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.     

Melanosomes, melanocytes and keratinocytes in the human epidermis in incontinentia pigmenti

A functioning epidermal melanin unit implies a melanocyte capable of transferring melanosomes to keratinocytes; this requires not only melanocytes with adequate dendrites but also "receptive" keratinocytes. Skin with incontinentia pigmenti was examined by electron microscopy. Premelanosomes were occasionally found within keratinocytes and deposits of extracellular granular material that came from vacuolar degeneration of keratinocytes adjacent to melanocytes.     

Ultrastructure of normals and castrates and the effects of testosterone and ultraviolet (UVL-B) irradiation on scrotal skin of rats.

The ultrastructure of the testosterone dependent epidermal melanocyte system of the scrotal skin of normals and castrates, with and without testosterone replacement therapy, and UVL-B (280-315 nm) radiation in black Long Evans rats is reported. UVL-B increases melanocyte activity, melanosome forming apparatus, (size of Golgi zone and RER, and quantity of cytoplasmic vesicles, dendrites, and stages of melanosomes) in normals and in castrates. Testosterone replacement therapy to castrates is not a prerequisite for stimulation by UVL-B, but it enhances the effects of UVL-B without restoring normalcy as melanosome packaging into complexes predominates. After UVL-B stimulation of normals or castrates, melanocyte dendrites are observed more often. Melanocyte dendrites of skin of castrated rats are observed less often than in normals, but with testosterone replacement therapy, the dendrites become more numerous. Melanosomes donated to keratinocytes are mostly located as singles in normals and as complexes in castrates. After UVL-B, castration, or testosterone replacement therapy, the melanosomes are packaged in keratinocytes in complexes larger than in normals. In the epidermis of long term castrates (9-109 days), non-specific clear cells are observed and Langerhans cells containing melanosomes; we did not observe them in normals. Melanocytes of castrates have a reduced melanosome forming apparatus. The dermis of castrates contains many dermal melanocytes in the superficial dermis with melanosomes in several stages of formation. These cells are not apparent in normals at this location in the dermis. Testosterone replacement therapy and/or UVL-B administered to castrates does not restore the epidermal melanocyte system nor the dermis to precastration ultrastructural appearance; castration has a permanent altering effect as melanosomes are packaged into complexes.     

Molecular motors and their role in pigmentation.

Skin pigmentation is orchestrated through a series of complementary processes. After migration of melanoblasts out of the neural crest to epidermis and hair follicle, these cells mature into melanocytes. Differentiated melanocytes produce melanin in specialized organelles, the melanosomes. Moreover, the cytoplasm of melanocytes branches into extensions, the dendrites. Via the tips of these dendrites they donate their mature melanosomes to the keratinocytes resulting in skin pigmentation. Thus, one essential part of the process of pigmentation is the translocation of melanosomes from their site of origin in the perinuclear cytoplasm towards the dendrite tips. Motor proteins are molecules which use the energy derived from ATP hydrolysis to move along cytoskeletal elements, either actin filaments or microtubules, to transport their cargo, which can be organelles, vesicles or chromosomes. This review describes the different classes of microtubule-based and actin-based motor proteins with their characteristics and functional importance in cell biology and organelle transport. Some of them will be highlighted and several recent studies in mammalian pigment cells indicating their role in pigment granule transport will be discussed. As a result of these data and previous suggestions, a model will be proposed for the possible cooperation of both systems in melanosome movement.     

Role of keratinocyte‐derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes

Melanocytes characterized by the activities of tyrosinase, tyrosinase‐related protein (TRP)‐1 and TRP‐2 as well as by melanosomes and dendrites are located mainly in the epidermis, dermis and hair bulb of the mammalian skin. Melanocytes differentiate from melanoblasts, undifferentiated precursors, derived from embryonic neural crest cells. Because hair bulb melanocytes are derived from epidermal melanoblasts and melanocytes, the mechanism of the regulation of the proliferation and differentiation of epidermal melanocytes should be clarified. The regulation by the tissue environment, especially by keratinocytes is indispensable in addition to the regulation by genetic factors in melanocytes. Recent advances in the techniques of tissue culture and biochemistry have enabled us to clarify factors derived from keratinocytes. Alpha‐melanocyte‐stimulating hormone, adrenocorticotrophic hormone, basic fibroblast growth factor, nerve growth factor, endothelins, granulocyte‐macrophage colony‐stimulating factor, steel factor, leukemia inhibitory factor and hepatocyte growth factor have been suggested to be the keratinocyte‐derived factors and to regulate the proliferation and/or differentiation of mammalian epidermal melanocytes. Numerous factors may be produced in and released from keratinocytes and be involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes through receptor‐mediated signaling pathways.     

