An In Vivo Mouse Model of Human Skin Substitute Containing Spontaneously Sorted Melanocytes Demonstrates Physiological Changes after UVB Irradiation

An In Vivo Mouse Model of Human Skin Substitute Containing Spontaneously Sorted Melanocytes Demonstrates Physiological Changes after UVB Irradiation Akira Hachiya,w Penkanok Sriwiriyanont,z Eiko Kaiho, Takashi Kitahara, Yoshinori Takema, and Ryoji Tsuboiw

Kao Biological Science Laboratories, Haga, Tochigi, Japan; wDepartment of Dermatology, Tokyo Medical University, Nishishinjuku, Shinjuku-ku, Tokyo , Japan;

zThe Skin Sciences Institute, Cincinnati Children’s Hospital Research Foundation, Cincinnati, Ohio, USA

Human skin substitutes (HSS) have been developed for repairing burns and other acute or chronic wounds. But although the clinical utility of HSS is well known, scant attention has been paid to their cosmetic properties, especially with regard to color compatibility with the patient’s complexion. In this study, we generated an HSS from mixed cell slurries containing keratinocytes and fibroblasts with and without melanocytes on the back of severe combined immunodeficient mice by means of a spontaneous cell-sorting technique. At 16 wk after grafting, Caucasian donor-derived HSS with melanocytes were macroscopically clearly darker than those without melanocytes, and a more darkly pigmented HSS was produced when cells from donors of African descent were seeded. Immunohistochemistry of c-kit, S-100, and HMB45, as well as Fontana–Masson staining and transmission electron microscopy (TEM) demonstrated that melanocytes spontaneously localized to the basal layer. Melanosome transfer to keratinocytes was correctly reorganized, and melanin was evenly dispersed in the basal and suprabasal layers. Colorimetric analysis showed a significantly lower L-value by day 14 following irradiation with 120 mJ per cm2 ultraviolet-B (UVB) (po0.01), whereas epidermal thickness increased by 50% 1 d after exposure (po0.01), indicating a normal physiological response to UVB irradiation. These findings suggest that HSS with spontaneously sorted melanocytes offer a means of treating both the structural and cosmetic aspects of skin conditions and trauma, such as pigmentary disorders and skin wounds, by allowing manipulation of the color and population of donor melanocytes.

Key words: human melanocytes/human skin substitute/pigmentation/spontaneous sorting/UVB J Invest Dermatol 125:364 – 372, 2005

Many attempts have been made to repair burns and other acute or chronic wounds using human skin substitutes (HSS) prepared from autologous or allogeneic cultured keratinocytes (Green et al, 1979; Bell et al, 1981; Bell and Rosenberg, 1990; Beumer et al, 1993; Boyce et al, 1996; Berthod and Damour, 1997). Initial efforts in this field focusing on developing suitable substitutes for the epidermis met with mixed results in clinical use (Green et al, 1979; Hefton et al, 1983; Gallico et al, 1984). Conventional HSS have been prepared by superimposing a sheet of partially differentiated keratinocytes on cadaver dermis (Krejci et al, 1991; Ben-Bassat et al, 1992), a dermal equivalent of bovine collagen and chondroitin sulfate, or a collagen–glycosaminoglycan matrix containing fibroblasts (Burke et al, 1981; Hansbrough et al, 1989; Cooper and Hansbrough, 1991; Boyce et al, 1993). Most of these conventional models use two separate skin layers, the epidermis and the dermis, indicating the need to engineer a dermal–epidermal

junction between the two independently formed layers. Cultured autografts, however, have been found wanting in several respects, including initial graft ‘‘take,’’ susceptibility to infection, adherence to the graft bed, long-term graft stability, and prevention of wound contraction under circumstances where cultured epidermal autografts of cultured keratinocytes are created in large quantities from small donor biopsies (Cuono et al, 1986; Madden et al, 1986; De Luca et al, 1989). In addition, some of the anatomical and physiological weaknesses of cultured cells used to treat wounds may be attributed to deficiencies in the nutrient content of the medium used, especially of vitamins (Bettger and Ham, 1982; Bettger and McKeehan, 1986). It was recently reported that the inherent cell adhesive peculiarities of keratinocytes and dermal fibroblasts present an opportunity to reconstitute full-thickness human skin by sorting out the heterogeneous cell population (Wang et al, 2000), suggesting a novel approach to reconstituting HSS. This novel technique involves pouring a cell slurry containing keratinocytes and fibroblasts into silicone chambers implanted directly on the muscle fascia of severe combined immunodeficient (SCID) mice. When the authors compared

Abbreviations: ET-1, endothelin-1; HSS, human skin substitutes; MED, minimal erythema dose; OCT, optical coherence tomography; SCF, stem cell factor; SCID, severe combined immunodeficiency; TEM, transmission electron microscopy; UVB, ultraviolet-B

Copyright r 2005 by The Society for Investigative Dermatology, Inc.

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the cell-sorted cultured HSS with the conventional composite skin model, the former demonstrated greater numbers of keratin-intermediate filaments within basal keratinocytes connected to hemidesmosomes and collagen filaments, and more numerous connections of anchoring fibrils in the dermis to the lamina densa of the basement membrane. Besides the fundamental improvements in methodology, improvements in the nutritional quality of the culture medium are also reported to have made HSS more effective when grafted onto athymic mice. For instance, it has been suggested that the addition of vitamin C (ascorbic acid) develops the functional epidermal barrier earlier, reduces wound contraction, and increases HLApositive substitutes after grafting (Gunzler et al, 1988; Saika et al, 1991; Quarles et al, 1992; Boyce et al, 2002). As described above, several trials have been conducted to refine HSS. Scant attention has been paid to the cosmetic aspect of HSS, however, especially with regard to color compatibility with the patient’s complexion. So far, only ex vivo reconstructions of the epidermis with keratinocytes and melanocytes have been reported (Bessou et al, 1995; Bessou-Touya et al, 1998; Cario-Andre et al, 1999). For this reason, mice with melanocytes functioning as they do in normal human skin in situ provide an attractive model for preparing HSS using the new spontaneous cell-sorting technique. Objective evaluations are essential for assessing the function of the HSS. All the previous evaluations, however, were performed just 2–4 wk after grafting, a stage at which reconstruction of the epidermal structure was not yet complete, and scabs were frequently observed in our previous studies (data not shown). If the color of HSS could be regulated precisely, such a technique would be valuable in the treatment of hyper- and hypo-pigmentary disorders, such as senile fleck and vitiligo, in addition to the restoration of wounds. In this study, we examined whether cultured human melanocytes in a cell slurry are sorted to the basal layer in the epidermis with the use of spontaneous cell-sorting methods and if so, whether this results in pigmentation in HSS comparable to the pigmentation features of the original donor skin. We further examined whether exposure of HSS containing melanocytes to ultraviolet-B (UVB) irradiation would stimulate melanogenesis as it does in normal human skin in situ. Here, we report that melanocytes spontaneously sorted to the basal layer of the epidermis in HSS prepared

