Culture of amelanotic melanocytes derived from human fetal hair follicles

Human melanocyte stem cells (MSCs) or melanoblasts are not well-investigated owing to the devoid of suitable culture system. Establishing cell lines of MSCs and/or their progenies from human hair follicles will provide a better opportunity to satisfy clinical needs and to enable a deeper understanding of hair-related diseases. In the present study, we cultured melanocytes derived from human fetal hair follicles, perform immunocytochemistry and Fontana Masson staining on them, and employed atomic force microscopy (AFM) and scanning electron microscopy to observe their subtle morphologies. The results show that the cultured melanocytes have a bipolar or tripolar appearance, which obviously differ from cultured epidermal melanocytes. Compared to cells derived from adult human hair follicles, these cells display a high proliferative capability and exhibit a clonal growth behavior. At the second passage, all these cells were positive for immunocytochemical staining with the NKI/beteb monoclonal antibody and Fontana Masson staining. Under AFM, the cells exhibited rounded, oval, triangular, or quadrangular perikarya, from which two or three dendrites arose. The dendritic arbor was not homogeneous but appeared as spindle-shaped dendritic swellings, knob-like processes, without any filopodia arising from the dendrites or the cell body. Without using a feeder layer, we successfully obtained the clonal growth of melanocytes from human fetal HFs, suggesting that the medium was suitable for the growth of MSCs and their progenies.     

In vitro dedifferentiation of melanocytes from adult epidermis

In previous work we described a novel culture technique using a cholera toxin and PMA-free medium (Mel-mix) for obtaining pure melanocyte cultures from human adult epidermis. In Mel-mix medium the cultured melanocytes are bipolar, unpigmented and highly proliferative. Further characterization of the cultured melanocytes revealed the disappearance of c-Kit and TRP-1 and induction of nestin expression, indicating that melanocytes dedifferentiated in this in vitro culture. Cholera toxin and PMA were able to induce c-Kit and TRP-1 protein expressions in the cells, reversing dedifferentiation. TRP-1 mRNA expression was induced in dedifferentiated melanocytes by UV-B irradiated keratinocyte supernatants, however direct UV-B irradiation of the cells resulted in further decrease of TRP-1 mRNA expression. These dedifferentiated, easily accessible cultured melanocytes provide a good model for studying melanocyte differentiation and possibly transdifferentiation. Because melanocytes in Mel-mix medium can be cultured with human serum as the only supplement, this culture system is also suitable for autologous cell transplantation.     

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.     

Reconstruction of a tissue-engineered skin containing melanocytes

The objective of this study was to establish a new method for reconstruction of a tissue-engineered skin containing melanocytes by employing tissue engineering. The keratinocytes, melanocytes and dermal fibroblasts were isolated and purified from human foreskin biopsies. Then the cells were used to construct a tissue-engineered skin containing melanocytes. The localization of melanocytes in the tissue-engineered skin was detected by DOPA staining, S-100 immunohistochemical staining and transmission electron microscope (TEM). The results showed that the melanocytes could be detected in the basal layer of the constructed skin and the melanocytes showed dendritic morphology. Moreover the constructed skins were used to repair the athymic mice skin defects. Animal experiment results indicated that the skin equivalents could successfully repair full thickness skin defects in athymic mice and generated black skins by 6weeks after grafting. Melanocytes located in the basal layer of the athymic mice skin could also be detected by using the S-100 immunohistochemical staining. Our established method is useful to repair the full-thickness skin defects.     

Novel application of a fibrin cell block method to evaluate melanocytic cell populations

Confirming melanocytic lineage and purity is important for experiments using cultured human melanocytes. The objective of this study was to develop a simple, reliable method to evaluate and archive cultured melanocytic cells. Melanocytes were isolated from adult skin biopsies or from neonatal foreskins using standard culturing methods. Fibrin cell blocks (FCBs) were prepared from cultured cells at passages two and six. Fibrin blocks were paraffin-embedded and sectioned for immunohistochemical (CD68, Melan-A, and HMB-45) and H & E staining. Flow cytometry was performed (Melan-A) at passage six. A mixing experiment with cultured melanocytes and fibroblasts was performed and cell population purity was determined by manual counts of positively staining cells in the FCBs and by flow cytometry. The FCB method of evaluating population purity was validated experimentally and by correlation with flow cytometry results. Preparation of a FCB followed by immunohistochemical staining is an easy and inexpensive way to confirm melanocytic lineage, estimate population purity, and provide a permanent archive of cultured cells.     

