete absence of melanin pigment. These results suggested that hairless dogs should be a useful animal model for investigating the effects and cutaneous toxicity of depigmenting agents.     

Melanocytes in Human Embryonic and Fetal Skin: A Review and New Findings

Melanocytes account for approximately 5–10% percent of the cells in adult epidermis. Unlike the ectodermally derived keratinocytes, they originate in the neural crest and migrate into the epidermis early in development. There has been an interest in melanocytes in developing human skin since the late 1800s, when concentrated pigmented cells were identified in the sacro-coccygeal skin of Japanese fetuses. This observation led to speculation and subsequent investigation about the racial nature of the melanocytes in this site (the Mongolian spot), the presence of melanocytes in fetuses of other races, the timing of appearance of these cells in both the dermis and epidermis, and their origin. The early investigators relied primarily on histochemical methods that stained either the premelanosome or the pigmented melanosome, or relied upon the activity of tyrosinase within the melanosome to effect the DOPA reaction. Studies by electron microscopy added further documentation to the presence of melanocytes in the skin by resolving the structure of the melanosome regardless of its state of pigmentation. All of these methods recognized, however, only differentiated melanocytes. The thorough investigations of melanocytes in the skin from a large number of black embryos and fetuses by Zimmerman and colleagues between 1948 and 1955 provided insight into the time of appearance of melanocytes in the dermis (10–11 weeks' menstrual age) and the epidermis (11–12 weeks) and revealed the density of these cells in both zones of the skin of several regions of the body. The precise localization of the melanocytes in the developing hair follicles was contributed by the studies of Mishima and Widlan (J Invest Dermatol 1966; 46:263–277). More recently, monoclonal antibodies have been developed that recognize common oncofetal or oncodifferentiation antigens on the surface or in the cytoplasm of melanoma cells and developing melanocytes (but not normal adult melanocytes). These antibodies recognize the cells irrespective of the presence or absence of melanosomes or their activity in the synthesis of pigment and therefore are valuable tools for re-examining the presence, density, and distribution patterns of melanocytes in developing human skin. Using one of these antibodies (HMB-45), it was found that dendritic melanocytes are present in the epidermis between 40 and 50 days estimated gestational age in a density comparable with that of newborn epidermis and are distributed in relatively non-random patterns. A number of questions about the influx of cells into the epidermis, potential reservoirs of melanoblasts retained within the dermis, division of epidermal melanocytes, and the interaction of melanocytes and keratinocytes during development remain unresolved. The tools now appear to be available, however, to begin to explore many of these questions.     

Hairless pigmented guinea pigs: a new model for the study of mammalian pigmentation

A stock of hairless pigmented guinea pigs was developed to facilitate studies of mammalian pigmentation. This stock combines the convenience of a hairless animal with a pigmentary system that is similar to human skin. In both human and guinea pig skin, active melanocytes are located in the basal layer of the interfollicular epidermis. Hairless albino guinea pigs on an outbred Hartley background (CrI:IAF/HA(hr/hr)BR; designated hr/hr) were mated with red-haired guinea pigs (designated Hr/Hr). Red-haired heterozygotes from the F1 generation (Hr/hr) were then mated with each other or with hairless albino guinea pigs. The F2 generation included hairless pigmented guinea pigs that retained their interfollicular epidermal melanocytes and whose skin was red-brown in color. Following UV irradiation, there was an increase in cutaneous pigmentation as well as an increase in the number of active epidermal melanocytes. An additional strain of black hairless guinea pigs was developed using black Hr/Hr animals and a similar breeding scheme. These two strains should serve as useful models for studies of the mammalian pigment system.     

Pigmented spots in the wool-bearing skin on white merino sheep induced by ultraviolet light

Black-grey pigmented skin spots, some of which contained pigmented wool fibres, were observed in a flock of 8.5-year-old white Merino ewes. The spots were concentrated along the backline and increased in number following shearing, suggesting exposure to sunlight to be of importance in the development of these non-congenital pigmented skin spots in genetically white Merino sheep. To test the effect of ultraviolet light, white Merino sheep, ranging in age from 3 to 8 years, had a closely clipped midside area of wool-bearing skin irradiated on each of 28 consecutive days. Pigmented skin spots developed in 6 of the 16 white Merino sheep irradiated. Spots first appeared after 10 days of irradiation, the number subsequently increasing with time, and two skin spots were found to contain sparse numbers of black-grey pigmented wool fibres. Histological examination showed both the naturally occurring and irradiation-induced pigmented skin spots resulted from an increase in both number and activity of melanocytes localized along the epidermal-dermal border of the epidermis. With time, the melanocytes were observed to have entered, to varying depths, the outer-root sheath of follicles still producing white wool fibres. These ultraviolet-light-induced changes to epidermal melanocytes in white Merino sheep presumably occur due to alterations within the local tissue environment in which the melanocytes lie.     