The Mechanism of Melanocyte Dendrite Formation: The Impact of Differentiating Keratinocytes

In human epidermis one dendritic melanocyte interacts with about 36 keratinocytes and supplies them with melanin. In contrast to the vivo situation melanocytes in culture are far less dendritic. In the present study different culture systems were tested in order to observe the mechanism of melanocyte dendrite formation. In particular, we focused on the role of keratinocytes in this process. Time lapse studies revealed that only differentiated keratinocytes enhance melanocyte dendricity. Differentiated keratinocytes form connected cell sheets, which attach to part of the melanocyte plasma membrane. By contraction and retraction of keratinocyte units, new dendrites were drawn out from the melanocytes. Melanocytes remain passive during this process, which is indicated by the observation that sometimes extended dendrites could not withstand the tension and shear.     

Rac and rho: the story behind melanocyte dendrite formation

Melanocyte dendrites are hormonally responsive actin and microtubule containing structures whose primary purpose is to transport melanosomes to the dendrite tip. Melanocyte dendrites have been an area of intense interest for melanocyte biologists, but it was not until recently that we began to understand the mechanisms underlying their formation. In contrast with melanogenesis, for which numerous mutations in pigment producing genes and mouse models have been identified, a genetic defect resulting in impaired dendrite formation has not been found. Therefore, much of the insight into melanocyte dendrites has come from electron microscopy or in vitro culture systems of normal human and murine melanocytes as well as melanoma cell lines. The growth factors that regulate the formation of melanocyte dendrites have been thoroughly studied and it is clear that multiple signalling systems are able to stimulate, and in some cases inhibit, dendrite formation. Recent data points to the Rho family of small guanosine triphosphate (GTP)-binding proteins as master regulators of dendrite formation, particularly Rac and Rho. In this review I will summarize the progress scientists have made in understanding the structure, hormonal regulation and molecular mediators of melanocyte dendrite formation.     

Characterization of Melanogenesis in Normal Human Epidermal Melanocytes by Chemical and Ultrastructural Analysis

Chemical and ultrastructural studies were conducted to define the relationship between type of melanogenesis and fine structures of melanosomes in normal human epidermal melanocytes. Chemical analysis of epidermal melanin demonstrated that the ratio of eumelanin/pheomelanin varied individually, ranging from 1.31 to exclusively eumelanic. Ultrastructural analysis of fine structures of melanosomes revealed that spheroid melanosomes were frequently observed in melanocytes of the epidermis whose eumelanin/pheomelanin ratio was less than 5. Conversely, ellipsoid melanosomes predominated in melanocytes of the epidermis whose ratio was more than 10. On the basis of these findings, it seems reasonable to conclude that 1) normal human epidermal melanocytes synthesize both eumelanin and pheomelanin and 2) pheomelanin synthesis may be characterized by the presence of spheroid melanosomes whereas eumelanin synthesis is ascribed to ellipsoid melanosomes.     

Effect of bromocriptine on the larval skin of the green toad, Bufo viridis viridis leurenti

In the premetamorphic larval green toad, B. viridis viridis, as in other anurans, the skin is made up of a fibrous dermis and an epidermis of stratified epithelium. The effects of bromocriptine, an antiprolactin drug, on the premetamorphic skin of B. viridis viridis was examined. Bromocriptine, dissolved in rearing water at four different concentrations, induced a number of changes in the skin of treated tadpoles. In rough sequence of appearance, these changes include: retraction ofthe melanocyte dendrites, synchronous burst ofthe apical vesicles of the superficial epithelial cells, gradual disappearance of the melanosomes from the epithelial cells and widening of the intercellular spaces. In addition, macrophages appeared in the superficial dermis amongst the retracted melanocytes. White crystals were observed on the skin surface and similar crystals were ingested by the macrophages. Prolonged treatment with bromocriptine resulted in hypertrophy and extraction of some epidermal cells. Deep melanocytes of the mesenteries were not affected by bromocriptine-treatment indicating that the drug did not penetrate deep into the tadpole tissue. Whether the macrophages observed in the dermis were recruited from deeper tissues or were converted melanocytes is another issue in need of study.     