Up‐Regulation and Redistribution of Bax in Ultraviolet B‐Irradiated Melanocytes

Ultraviolet B (UVB) radiation may activate or deteriorate cultured human epidermal melanocytes, depending on the doses and culture conditions. It is also reported that cultured human epidermal melanocytes derived from different pigmentary phenotypes showed different responses to UVB radiation. In this study, we examined whether apoptosis of melanocytes can be induced by physiologic doses of UVB irradiation using cultured human epidermal melanocytes derived from oriental males of skin types III and IV. Propidium iodide staining for DNA condensation and flow cytometric analyses demonstrated the apoptotic cell death of melanocytes following UVB irradiation (0–30 mJ/cm²). The levels of p53, Bax, and Bcl‐2, determined by immunoblotting, revealed a dose‐dependent increase in p53 and Bax, but the level of Bcl‐2 remained unchanged. Confocal microscopic examination showed that Bax moved from a diffuse to a punctate distribution after UVB irradiation. However, there were no changes in the pattern of distribution of Bcl‐2. These data suggest that the high constitutional level of Bcl‐2 may protect melanocytes from UVB‐induced injury, and that apoptotic death of melanocytes may be induced by the elevation and redistribution of Bax.     

人KRT2促进体外培养的羊驼皮肤黑色素细胞的黑色素生成

基因表达谱显示,绵羊角蛋白2（keratin2, Krt2）的mRNA在不同毛色皮肤的表达不同,暗示Krt2 基因可能对皮肤黑色素生成有一定的影响。为探索角蛋白2对体外培养的羊驼皮肤黑色素细胞黑色素生成的影响,首先采用PCR扩增产物测序,结合DNAMAN 软件比对分析发现,羊驼Krt2 编码序列（cDNA）与NCBI公布的人KRT2高度同源（91%）;将人KRT2添加于培养的羊驼皮肤黑色素细胞,观察人KRT2对羊驼皮肤黑色素细胞黑色素的生成作用。免疫组织化学显示,外源性的人KRT2处理羊驼皮肤黑色素细胞72 h后,黑色素细胞的细胞质中Krt2表达增强。实时定量PCR及Western 印迹实验揭示,与胎牛血清清蛋白处理的羊驼皮肤黑色素细胞比较,1 ng/mL、10 ng/mL和100 ng/mL人KTR2处理的羊驼皮肤黑色素细胞酪氨酸酶（Tyr）、酪氨酸相关蛋白-1（Tyrp1）、小眼畸形相关转录因子（Mitf）基因表达明显上调（P <0.05）;尤其在添加10 ng/mL KTR2的细胞中,3个基因的mRNA相对表达水平升高尤其显著,分别是对照细胞的4倍、10倍和12.9倍（P<0.01）,蛋白质相对表达水平分别是对照细胞的2倍、2.1倍和1.7倍（P<0.01）。分光光度法测量A490结果证明,1 ng/mL、10 ng/mL和100 ng/mL人KTR2处理的羊驼皮肤黑色素细胞产生的黑色素含量分别是对照细胞的1.3倍（P <0.05）、1.8倍（P <0.01）和1.5倍（P <0.05）。上述结果说明,人KTR2处理可通过刺激羊驼皮肤黑色素细胞黑色素合成相关信号通路,促进黑色素的合成。     

Up-Regulation and redistribution of Bax in ultraviolet B-irradiated melanocytes

Ultraviolet B (UVB) radiation may activate or deteriorate cultured human epidermal melanocytes, depending on the doses and culture conditions. It is also reported that cultured human epidermal melanocytes derived from different pigmentary phenotypes showed different responses to UVB radiation. In this study, we examined whether apoptosis of melanocytes can be induced by physiologic doses of UVB irradiation using cultured human epidermal melanocytes derived from oriental males of skin types III and IV. Propidium iodide staining for DNA condensation and flow cytometric analyses demonstrated the apoptotic cell death of melanocytes following UVB irradiation (0-30 mJ/cm2). The levels of p53, Bax, and Bcl-2, determined by immunoblotting, revealed a dose-dependent increase in p53 and Bax, but the level of Bcl-2 remained unchanged. Confocal microscopic examination showed that Bax moved from a diffuse to a punctate distribution after UVB irradiation. However, there were no changes in the pattern of distribution of Bcl-2. These data suggest that the high constitutional level of Bcl-2 may protect melanocytes from UVB-induced injury, and that apoptotic death of melanocytes may be induced by the elevation and redistribution of Bax.     