Figure 1 Macroscopic observation of human skin substitutes (HSS) containing melanocytes. A mixed cell slurry containing approximately 6
106 keratinocytes and 6
106 fibroblasts with or without 1
106 melanocytes was poured into a chamber implanted onto the backs of severe combined immunodeficiency (SCID) mice as detailed in ‘‘Materials and methods’’. Photographs were taken approximately 16 wk after grafting. (A) Caucasian donor-derived HSS without melanocytes, (B) Caucasian donor-derived HSS with melanocytes, and (C) African descent donor-derived HSS with melanocytes. Scale bar ¼ 5 mm.

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on the back of SCID mice produced pigmentation comparable to the pigmentation features of the original donor skin, and that melanin synthesis and the epidermal thickness in these substitutes increase significantly after UVB exposure.

Results Cultured human melanocytes that spontaneously sorted to the epidermal basal layer of the HSS synthesized melanin, transferred melanosomes to keratinocytes, and produced pigmentation corresponding to the original complexion of the donor’s skin. In order to prove the hypothesis that HSS would become pigmented if the preparation included human melanocytes, we began by observing the surface of the HSS macroscopically. Approximately 16 wk after grafting, a certain portion of the HSS was retained on the tissue surface without scabbing and displayed a sheen very similar to that of human skin. Caucasian donor-derived HSS with melanocytes were macroscopically clearly darker than those without melanocytes (Fig 1A and B). In addition, more darkly pigmented HSS was produced when cells from donors of African descent were seeded in the same population and in the same ratio as that of the Caucasian donors (Fig 1C), indicating that the HSS produced pigmentation corresponding to the original complexion of the donor. The localization of melanin and melanocytes in the HSS with Caucasian donor-derived cells was then histologically examined using immunohistochemical staining with antibodies to the melanocyte-specific antigens, c-kit, S-100 and HMB45, Fontana–Masson staining, and transmission electron microscopy (TEM) analysis. The HSS without melanocytes, prepared from the mixed cell suspension with keratinocytes and fibroblasts demonstrated a normal skin structure with epidermal and dermal compartments (Fig 2A and B). The reconstructed epidermis consisted of basal, spinous, granular, and horny layers as in normal human skin (Fig 2A and B). The HSS containing melanocytes, prepared from the mixed cell suspension with three different types of cell, also led to the formation of both a normal epidermal structure as well as a reconstructed dermis (Fig 2C–G). In addition, it had a prominent rete ridge formation and acanthotic epidermis compared with the HSS without melanocytes. Immunoreactivity to each of the three antibodies mentioned above clearly showed that most melanocytes added to the suspension spontaneously sorted to the basal layer, comparatively few to the suprabasal layer, of the reconstructed epidermis (Fig 2C–E). The melanocytes were individually distributed mainly at the tips of the rete ridge of the epidermis. Meanwhile, neither immunoreactivity to the antibodies in the HSS without melanocytes nor immunoreactivity to control non-specific IgG in the HSS with melanocytes was positively observed (Fig 2A, B, F and G). As shown in Fig 2D, S100-positive cells were also detected in the dermis. They seemed to be mouse Langerhans cells migrating from SCID mouse as suggested by the fact that the S-100 antibody was also reported as recognizing mouse dendritic cells, and the fact that the cells themselves were also positive to another antibody BM8, anti-mouse F4/80 antigen (data not shown). Fontana–Masson staining showed that melanin

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Figure 2 Sorted melanocytes and melanin were observed at the basal layer of human skin substitutes (HSS). Immunohistochemical analysis was performed using antibodies to the melanocyte-specific antigens. In Caucasian donor-derived HSS without melanocytes, immunostaining was performed with anti-c-kit (A) and normal rabbit IgG (B). In Caucasian donor-derived HSS with melanocytes, immunostaining was performed with anti-c-kit (C), anti-S-100 (D), HMB45 antibody (E), normal rabbit IgG (F), and normal mouse IgG (G). Melanin was stained in the HSS by Fontana–Masson staining 16 wk after grafting. (H) Without melanocytes and (I) with melanocytes. Scale bar ¼ 50 mm.

was evenly dispersed in contiguous cells of the basal and suprabasal layers in the HSS with melanocytes, whereas it was not observed in the HSS without melanocytes (Fig 2H and I), suggesting that melanosome transfer is correctly reorganized in the reconstructed HSS. In the substitutes containing melanocytes, TEM analysis further demonstrated that melanosomes were transferred from melanocytes to keratinocytes (Fig 3A and B). Clusters of melanosomes in the keratinocytes were distributed from the peripheries of the cytoplasm to the immediate vicinity of the nucleus. UVB irradiation increased melanogenesis and induced acanthosis in HSS with melanocytes It has been reported that melanogenesis is stimulated in the basal layer of the human epidermis as a result of UVB irradiation (Pathak and Fanselow, 1983). In order to examine whether this response occurred in the HSS containing spontaneously sorted melanocytes, a certain area of the HSS was exposed to UVB at a dose of 120 mJ per cm2, corresponding to approximately 2 MED (minimal erythema dose) in Caucasian skin. When Fontana–Masson staining was carried out 14 d after irradiation, melanin granules were significantly observed in the UVB-exposed region of the Caucasian donor-derived HSS with melanocytes (Fig 4A), whereas melanin granules were abundantly seen in HSS derived from donors of African descent, even in portions shielded from UVB, and no more increase in melanogenesis was observed in the African descent-derived HSS after UVB irradiation (Fig 4B). At a higher magnification (Fig 4C and D), melanin bodies were observed mainly in the upper portion of the cytoplasm of the basal and suprabasal keratinocytes (supranuclear caps), identical to the phenomenon observed in the normal human epidermis in situ (Kobayashi et al, 1998). For quantitative

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Figure 3 Melanosomes were found clustering in the peripheries of keratinocyte nuclei in Caucasian donor-derived human skin substitutes (HSS). Transfer of melanosomes from melanocytes to keratinocytes was examined by transmission electron microscopy. (A) Caucasian donor-derived HSS without melanocytes, (B) Caucasian donor-derived HSS with melanocytes. White and black bars on the left margin of each figure indicate the extent of epidermal keratinocytes and dermal fibroblasts, respectively. Clusters of melanosomes in keratinocytes were distributed from the peripheries of the cytoplasm to the immediate vicinity of each keratinocyte nucleus shown. Scale bar ¼ 2 mm. KC, keratinocyte cytoplasm; KN, keratinocyte nucleus; MS, melanosomes; MC, melanocyte cytoplasm.

assessment of skin pigmentation, a colorimetric analysis was performed. In the group of Caucasian donor-derived HSS with melanocytes, the L value had significantly decreased by day 14 (po0.01), with the HSS becoming darker, whereas the L value in Caucasian donor-derived HSS without melanocytes had not changed (Fig 5).