Approaches to Repigmentation of Vitiligo Skin: New Treatment with Ultrasonic Abrasion,Seed‐Grafting and Psoralen Plus Ultraviolet A Therapy

Vitiligo vulgaris is a common disease throughout the world although its pathogenesis is not yet known. The most frequent treatment used for vitiligo is PUVA (psoralen plus ultraviolet A) and topical steroids but against stable refractory vitiligo, various other surgical techniques have been developed such as autografting, epidermal grafting with suction blisters, epithelial sheet grafting, and transplantation of cultured melanocytes. We have discovered a new method using ultrasonic abrasion, seed‐grafting and PUVA therapy. The ultrasonic surgical aspirator abrades only the epidermis of recipient sites. This easily and safely removes only the epidermis, even on spotty lesions or intricate regions which are difficult to remove using a conventional motor‐driven grinder or liquid nitrogen. Epidermal seed‐grafting can cover more area than sheet‐grafting, and subsequent PUVA treatment can enlarge the area of pigmentation with coalescence of adjacent grafts. In this article, we provide a general overview of the current surgical therapies including our method for treating stable refractory vitiligo.     

A variation of pigmentation in the glabrous skin of dogs

The usual pigmentation pattern in mammalian skin consists of fixed melanocytes in the basal layer of the epidermis, supplying keratinocytes with melanosomes. We observed that the glabrous skin (rhinaria and footpads) of dogs deviates from this pattern. In dogs, melanocytes are found in both the dermis and epidermis. The epidermal melanocytes are situated in the intercellular spaces of the basal and spinous layers. They are characterized by a quantity of cytoplasm containing a centriole, also developing melanosomes, and in some cases annulate lamellae. There is a high frequency of closely apposed melanocytes in the epidermis. Melanosomes in different stages of formation are also abundant. The morphology of the glabrous skin of dogs suggests transport of melanocytes from the dermis into the epidermis and formation of melanosomes in the epidermis. A distributed and intense pigment formation may be necessary to achieve the black noses of many dog breeds and wild canids, as well as dark footpads despite heavy abrasion and rapid skin renewal.     

Approaches to repigmentation of vitiligo skin: new treatment with ultrasonic abrasion,seed-grafting and psoralen plus ultraviolet A therapy

Vitiligo vulgaris is a common disease throughout the world although its pathogenesis is not yet known. The most frequent treatment used for vitiligo is PUVA (psoralen plus ultraviolet A) and topical steroids but against stable refractory vitiligo, various other surgical techniques have been developed such as autografting, epidermal grafting with suction blisters, epithelial sheet grafting, and transplantation of cultured melanocytes. We have discovered a new method using ultrasonic abrasion, seed-grafting and PUVA therapy. The ultrasonic surgical aspirator abrades only the epidermis of recipient sites. This easily and safely removes only the epidermis, even on spotty lesions or intricate regions which are difficult to remove using a conventional motor-driven grinder or liquid nitrogen. Epidermal seed-grafting can cover more area than sheet-grafting, and subsequent PUVA treatment can enlarge the area of pigmentation with coalescence of adjacent grafts. In this article, we provide a general overview of the current surgical therapies including our method for treating stable refractory vitiligo.     

Dermal melanocytosis in Japanese monkeys (Macaca fuscata)

The skin of Japanese monkeys (Macaca fuscata) shows diffuse discolorations resembling human dermal melanocytosis. Very few laboratory animals have melanocytes in the dermis. The purpose of this study was to clarify the dermatologic characteristics of Japanese monkeys in terms of gross appearance, skin color, and histopathologic findings. A colorimeter was used to record the skin colors of pigmented and nonpigmented sites. Tissue specimens obtained from both types of sites were examined histopathologically. All animals examined had pigmented sites on their bodies, and the discolorations extended over 25% to 33% of the body surface. The colorimeter could detect differences in skin color due to dermal melanocytosis. All parameters of the colorimetric systems used (Yxy, L*a*b*, and L*C*h* systems) demonstrated significant differences between pigmented and nonpigmented sites. In pigmented sites, the epidermis lacked melanocytes, but the dermis had numerous melanocytes with abundant melanin. Activated melanocytes with well-developed dendrites were distributed throughout the upper part of the dermal layer. Melanocytes were not arranged in clusters, and elastic and collagen fibers in the dermis showed no histological abnormalities. Nonpigmented sites lacked melanin granules in both the epidermis and dermis. This study revealed that gross dermal melanocytosis correlated well with colorimetric results and histopathologic findings. These findings suggest that the pigmentation of Japanese monkeys is equivalent to dermal melanocytosis in humans, to the end that Japanese monkeys may be a useful animal model for investigating dermal melanogenesis.     