Isolation and culture of amelanotic melanocytes from human hair follicles

We report a method to establish amelanotic melanocytes (AMMC) in culture and we investigate the effects of various components in the culture medium. Normal human scalp from cadaver donors was transected 1 mm below the epidermis, and hair follicles in the remaining dermis were isolated by a two-step enzyme treatment. The individual hair follicles were washed exhaustively and suspensions of hair follicle cells were prepared and cultured in Eagle's minimum essential medium supplemented with 12-o-tetradecanoyl-phorbol-13-acetate (TPA), cholera toxin and keratinocyte serum-free medium (K-SFM). Geneticin was used to eliminate contaminating fibroblasts. Proliferation of AMMC was observed after addition of TPA and K-SFM including bovine pituitary extract (BPE) into the culture medium. Cell type was determined by staining with monoclonal antibodies, NKI/beteb and HMB-45, which recognize premelanosomal and melanosomal antigens, respectively. The AMMC were also examined using transmission electron microscopy. Treatment with geneticin eliminates the majority of fibroblasts and does not impair the growth of keratinocytes or AMMC. After contaminating fibroblasts and keratinocytes were removed, two distinct cell morphologies remained: (1) large, dendritic and deeply pigmented cells, which did not proliferate and which disappeared by the third passage, and (2) small bipolar cells, which initially were unpigmented and proliferated very rapidly. We observed that TPA at various concentrations stimulated the proliferation of the cells, and at high concentrations could induce the formation of multiple dendrites. K-SFM including BPE accelerated the proliferation of the cells in a dose-dependent manner. After passage 3, almost all cells expressed premelanosomal and melanosomal antigens, recognized by NKI/beteb and HMB-45, respectively. Active mitochondria, abundant rough endoplasmic reticulum, Golgi complexes, ribosomes and melannosomes (predominantly in stages I, II or III with some at stage IV in some AMMC) were observed ultrastructurally in the cytoplasm of the cultured cells.     

A ROCK inhibitor promotes keratinocyte survival and paracrine secretion,enhancing establishment of primary human melanocytes and melanocyte–keratinocyte co‐cultures

Primary melanocytes isolated from skin and expanded in culture have been widely used for laboratory research and clinical applications. The conventional method to isolate primary melanocytes from skin usually requires about 3–4 weeks of culture for melanocytes to grow sufficiently to passage. Considering that melanocytes comprise only 3%–7% of epidermal cells in normal human skin, it would be extremely helpful to increase the isolation efficiency and shorten the initial culture time to quickly meet various application needs. Here, we report that adding Y‐27632, a Rho kinase inhibitor, into the initial culture medium for 2 days can dramatically increase the yield of melanocytes. We found that Y‐27632 can promote keratinocyte attachment and survival in the melanocyte culture system, resulting in not only better recovery, but also increased proliferation of melanocytes by a paracrine signaling pathway. More specifically, Y‐27632 significantly induced keratinocyte expression of stem cell factor, which played an important role in enhancing the growth of melanocytes. In summary, Y‐27632 could profoundly enhance the yield of primary melanocytes in the initial culture through paracrine effects on keratinocytes.     

Rapid culture of human keratinocytes in an autologous,feeder-free system with a novel growth medium

Background aimsThe ability to culture human keratinocytes is beneficial in the treatment of skin injury and disease, as well as for testing chemicals in vitro as a substitute for animal testing.ResultsWe have identified a novel culture medium for the rapid growth of keratinocytes from human skin. “Kelch's medium” supports keratinocyte growth that is as rapid as in the classical Rheinwald and Green method, but without the need for cholera toxin or xenogeneic feeder cells. It enables keratinocytes to out-compete co-cultured autologous fibroblasts so that separation of the epidermis from the dermis is no longer required before keratinocyte culture. Enzymatic digests of whole human skin can therefore be used to generate parallel cultures of autologous keratinocytes, fibroblasts and melanocytes simply by using different cell culture media.ConclusionsThis new keratinocyte medium and the simplified manufacturing procedures it enables are likely to be beneficial in skin engineering, especially for clinical applications.     

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.     