Figure 4 Ultraviolet-B (UVB) irradiation increased melanin synthesis in Caucasian donor-derived human skin substitutes (HSS). Enhancement of melanin synthesis was observed in the Caucasian HSS by Fontana– Masson staining 14 d after UVB irradiation. (A) Caucasian donor-derived HSS. The arrow indicates the border of the region exposed to UVB. The left side shows the non-exposed area and the right side shows the exposed area. Scale bar ¼ 50 mm. (B) HSS derived from donors of African descent. The arrow indicates the border of the region exposed to UVB. The left side shows the non-exposed area and the right side shows the exposed area. Scale bar ¼ 50 mm. (C) Caucasian donor-derived HSS at higher magnification. Melanin granules were observed in the upper portion of the cytoplasm of basal and suprabasal keratinocytes (supranuclear caps) in the HSS following UVB exposure. Scale bar ¼ 10 mm. (D) HSS derived from donors of African descent at higher magnification. Melanin granules were abundantly seen in the upper portion of the cytoplasm of basal, suprabasal, and spinous layers of keratinocytes in the HSS without UVB exposure. Scale bar ¼ 10 mm.

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Figure 5 Intensity of pigmentation in Caucasian donor-derived human skin substitutes (HSS) at various time points following ultraviolet-B (UVB) irradiation. The intensity of UVB-induced pigmentation, measured by using a colorimeter, was expressed as an L value. The open circle at each day represents the average value of the HSS without melanocytes, whereas the gray circles represent the average values of the HSS with melanocytes. The values represent the mean  SD from four individual HSS. po0.05, po0.01.

Epidermal hyperplasia and hypertrophy are kinds of skin response frequently observed after UVB exposure (Kligman, 1986). In order to determine whether these processes also occur in HSS, epidermal thickness was measured with the optical coherence tomography (OCT) technique. In brief, the layered skin structures such as the horny layer, epidermis, and dermis were visualized by means of refractive index evaluation in the non-invasive OCT technique (Knuttel and Boehlau-Godau, 2000). Typical images of the scanned HSS are shown in Fig 6. An increase in the epidermal thickness (acanthosis) was apparent 1 d after UVB exposure and progressed until day 14. The average epidermal thickness of the four Caucasian donor-derived HSS with melanocytes (Fig 7) was about 80 mm before UVB irradiation, approximating closely the thickness of normal human epidermis in situ. One day after UVB exposure, the epidermal thickness increased by about 50% (po0.01), and acanthosis continued to day 14. Increase in stem cell factor (SCF) and endothelin-1 (ET-1) expression seen in UVB-induced pigmentation in HSS with melanocytes It has been reported that SCF and ET-1 are upregulated at the gene and protein levels in the human epidermis 3–5 and 5–10 d after 2 MED UVB irradiation, respectively, and that they play a central role in UVBinduced pigmentation (Imokawa et al, 1995; Hachiya et al, 2001, 2004). In order to determine whether these cytokines also play a role in the HSS containing spontaneously sorted melanocytes, the entire surface area of the HSS composed of Caucasian donor-derived cells was exposed to UVB as

Figure 6 Ultraviolet-B (UVB)-irradiation induced acanthosis of the Caucasian donor-derived human skin substitutes (HSS). Photographs were taken using the optical coherence tomography technique. The area lying between the arrows represents the epidermal thickness of the HSS. (A) Before UVB exposure; (B) 1 d after UVB exposure; (C) 2 d after UVB exposure; and (D) 14 d after UVB exposure. Scale bar ¼ 100 mm.

previously stated, and the mRNA transcript expression of SCF and that of preproendothelin-1 (prepro-ET-1) and tyrosinase was examined 3 and 10 d after the exposure, respectively. Real-time quantitative RT-PCR analysis of UVB-exposed epidermis demonstrated that the expression of SCF mRNA transcripts had significantly (po0.05) increased 3 and 5 d (data not shown) after irradiation compared with the non-irradiated epidermis (Fig 8A). On the other hand, the expression of prepro-ET-1 and tyrosinase mRNA transcripts in the UVB-exposed epidermis significantly (po0.05) increased 10 days after irradiation, compared with the non-exposed epidermis (Fig 8B and C). Similar phenomena were observed in UVB-irradiated human skin (Hachiya et al, 2004).

Discussion The spontaneous cell-sorting technique was reported to be a distinct improvement upon the conventional types of HSS aimed at developing a basement membrane zone, especially the dermal–epidermal junction, and increasing the

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Figure 7 Epidermal thickness of the Caucasian donor-derived human skin substitutes (HSS) at various time points following ultraviolet-B (UVB) irradiation. The thickness of the epidermis was measured by using photographs taken by means of the optical coherence tomography technique as detailed in Materials and methods. The open circle at each day represents the average value of the HSS after UVB irradiation, whereas the open squares represent the average values of the non-irradiated HSS. The values for UVB-irradiated HSS and non-irradiated HSS represent the mean  SD from four and seven individual HSS, respectively. po0.05, po0.01.

numbers of intermediate filaments (Wang et al, 2000). HSS manufactured by using the cell-sorting technique also form rete ridges in the reconstituted epidermis, compared with the flat epidermis found in the conventional HSS. Although skin equivalents can be used with skin of various complexions, prior to this study they had not been designed and tested with respect to the intensity and evenness of skin color. Where conventional types of HSS used on athymic mice are concerned, the ratio of the pigmented area and the colorimetric value were reported to increase proportionally to the number of melanocytes added onto a collagen– glycosaminoglycan substrate harboring fibroblasts (Swope et al, 2002). Although such a report might be useful in providing insights into possible treatments for pigmented skin, the conventional technique itself requires refinement and closer assessment for wider clinical application, since the authors only consider the proportion of pigmented area to the number of melanocytes. Based on the report of Wang et al (2000), the basement membrane zone of HSS in the conventional model needs to be reinforced as a result of an epidermis and dermis grown separately and later sandwiched together. In addition to the lack of consideration vis-a`-vis the complexion of the patient receiving the graft, assessments of the previous HSS were made while their appearance was still disordered, 2–4 wk after grafting. Even in the ex vivo reconstruction of the epidermis with keratinocytes and melanocytes, the appraisals were performed a few weeks after seeding, and the functions of the reconstituted epidermis