The epidermis as a graft

Laboratory epidermal autotransplantation was performed on the surface of a full-thickness skin defect using mongrel female rats. Epidermal graft represented suction blister roofs, formed as the result of the donor skin site treatment with lowered up to -0.6 kg/cm2 pressure. It contained all epidermal cell layers. Following 1, 7 and 28 days after the transplantation recipient bed sites containing grafted epidermis were excised and histological study war performed. It was demonstrated that epidermal graft received by the method described was able to grow as well as to differentiate on the surface of a full-thickness skin defect.     

Mitotic activity in non-neoplastic melanocytes in vivo as determined by histochemical, autoradiographic, and electron microscope studies

Mitotic figures were demonstrated in the differentiated melanocytes of normal epidermal and nonepidermal tissues without the presence of external stimuli. These dividine melanocytes were present in human and mouse skin, mouse hair, chick feathers, and embryonic chick retinal pigment epithelium. In normal adult human epidermis, dividing melanocytes, though rare, were found in the nonstimulated areas. L-3,4- dihydroxyphenylalanine reaction on the melanocytes during mitosis demonstrated activity of the melanin-forming enzyme, tyrosinase, and ultrastructural studies demonstrated the characteristic melanosomes in variour stages of maturation. Other ultrastructural characteristics of the melanocytes during mitosis, except for the Golgi apparatus, which was smaller and less complex, were similar to those seen in well- differentiated nondividing melanocytes. Autoradiographic studies of thymidine incorporation into mouse skin indicated that 0.7% of epidermal melanocytes, when slightly stimulated, are in the S phase. Thus, in vivo differentiation of non-neoplastic melanocytes (to produce pyrosinase and melanosomes) does not preclude their replication by mitotic division.     

Clinical interest of cutaneous models reproducedin vitro for severe burn treatment: histopathological and ultrastructural study

The healing of minimal skin lesions is usually obtained by epidermal migration and proliferation from peripheral wound margins. However, cutaneous grafts or reconstituted skin are necessary for severe injuries. Various models have recently been reproduced for this purpose. The aim of this work is to report the histopathologic evolution of burn lesions treated two years ago by autologous epidermis (Genzyme Tissue Repair, Boston, USA). Fifteen patients with severe burns (more than 80% of surface) have been treated. These observations have been based exclusively on biopsies of grafted wounds. Cultured epidermis is rapidly fully differentiated after grafting with temporary hyperplasia and normal strata. At 18 months, rete ridges formation is present only in young patients. Melanocytes and Langerhans' cells repopulated grafts rapidly. The use of cultured epidermis nowadays represents an important improvement in burn treatment.Abbreviations CEA cultured epidermal autologous sheets - TBSA total burn surface area     

Organotypic Culture of Human Skin to Study Melanocyte Migration

An ex vivo model system was developed to investigate melanocyte migration. Within this model system, melanocytes migrate among other epidermal cells in the epibolic outgrowth of skin explants. This process is initiated by loss of contact inhibition of epidermal cells at the rim of the explants and by locally produced chemotactic factors. Punch biopsies provided explants of reproducible diameter. Optimal culture conditions include medium consisting of Dulbecco's Minimal Essential Medium containing 10% inactivated normal human serum and placement of explants epidermal side up at the air-liquid interphase. Within 7 days, epidermal cells completely surround the explant. Approximately 3 days after the onset of keratinocyte migration, melanocytes distribute themselves within the newly formed epidermis. Throughout the 7-day culture period, melanocytes and keratinocytes show maintenance of subcellular morphology, and the dermo-epidermal junction remains intact. Melanocyte migration was quantified using immunoperoxidase staining in combination with light microscopy and computer-aided image analysis. Preliminary results using the model system to compare migration in control and nonlesional vitiligo skin indicate that no inherent migration defect is responsible for impaired repigmentation of vitiligo lesions. The organotypic culture model system allows for investigations on melanocytes within their environment of autologous epidermal and dermal components, closely resembling in vivo circumstances in human skin.     