Roscovitine inhibits differentiation and invasion in a three-dimensional skin reconstruction model of metastatic melanoma

The aim of this study was to investigate the therapeutic potential of a cyclin-dependent kinase inhibitor, roscovitine, in cultured melanoma cells and a three-dimensional skin reconstruction model of metastatic melanoma. The modulatory effects of roscovitine on the growth and survival of normal melanocytes and cultured melanoma cell lines were tested. Additionally, we investigated the potential of roscovitine to regulate the growth and differentiation of a metastatic melanoma cell line (A375) in a three-dimensional skin reconstruction culture consisting of A375 cells admixed with normal human keratinocytes embedded within a collagen-constricted fibroblast matrix. We show that roscovitine is able to induce apoptosis in the melanoma cell lines A375, 888, and 624 but not in normal human cultured epithelial melanocytes. The degree of apoptosis within these cell lines correlated with the accumulation of p53 protein and concomitant reduction of X-linked inhibitor of apoptosis protein, with no change in the proteins Bcl-2 and survivin. We also found that roscovitine inhibited the growth and differentiation of A375 melanoma cells within the dermal layer of the skin. The results of this study show that roscovitine has the potential to inhibit the differentiation and invasion of metastatic melanoma and may be useful as a therapy for the treatment of patients with metastatic melanoma.     

Production of progenitor cells from primary human epithelial cell monolayer cultures

Primary keratinocytes derived from human epidermis are widely used in tissue engineering and regenerative medicine. An important aspect in clinical applications is the preservation of human skin keratinocyte stem cells. However, it is difficult to expand the number of human skin keratinocyte stem cells, which are undifferentiated and highly proliferative in culture by using standard cell culture methods. It is even more difficult to identify them, since universal specific markers for human skin keratinocyte stem cells have not been identified. In this paper, we show a method to produce a large number of primary progenitor human skin keratinocytes by using our novel culture techniques. Primary human skin keratinocyte monolayers are cultured using twice the volume of medium without serum and lacking essential fatty acids. Once the cells reach 70–80% confluence, they begin to float up into the overlying medium and are called “epithelial pop-up keratinocytes (ePUKs)” allowing the cells to be passaged without the use of trypsin. We analyzed the properties of ePUKs by cell size, cell viability, immunocytofluorescence biomarker staining, and cell cycle phase distribution by fluorescence-activated cell sorting (FACS). Our results showed that these ePUKs appear to be progenitor epithelial cells, which are small in size, undifferentiated, and have a high proliferative capacity. We believe that ePUKs are suitable for use in medical applications requiring a large number of primary human progenitor skin keratinocytes.     

Role of leukemia inhibitory factor in the regulation of the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture

Mouse epidermal melanoblasts/melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanoblast/melanocyte-proliferation medium supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Leukemia inhibitory factor (LIF) supplemented to the medium from initiation of primary culture increased the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes. Pure cultured primary melanoblasts or melanocytes were further cultured with the medium supplemented with LIF from 14 days (keratinocyte depletion). LIF stimulated the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes in the absence of keratinocytes. Moreover, anti-LIF antibody supplemented to the medium from initiation of primary culture inhibited the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes. These results suggest that LIF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture in cooperation with cAMP elevator and bFGF.     

Convenient methodology for extraction and subsequent selective propagation of mouse melanocytes in culture from adult mouse skin tissue

Mouse melanoma B16-BL6 cells are useful cells for cancer metastatic studies. To understand the metastatic principle at molecular levels, it is necessary to carry out experiments in which cancer cells and their normal counterparts are compared. However, unlike normal human melanocytes, preparation of normal mouse melanocytes is quite difficult due to the lack of marketing and insufficient information on an established protocol for primary culture of mouse melanocytes. In this study, we aimed to establish a convenient method for primary culture of mouse melanocytes on the basis of the protocol for human melanocytes. The main obstacles to preparing pure mouse melanocytes are how to digest mouse skin tissue and how to reduce the contamination of keratinocytes and fibroblasts. The obstacles were overcome by collagenase digestion for skin specimens, short time trypsinization for separating melanocytes and keratinocytes, and use of 12-O-Tetradecanoylphorbol 13-acetate (TPA) and cholera toxin in the culture medium. These supplements act to prevent the proliferation of keratinocytes and fibroblasts, respectively. The convenient procedure enabled us to prepare a pure culture of normal mouse melanocytes. Using enriched normal mouse melanocytes and cancerous B16-BL6 cells, we compared the expression levels of melanoma cell adhesion molecule (MCAM), an important membrane protein for melanoma metastasis, in the cells. The results showed markedly higher expression of MCAM in B16-BL6 cells than in normal mouse melanocytes.