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Figure 8 Increase in stem cell factor (SCF), endothelin-1 (ET-1), and tyrosinase expression in the Caucasian donor-derived human skin substitutes (HSS) after 2 MED (minimal erythema dose) of ultraviolet-B (UVB) irradiation. The transcript expression of SCF, prepro-ET-1 and tyrosinase in HSS after UVB irradiation was examined using real-time quantitative RT-PCR normalized against glyceraldehyde-3-phosphate dehydrogenase as detailed in Materials and methods. SCF mRNA transcripts’ expression and prepro-ET-1 and tyrosinase mRNA transcripts expression were measured 3 and 10 d after the irradiation, respectively. The values for UVB-irradiated HSS and non-irradiated HSS represent the mean  SD from six individual HSS. po0.05.

were analyzed only from the viewpoint of pigmentation and photoprotection, whereas any possibilities for wider practical application were not proposed (Bessou et al, 1995; Bessou-Touya et al, 1998; Cario-Andre et al, 1999). This study was therefore conducted in order to manufacture HSS containing human melanocytes by using the spontaneous cell-sorting method, and to evaluate them over a 12wk period following grafting, after the epidermal structure was better defined. We found that human melanocytes were spontaneously sorted to the basal layer of the reconstituted epidermis without interfering with neighboring keratinocytes and fibroblasts, and produced melanin that was then transferred to contiguous keratinocytes. Compared with HSS derived from Caucasian donors, those derived from donors of African descent showed darker skin pigmentation corresponding to the donors’ original complexion. Furthermore, increases of melanin and acanthosis in the HSS from Caucasian donors were found after UVB exposure. The sum of these findings strongly suggests that the technique of forming HSS with human melanocytes would be very useful for treating skin wounds or pigmentary disorders in patients of various complexions by allowing control over the color and the population of donor melanocytes. One of the most important questions raised in the course of producing HSS containing melanocytes was whether stem cells or transit-amplifying cells were involved in the reconstitution of HSS and whether cultured melanocytes and keratinocytes could rejuvenate to stem-like cells during the formation of HSS. Keratinocytes derived from newborn mouse skin were reported to be able to generate epidermis, hair, and sebaceous glands if first mixed with newborn dermal fibroblasts, implanted at a site where mouse skin has been surgically removed and then grafted onto the back of a

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nude mouse (Lichti et al, 1993; Weinberg et al, 1993). It was also reported that stem cells derived from the bulge area of hair follicles generated two distinct populations: the first layer to form retained basal lamina contact, whereas the second, located in the suprabasal layer, arose only after the start of the first postnatal hair cycle. Both were found to be capable of creating an epidermis, hair follicles, and sebaceous glands in grafts (Blanpain et al, 2004). It may be interesting to trace the fate of reconstituted HSS in our model with regard to the age and site of the donor skins. Stem cells of keratinocytes located at the tips of dermal papilla express high levels of b1-integrin and g-catenin, and low levels of E-cadherin, b-catenin, and plakoglobin (Jones et al, 1995; Mole`s and Watt, 1997; Watt, 1998). Consistent with the previous reports are the high levels of E-cadherin expression observed only at the tips of rete ridges in the HSS (data not shown), suggesting the plausible involvement of stem-like cells in HSS. Regarding melanoblasts and melanocyte stem cells, the cells expressing c-kit and dopachrome tautomerase were reported to exist in the bulge region of the hair follicle, a specific environment known as the niche, and that a portion of amplifying stem-cell progeny can migrate out of the niche and retain sufficient regenerative capability to function as stem cells after repopulating other vacant niches (Nishimura et al, 2002). In our experiments, c-kit-, S-100-, and HMB45-positive cells were found mainly at the tips of the rete ridges in the epidermis. Some c-kit positive cells might be melanoblasts or melanocyte stem-like cells since c-kit expression is considered to be a marker of melanoblasts, whereas HMB45-positive expression is considered to be a marker of differentiated melanocytes (Yoshida et al, 1996). It was also reported that melanogenic sublineages invariably diverge only from the ckit-expressing cells in the neural tube, indicating the high likelihood of c-kit expressed cells acting as a marker of melanocyte stem cells (Luo et al, 2003). Involvement of melanocyte stem-like cells in HSS also remains to be clarified by treating the mixed cell slurry with a c-kit neutralizing antibody. HSS with melanocytes might be a useful tool for studying melanocyte stem-like cells and the homeostasis of the skin, as melanocyte stem cells and their niche in the epidermis have not yet received due attention. It would be very interesting to examine the mechanism(s) underlying UVB-induced pigmentation in HSS. It was recently suggested that SCF/c-kit signaling and ET-1/endothelin B receptor (ETBR) linkage are predominantly involved in the early and late phases of UVB-induced human pigmentation, respectively, with the activity of the former stimulating the signaling of the latter by increasing ETBR expression in melanocytes (Hachiya et al, 2001, 2004). When human forearm skin was exposed to UVB radiation at 2 MED, the expression of SCF mRNA transcripts and protein was significantly enhanced at 3 days post-irradiation. This enhancement was followed by the upregulation of prepro-ET-1 mRNA transcripts and ET-1 protein at 7–10 d post-irradiation, together with an increase in the expression of tyrosinase mRNA transcripts and protein, as well as pigmentation. SCF, also known as kit ligand, or steel factor, is encoded by the steel (Sl ) locus, and its receptor c-kit is encoded by the dominant white spotting (W ) locus. Mutations in either of those loci produce very similar phenotypes