Restoration of cutaneous pigmentation by transplantation to mice of isogeneic human melanocytes in dermal–epidermal engineered skin substitutes

Autologous engineered skin substitutes (ESS) containing melanocytes (hM) may restore pigmentation and photoprotection after grafting to full‐thickness skin wounds. In this study, normal hM were isolated from discard skin, propagated with or without tyrosinase inhibitors, cryopreserved, recovered into culture, and added to ESS (ESS‐P) before transplantation. ESS‐P were incubated in either UCMC160/161 or UCDM1 medium, scored for hM densities, and grafted to mice. The results showed that sufficient hM can be propagated to expand donor tissue by 100‐fold; incubation of hM in tyrosinase inhibitors reduced pigment levels but did not change hM recovery after cryopreservation; hM densities in ESS‐P were greater after incubation in UCDM1 than UCMC160 medium; hM were localized to the dermal–epidermal junction of ESS‐P; and UCDM1 medium promoted earlier pigment distribution and density. These results indicate that hM can be incorporated into ESS‐P efficiently to restore cutaneous pigmentation and UV photoprotection after full‐thickness skin loss conditions.     

Eumelanin and Phaeomelanin Contents of Human Epidermis and Cultured Melanocytes

There are two chemically distinct types of melanin: the red-yellow phaeomelanins and the brown-black eumelanins. While both melanins have been detected in human epidermis and cultured melanocytes, it is unknown how the phaeomelanin/eumelanin ratio in human melanocytes maintained in vitro relates to that in the epidermis from which they were isolated. This study uses high-performance liquid chromatography to quantify the eumelanin and phaeomelanin contents of epidermis and/or cultured melanocytes from 12 Europeans with lightly pigmented skin and 9 non-Europeans with more deeply pigmented skin. Epidermis from non-Europeans contained the highest levels of both eumelanin and phaeomelanin and had the lowest phaeomelanin/eumelanin ratios. In contrast, while cultured melanocytes from non-Europeans also had higher levels of eumelanin and phaeomelanin than melanocytes from Europeans, there was no difference in the phaeomelanin/eumelanin ratios in the two groups. However, the phaeomelanin/eumelanin ratios were higher in the cultured melanocytes than in the corresponding epidermis so that while eumelanin was the predominant melanin in the epidermis, phaeomelanin was the major melanin in the cultured melanocytes. These observations may have important implications for the use of cultured human melanocytes in the study of melanogenesis in man.     

Testosterone regulation of pigmentation in scrotal epidermis of the rat

Summary The effects of testosterone on melanocyte number, morphology, melanin content and tyrosinase activity were studied in epidermis from several body regions of the black-pelted Long-Evans rat. Determinations were made in epidermal sheets processed for histochemical analysis by incubation in the presence of the melanin precursor, 3,4-dihydroxyphenylalanine (DOPA). Melanin content, cell volume, dendritic branching and tyrosinase activity of scrotal epidermal melanocytes all decreased progressively with time following castration. Daily testosterone injection, begun 14 days after castration, increased tyrosinase activity in 4 days, and dendritic branching in 6 days, of treatment; melanin content, cell volume and enzyme activity were restored to normal intact levels within 14 days of treatment, at which time newly synthesized melanin was evident in keratinocytes. The total number of scrotal epidermal melanocytes was not changed by castration or testosterone administration. Neither castration nor testosterone replacement affected any parameter of epidermal melanocytes in preputial, perianal or eyelid skin which, together with the scrotum, are the animals' only pigmented areas. Androgen control of epidermal pigmentation in the male rat is therefore specific for the scrotum and is manifested through regulation of melanin synthesis in stable populations of melanocytes rather than through increases in numbers of melanocytes.This work was supported in part by research grant no. HD 00446, and training grant no. HD 00152, from the Institute of Child Health and Human Development, Public Health Service.     

Demonstration of the intermediate cells of Billingham and Medawar in the epidermis of Macaca mulatta and rhesus monkey.

The digest of the pure epidermis preparation from Macaca mulatta demonstrated three types of dendritic cells i.e. (1) dopa-positive melanocytes, (2) dopa-negative Langerhans' cells and (3) weakly dopa-positive intermediate cells of Billingham and Medawar. The last type of cell was found to have characteristics being intermediate between those of the melanocytes and of the Langerhans' cell. A similar digest prepared from the epidermis of the rhesus monkey demonstrated isomorphous dendritic cells which were all dopa-negative. Here also the intermediate cells of Billingham and Medawar with characteristics intermediate between those of the melanocytes and of the Langerhans' cells were identified.