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characterized by the loss of neural crest-derived pigment cells, hematopoietic stem cells, and primordial germ cells (Matsui et al, 1990; Orr-Urtreger et al, 1990; Bernstein et al, 1991; Williams et al, 1992; Besmer et al, 1993; Halaban and Moellmann, 1993; Galli et al, 1993). ET-1 itself was first identified as a 21-residue peptide with potent vasoconstrictive properties in the culture media of porcine endothelial cells (Yanagisawa et al, 1988). ET isopeptides, approximately 200-residue inactive prepropolypeptides (preproendothelins) that are encoded by distinct genes (Inoue et al, 1989), are reported to have hormonal regulatory properties in various types of cells, including melanocytes, and in target organs via a receptor-mediated biochemical mechanism (Brenner et al, 1989; Resink et al, 1989; Reynolds et al, 1989; Watanabe et al, 1989). Therefore, it was essential to determine whether the mechanism(s) including SCF/c-kit and ET-1/ETBR signaling existed in the HSS. Real-time quantitative RT-PCR analysis of UVB-exposed epidermis in Caucasian donor-derived HSS containing melanocytes demonstrated that the expression of SCF and prepro-ET-1 mRNA transcripts had significantly increased 3 and 10 d after irradiation, respectively, compared with results in the non-irradiated epidermis. The increase in SCF expression seems to precede the increase in ET-1 and tyrosinase expression in HSS following UVB exposure. The factor accounting for the delay in the expression of tyrosinase is thought to be the involvement of microphthalmiaassociated transcription factor (MITF). It has been observed that MITF expression is regulated by SCF through the mitogen-activated protein kinase pathway, and that MITF stimulates tyrosinase mRNA transcripts expression in melanocytic cell lines (Bertolotto et al, 1998; Hemesath et al, 1998). These findings suggest that our melanocyte-containing HSS behave like natural human skin in situ, making them an attractive biological model for skin research. UVB irradiation causes a variety of biological effects in the skin including sunburn, thickening of the epidermis, and increase of melanin (Kligman, 1986). In order to protect skin from UV exposure, supranuclear melanin caps are formed in keratinocytes to absorb incoming UV photons (Montagna and Carlisle, 1991). These were also observed in the keratinocytes of the HSS and were enhanced by UVB irradiation in Caucasian donor-derived HSS, whereas they were abundantly seen in the HSS derived from donors of African descent without UVB irradiation, suggesting another common feature shared with normal human skin in situ. It has been thought that the more darkly complected African skin is more resistant to the deleterious effects of UV irradiation such as sunburn, photoaging, and skin carcinogenesis than Caucasian skin because the induction of DNA photodamage, the influx of neutrophils, activation of proteolytic enzymes, and diffuse keratinocyte activation and IL-10 expression are interrupted by the higher melanin content and different melanosomal distribution within the epidermis (Rijken et al, 2004). In keeping with these observations, no increase in melanogenesis was observed in the African descent-derived HSS after UVB irradiation. It seemed that a higher amount of melanin and its distribution in the epidermis precluded further induction of pigmentation following UVB exposure in our models. In addition to the measurement of UVB-induced pigmentation, the use of the non-

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invasive OCT technique made it possible to measure the thickness of the epidermis periodically. Acanthosis was recorded one day after the UVB irradiation and continued for 14 days. Both the acanthosis and increase in melanin observed in HSS after UVB exposure are identical to the reactions observed in normal human skin, and might have a practical application in suggesting the type of ingredient to be used in cosmetics for absorbing or reflecting UV irradiation. In our study, cells derived from Caucasian donors and donors of African descent were used to reconstitute HSS. HSS derived from donors of African descent produced more melanin in the epidermis compared with those derived from Caucasian donors, and were darkly pigmented, in keeping with the original skin color of the donor. Our findings are in agreement with the previous paper showing that the most lightly pigmented skin types, including the Caucasian, have approximately half as much epidermal melanin as the most darkly pigmented skin types such as the African and Indian (Alaluf et al, 2002). In addition, it has also been suggested that the racial factor determines the distribution patterns of melanosomes in keratinocytes in a mono-layered co-culture system including keratinocytes and melanocytes as in actual human skin (Minwalla et al, 2001; Thong et al, 2003). Although the precise mechanism of melanosome distribution in keratinocytes of HSS should be identified, improvements in reconstituting colored HSS would be useful in treating patients of various complexions who suffer from hyper- and hypo-pigmentary disorders such as senile fleck and vitiligo, as well as from discoloration of skin surface due to burns and other acute or chronic wounds. Among these conditions, vitiligo, an acquired hypo-pigmentary skin disorder characterized by a progressive loss of functional melanocytes, would be the best target of treatment using melanocyte transplants (Cui et al, 1991; Le Poole et al, 1993). We are convinced that the techniques presented here on HSS containing melanocytes will prove beneficial for improving the quality of the surgical treatment of vitiligo. Our findings provide new insights into the development of pigmented HSS for clinical and research uses, and we hope that a fundamental understanding of the mechanisms of cell sorting and differentiation will be advanced by the model we have presented.

Materials and Methods Materials Normal human keratinocytes and melanocytes from Caucasian donors and melanocyte medium M154 were obtained from Cascade Biologics (Portland, Oregon). Normal human fibroblasts from Caucasian donors and pentobarbital sodium were purchased from Dainippon Pharmaceutical (Osaka, Japan). Polyclonal rabbit anti-sera against human c-kit were purchased from Immuno-Biological Laboratories (IBL, Gunma, Japan). Antibodies to S-100 were purchased from the Nichirei Corporation (Tokyo, Japan), and antibodies to HMB45 were from DakoCytomation (Kyoto, Japan). Serum-free keratinocyte medium (SFM), bovine pituitary extract (BPE), epidermal growth factor (EGF), Dulbecco’s modified Eagle’s medium (DMEM), and Dulbecco’s phosphatebuffered saline (PBS) were purchased from Gibco Laboratories (Grand Island, New York). All other chemicals were of reagent grade.

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Cell cultures Cells from donors of African descent were prepared from neonatal foreskin at the time of circumcision at Christ Hospital and University Hospital (Cincinnati, Ohio). The dermis and epidermis were separated by dispase treatment. Fibroblasts were isolated from the dermis by trypsinization, and were cultured in DMEM with 1
antibiotic-antimycotic and 10% fetal bovine serum. Keratinocytes and melanocytes were isolated from the epidermis and cultured in complete Epilife (Cascade Biologics) with PSA and complete M154 (supplemented with 1 ng per mL recombinant basic fibroblast growth factor (FGF), 5 mg per mL insulin, 0.5 mg per mL hydrocortisone, 10 ng per mL phorbol 12-myristate 13-acetate, 50 mg per mL streptomycin, and 0.2% (vol/vol) BPE), respectively. Normal human keratinocytes, fibroblasts, and melanocytes were maintained at 371C with 5% CO2 in modified SFM (supplemented with 5 ng per mL EGF and 50 mg per mL BPE), DMEM (containing 10% fetal bovine serum), and complete M154, respectively. Grafting cells onto SCID mice Animals were handled according to the guidelines of the Ethical Committee for Animal Experiments of Kao Biological Science Laboratories. SCID mice (BALB/cA-nu; scid Jic and NOD/Shi-scid Jic) were purchased from the Central Institute for Experimental Animals (Kanagawa, Japan) and maintained on a standard laboratory diet and water ad libitum. A mixed cell slurry containing approximately 6
106 keratinocytes, 6
106 fibroblasts, and 1
106 melanocytes was prepared for each mouse as described elsewhere (Wang et al, 2000). Briefly, the cells were harvested using 0.1% trypsin and were later neutralized with each conditioned medium containing 10% fetal bovine serum. The three types of cells were mixed in a 15 mL polystyrene conical centrifuge tube (Falcon, Becton Dickinson, Franklin Lakes, New Jersey), and centrifuged at 200
g for 5 min. Excess medium was removed by decantation, and the cell pellets were kept on ice until use. Prior to surgery, the mice were anesthetized with an intraperitoneal injection of pentobarbital sodium solution diluted 10fold. A hat-like silicone structure (CRD culture chambers, Renner, Darmstadt, Germany) consisting of a brim 0.6 cm in width and a chamber 1.2 cm in diameter was implanted into a surgically prepared circular wound 1.0 cm in diameter on the back of each SCID mouse. The chamber was then fixed in place by purse-string suture to the surrounding skin. Following the surgery, the mice were kept in an incubator at 371C until recovery from anesthesia. The mixed cell suspension was poured into the chamber through the 3.5 mm hole on top of the silicone chamber, so as to be applied directly onto the mouse muscle fascia. The upper part of the chamber was mechanically removed 1 wk after implantation to evaporate the chamber inside gradually. The lower part of the chamber including the brim was allowed to fall off spontaneously from the skin approximately 6 wk after implantation. The suture itself was removed 4 wk post-implantation after the skin surface had been allowed to dry further. Immunohistochemistry The HSS collected from SCID mice were fixed in formalin and embedded in paraffin. The immunoreactivity of melanocyte-specific proteins was assessed using antibodies to human c-kit, S-100, and HMB45. Localization of binding was visualized by using the OmniTags Plus universal streptavidin/biotin immunoperoxidase detection system with aminoethylcarbazole (AEC) (Thermo Shandon, Pittsburgh, Pennsylvania). Normal rabbit or mouse IgG (Sigma, St Louis, Missouri) was used as negative control. Fontana–Masson staining Melanin pigment was visualized using Fontana–Masson staining with an eosin counterstaining. TEM TEM was performed in order to confirm the transfer of melanosomes from melanocytes to keratinocytes visually. HSS grown on SCID mice for 16 wk were fixed in 0.1 M cacodylate buffer (pH 7.4) containing 2% paraformaldehyde and 2.5% glutaraldehyde. Samples were treated with 2% osmium tetroxide and 2% uranyl acetate, dehydrated, and then embedded in epoxy

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resin (EPOK812; Okenshoji, Tokyo, Japan) as described elsewhere (Hyatt, 1986). Ultra-thin sections were prepared using a Reichert–Nissei Ultracut N, mounted on TEM grids, and examined with an H-7000 transmission electron microscope (Hitachi Kyowa Engineering, Ibaraki, Japan). UVB irradiation UVB irradiation of the HSS on the SCID mice was performed approximately 16 wk after grafting. The entire surface or an area (approximately 25 mm2) of each HSS was irradiated with 120 mJ per cm2 UVB (FL20SE30 lamps, Toshiba, Tokyo, Japan), corresponding to approximately two times the MED for Caucasian skin (Parrish et al, 1981). Measurement of skin color The intensity of UVB-induced pigmentation in HSS, measured using a colorimeter (Nippon Denshoku Industries, Tokyo, Japan), was expressed as an L value. Measurement of epidermal thickness Epidermal thickness after UVB irradiation was measured by using the non-invasive OCT technique (ISIS Optronics GmbH, Mannheim, Germany), and the images were analyzed using Image-Pro Plus version 4.5.1. software (Media Cybernetics, Silver Spring, Maryland). The OCT technique has already been published elsewhere (Knuttel and Boehlau-Godau, 2000). The average thickness of the epidermis was calculated using the images of the HSS surfaces and the basement membrane zones generated by scanning. Real-time quantitative RT-PCR After UVB irradiation, the transcript expression of SCF, prepro-ET-1, a precursor of endothelin-1, and tyrosinase in melanocyte-containing HSS, was examined using real-time quantitative RT-PCR normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The HSS were immersed in RNAlater (Qiagen, Valencia, California) to prevent RNA degradation. The epidermis was enzymatically separated from the HSS by incubation in dispase. Total RNA from each epidermal sheet was prepared using an RNeasy micro kit (Qiagen). cDNA was then synthesized by reverse transcription of total RNA using oligo dT and Moloney murine leukemia virus reverse transcriptase. On-demand probes for SCF, prepro-ET-1, tyrosinase, and GAPDH in TaqMan Gene Expression Assays (Applied Biosystems, Foster City, California) were used. Real-time quantitative RTPCR with TaqMan Gene Expression Assays was performed in an ABI PRISM 7300 sequence detection system (Applied Biosystems). Statistics A non-parametric one-way ANOVA (Kruskal–Wallis test) was used to evaluate differences between the groups. Where appropriate, a non-parametric post hoc multiple comparison test (Steel–Dwass test) was performed to evaluate differences between the groups. A p valueo0.05 was considered statistically significant.

The authors thank S. Izawa and K. Tani for technical assistance. DOI: 10.1111/j.0022-202X.2005.23832.x Manuscript received January 13, 2005; revised March 28, 2005; accepted for publication April 11, 2005 Address correspondence to: Akira Hachiya, Kao Biological Science Laboratories, Haga, Tochigi 321-3497, Japan. Email: hachiya.ak [email protected]

References Alaluf S, Atkins D, Barrett K, Blount M, Carter N, Heath A: Ethnic variation in melanin content and composition in photo-exposed and photo-protected human skin. Pigment Cell Res 15:112–118, 2002 Bell E, Ehrlich HP, Sher S, et al: Development and use of a living skin equivalent. Plast Reconstr Surg 67:386–392, 1981

371

Bell E, Rosenberg M: The commercial use of cultivated human cells. Transplant Proc 22:971–974, 1990 Ben-Bassat H, Eldad A, Chaouat M, Livoff A, Ron N, Ne’eman Z, Wexler MR: Structural and functional evaluation of modifications in the composite skin graft: Cryopreserved dermis and cultured keratinocytes. Plast Reconstr Surg 89:510–520, 1992 Bernstein A, Forrester L, Reith AD, Dubreuil P, Rottapel R: The murine W/c-kit and steel loci and the control of hematopoiesis. Semin Hematol 28:138–142, 1991 Berthod F, Damour O: In vitro reconstructed skin models for wound coverage in deep burns. Br J Dermatol 136:809–816, 1997 Bertolotto C, Busca R, Abbe P, Bille K, Aberdam E, Ortonne JP, Ballotti P: Different cis-acting elements are involved in the regulation of TRP1 and TRP2 promoter activities by cyclic AMP: Pivotal role of M boxes (GTCATGTGCT) and of microphthalmia. Mol Cell Biol 18: 694–702, 1998 Besmer P, Manova K, Duttlinger R, Huang EJ, Packer A, Gyssler C, Bachvarova R: The kit-ligand (steel factor) and its receptor c-kit/W: Pleiotropic roles in gametogenesis and melanogenesis. Development 119:125–137, 1993 Bessou S, Surleve-Bazeille JE, Sorbier E, Taieb A: Ex vivo reconstruction of the epidermis with melanocytes and the influence of UVB. Pigment Cell Res 8:241–249, 1995 Bessou-Touya S, Picardo M, Maresca V, Surleve-Bazeille JE, Pain C, Taieb A: Chimeric human epidermal reconstructs to study the role of melanocytes and keratinocytes in pigmentation and photoprotection. J Invest Dermatol 111:1103–1108, 1998 Bettger WJ, Ham RG: The nutrient requirements of cultured mammalian cells. Adv Nutr Res 4:249–286, 1982 Bettger WJ, McKeehan WL: Mechanisms of cellular nutrition. Physiol Rev 66: 1–35, 1986 Beumer GJ, van Blitterswijk CA, Bakker D, Ponec M: Cell-seeding and in vitro biocompatibility evaluation of polymeric matrices of PEO/PBT copolymers and PLLA. Biomaterials 14:598–604, 1993 Blanpain C, Lowry WE, Geoghegan A, Polak L, Fuchs E: Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche. Cell 118:635–648, 2004 Boyce ST, Greenhalgh DG, Kagan RJ, et al: Skin anatomy and antigen expression after burn wound closure with composite grafts of cultured skin cells and biopolymers. Plast Reconstr Surg 91:632–641, 1993 Boyce ST, Supp AP, Harriger MD, Pickens WL, Wickett RR, Hoath SB: Surface electrical capacitance as a noninvasive index of epidermal barrier in cultured skin substitutes in athymic mice. J Invest Dermatol 107:82–87, 1996 Boyce ST, Supp AP, Swope VB, Warden GD: Vitamin C regulates keratinocytes viability, epidermal barrier, and basement membrane in vitro, and reduces wound contraction after grafting of cultured skin substitutes. J Invest Dermatol 118:565–572, 2002 Brenner BM, Troy JL, Ballermann BJ: Endothelium-dependent vascular responses. Mediators and mechanisms. J Clin Invest 84:1373–1378, 1989 Burke JF, Yannas IV, Quinby WC Jr, Bondoc CC, Jung WK: Successful use of a physiologically acceptable artificial skin in the treatment of extensive burn injury. Ann Surg 194:413–428, 1981 Cario-Andre M, Bessou S, Gontier E, Maresca V, Picardo M, Taieb A: The reconstructed epidermis with melanocytes: A new tool to study pigmentation and photoprotection. Cell Mol Biol (Noisy-le-grand) 45:931–942, 1999 Cooper ML, Hansbrough JF: Use of a composite skin graft composed of cultured human keratinocytes and fibroblasts and a collagen-GAG matrix to cover full-thickness wounds on athymic mice. Surgery 109:198–207, 1991 Cui J, Shen LY, Wang GC: Role of hair follicles in the repigmentation of vitiligo. J Invest Dermatol 97:410–416, 1991 Cuono C, Langdon R, McGuire J: Use of cultured epidermal autografts and dermal allografts as skin replacement after burn injury. Lancet 1:1123–1124, 1986 De Luca M, Albanese E, Bondanza S, et al: Multicenter experience in the treatment of burns with autologous and allogenic cultured epithelium, fresh or preserved in a frozen state. Burns 15:303–309, 1989 Galli SJ, Zsebo KM, Geissler EN: The kit ligand, stem cell factor. Adv Immunol 55:1–96, 1993 Gallico GG, O’Connor NE, Compton CC, Kehinde O, Green H: Permanent coverage of large burn wounds with autologous cultured human epithelium. N Engl J Med 311:448–451, 1984 Green H, Kehinde O, Thomas J: Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc Natl Acad Sci USA 76: 5665–5668, 1979 Gunzler V, Brocks D, Henke S, Myllyla R, Geiger R, Kivirikko KI: Syncatalytic inactivation of prolyl 4-hydroxylase by synthetic peptides containing the

372 HACHIYA ET AL unphysiologic amino acid 5-oxaproline. J Biol Chem 263:19498–19504, 1988 Hachiya A, Kobayashi A, Ohuchi A, Takema Y, Imokawa G: The paracrine role of stem cell factor/c-kit signaling in the activation of human melanocytes in ultraviolet B-induced pigmentation. J Invest Dermatol 116:578–586, 2001 Hachiya A, Kobayashi A, Yoshida Y, Kitahara T, Takema Y, Imokawa G: Biphasic expression of two paracrine melanogenic cytokines, stem cell factor and endothelin-1, in ultraviolet B-induced human melanogenesis. Am J Pathol 165:2099–2109, 2004 Halaban R, Moellmann G: White mutants in mice shedding light on humans. J Invest Dermatol 100:176S–185S, 1993 Hansbrough JF, Boyce ST, Cooper ML, Foreman TJ: Burn wound closure with cultured autologous keratinocytes and fibroblasts attached to a collagenglycosaminoglycan substrate. J Am Med Assoc 262:2125–2130, 1989 Hefton JM, Madden MR, Finkelstein JL, Shires GT: Grafting of burn patients with allografts of cultured epidermal cells. Lancet 2:428–430, 1983 Hemesath TJ, Price ER, Takemoto C, Badalian T, Fisher DE: MAPK links the transcription factor microphthalmia to c-kit signaling in melanocytes. Nature 391:289–301, 1998 Hyatt MA: Basic Techniques for Transmission Electron Microscopy. Orlando, FL: Academic Press, 1986; p 56–68 Imokawa G, Miyagishi M, Yada Y: Endothelin-1 as a new melanogen: Coordinated expression of its gene and the tyrosinase gene in UVB-exposed human epidermis. J Invest Dermatol 105:32–37, 1995 Inoue A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T, Goto K, Masaki T: The human preproendothelin-1 gene. Complete nucleotide sequence and regulation of expression. Proc Natl Acad Sci USA 86:2863–2867, 1989 Jones PH, Harper S, Watt FM: Stem cell patterning and fate in human epidermis. Cell 80:83–93, 1995 Kligman LH: Photoaging. Manifestations, prevention, and treatment. Dermatol Clin 4:517–528, 1986 Knuttel A, Boehlau-Godau M: Spatially confined and temporally resolved refractive index and scattering evaluation in human skin performed with optical coherence tomography. J Biomed Opt 5:83–92, 2000 Kobayashi N, Nakagawa A, Muramatsu T, et al: Supranuclear melanin caps reduce ultraviolet induced DNA photoproducts in human epidermis. J Invest Dermatol 110:806–810, 1998 Krejci NC, Cuono CB, Langdon RC, McGuire J: In vitro reconstitution of skin: Fibroblasts facilitate keratinocyte growth and differentiation on acellular reticular dermis. J Invest Dermatol 97:843–848, 1991 Le Poole IC, Das PK, van den Wijngaard RM, Bos JD, Westerhof W: Review of the etiopathomechanism of vitiligo: A convergence theory. Exp Dermatol 2:145–153, 1993 Lichti U, Weinberg WC, Goodman L, Ledbetter S, Dooley T, Morgan D, Yuspa SH: In vivo regulation of murine hair growth: Insights from grafting defined cell populations onto nude mice. J Invest Dermatol 101:124S–129S, 1993 Luo R, Gao J, Wehrle-Haller B, Henion PD: Molecular identification of distinct neurogenic and melanogenic neural crest sublineages. Development 130:321–330, 2003 Madden MR, Finkelstein JL, Staiano-Coico L, Goodwin CW, Shires GT, Nolan EE, Hefton JM: Grafting of cultured allogeneic epidermis on second- and third-degree burn wounds on 26 patients. J Trauma 26:955–962, 1986 Matsui Y, Zsebo KM, Hogan BLM: Embryonic expression of a hematopoietic growth factor encoded by the Sl locus and the ligand for c-kit. Nature 347:667–669, 1990 Minwalla L, Zhao Y, Le Poole IC, Wickett RR, Boissy RE: Keratinocytes play a role in regulating distribution patterns of recipient melanosomes in vitro. J Invest Dermatol 117:341–347, 2001

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Mole`s J-P, Watt FM: The epidermal stem cell compartment. Variation in expression levels of E-cadherin and catenins within the basal layer of human epidermis. J Histochem Cytochem 45:867–874, 1997 Montagna W, Carlisle K: The architecture of black and white facial skin. J Am Acad Dermatol 24:929–937, 1991 Nishimura EK, Jordan SA, Oshima H, et al: Dominant role of the niche in melanocyte stem-cell fate determination. Nature 416:854–860, 2002 Orr-Urtreger A, Avivi A, Zimmer Y, Givol D, Yarden Y, Lonai P: Developmental expression of c-kit, a proto-oncogene encoded by the W locus. Development 109:911–923, 1990 Parrish JA, Zaynoum S, Anderson RR: Cumulative effects of repeated subthreshold doses of ultraviolet radiation. J Invest Dermatol 76:356–358, 1981 Pathak MA, Fanselow DL: Photobiology of melanin pigmentation: Dose/response of skin to sunlight and its contents. J Am Acad Dermatol 9:724–733, 1983 Quarles LD, Yohay DA, Lever LW, Caton R, Wenstrup RJ: Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: An in vitro model of osteoblast development. J Bone Miner Res 7:683–692, 1992 Resink TJ, Scott-Burden T, Buhler ER: Enhanced responsiveness to angiotensin II in vascular smooth muscle cells from spontaneously hypertensive rats is not associated with alterations in protein kinase C. Biochem Biophys Res Commun 158:279–286, 1989 Reynolds EE, Mok LL, Kurokawa S: Phorbol ester dissociates endothelin-stimulated phosphoinositide hydrolysis and arachidonic acid release in vascular smooth muscle cells. Biochem Biophys Res Commun 160:868–873, 1989 Rijken F, Bruijnzeel PL, van Weelden H, Kiekens RC: Responses of black and white skin to solar-simulating radiation: Differences in DNA photodamage, infiltrating neutrophils, proteplytic enzymes induced, keratinocyte activation, and IL-10 expression. J Invest Dermatol 122:1448–1455, 2004 Saika S, Kanagawa R, Uenoyama K, Hiroi K, Hiraoka J: L-ascorbic acid 2-phosphate, a phosphate derivative of L-ascorbic acid, enhances the growth of cultured rabbit keratinocytes. Graefes Arch Clin Exp Ophthalmol 229:79– 83, 1991 Swope VB, Supp AP, Boyce ST: Regulation of cutaneous pigmentation by titration of human melanocytes in cultured skin substitutes grafted to athymic mice. Wound Repair Regen 10:378–386, 2002 Thong HY, Jee SH, Sun CC, Boissy RE: The patterns of melanosome distribution in keratinocytes of human skin as one determining factor of skin color. Br J Dermatol 149:498–505, 2003 Wang CK, Nelson CF, Brinkman AM, Miller AC, Hoeffler WK: Spontaneous cell sorting of fibroblasts and keratinocytes creates an organotypic human skin equivalent. J Invest Dermatol 114:674–680, 2000 Watanabe H, Miyazaki H, Kondoh M, et al: Two distinct types of endothelin receptors are present on chick cardiac membranes. Biochem Biophys Res Commun 161:1252–1259, 1989 Watt FM: Epidermal stem cells: Markers, patterning and the control of stem cell fate. Philos Trans R Soc Lond B Biol Sci 353:831–837, 1998 Weinberg WC, Goodman LV, George C, Morgan DL, Ledbetter S, Yuspa SH, Lichti U: Reconstitution of hair follicle development in vivo: Determination of follicle formation, hair growth, and hair quality by dermal cells. J Invest Dermatol 100:229–236, 1993 Williams DE, de Vries P, Namen AE, Widmer MB, Lyman SD: The steel factor. Dev Biol 151:368–376, 1992 Yanagisawa M, Kurihara H, Kimura S, et al: A novel peptide vasoconstrictor, endothelin, is produced by vascular endothelium and modulates smooth muscle Ca2 þ channels. Nature 322:411–415, 1988 Yoshida H, Kunisada T, Kusakabe M, Nishikawa S, Nishikawa SI: Distinct stages of melanocyte differentiation revealed by analysis of nonuniform pigmentation patterns. Development 122:1207–1214, 1996