Stimulation of the proliferation and differentiation of mouse pink-eyed dilution epidermal melanocytes by excess tyrosine in serum-free primary culture

The epidermal cell suspensions of the neonatal dorsal skin derived from wild type mouse at the pink-eyed dilution (p) locus (black, C57BL/10JHir-P/P) and their congenic mutant mouse (pink-eyed dilution, C57BL/10JHir-p/p) were cultured with a serum-free melanocyte growth medium supplemented with additional L-tyrosine (Tyr) from initiation of the primary culture. L-Tyr inhibited the proliferation of P/Pmelanocytes in a dose-dependent manner, whereas L-Tyr stimulated the proliferation of p/p melanoblasts and melanocytes regardless of dose. On the other hand, L-Tyr stimulated (P/P) or induced (p/p) the differentiation of epidermal melanocytes in a dose-dependent manner. In both P/P and p/p melanoblasts and melanocytes cultured with 2.0 mM L-Tyr for 14 days, slight increases in contents of eumelanin marker, pyrrole-2,3,5-tricarboxylic acid (PTCA) and pheomelanin marker, aminohydroxyphenylalanine (AHP) were observed. The average number of total melanosomes (stages I, II, III, and IV) per P/P melanocyte was not changed by L-Tyr treatment, but the proportion of stage IV melanosomes in the total melanosomes was increased. On the contrary, in p/p melanoblasts and melanocytes L-Tyr increased dramatically the number of stage II, III, and IV melanosomes as well as the proportion of stage III melanosomes. Contents of PTCA and eumelanin precursor, 5,6-dihydroxyindole-2-carboxylic acid (DHICA) of cultured media in p/p melanocytes were much more greatly increased than in P/P melanocytes. However, contents of AHP and pheomelanin precursor, 5-S-cysteinyldopa (5-S-CD) of cultured media in p/p melanocytes were increased in a similar tendency to P/Pmelanocytes. These results suggest that p/p melanocytes in the primary culture are induced to synthesize eumelanin by excess L-Tyr, but difficult to accumulate them in melanosomes.     

Changes in distribution pattern of cytoplasmic filaments in human melanocytes during ultraviolet-mediated melanin pigmentation. The role of the 100-Angstrom filaments in the elongation of melanocytic dendrites and in the movement and transfer of melanosomes

Human melanocytes characteristically contain 100-A filaments. These 100- A filaments shift from the perinuclear area to the center of the dendritic processes and are in close association with melanosomes during the different stages of UV-mediated melanin pigmentation. We suggest that these 100-A filaments in human melanocytes participate in the elongation of the dendrites and in the transfer of melanosomes.     

Developmental relocation of presynaptic terminals along distinct types of dendritic filopodia

Dendritic filopodia are long thin protrusions occurring predominantly on developing neurons. Data from different systems suggest a range of crucial functions for filopodia in central circuit formation, including steering of dendritic growth, branch formation, synaptogenesis, and spinogenesis. Are the same filopodia competent to mediate all these processes, do filopodia acquire different functions through development, or do different filopodial types with distinct functions exist? In this study, 3-dimensional reconstructions from confocal image stacks demonstrate the existence of two morphologically and functionally distinct types of filopodia located on the dendritic tips versus the dendritic shafts of the same developing motoneuron. During dendritic growth, both filopodial types undergo a process of stage-specific morphogenesis. Using novel quantification strategies of 3-dimensional co-localization analysis for immunocytochemically labeled presynaptic specializations along postsynaptic filopodia, we find that presynaptic terminals accumulate along filopodia towards the dendrites at both stable dendritic shafts and on growing dendritic tips. On tips, this is likely to reflect synaptotrophic growth of the dendrite. At stable shafts, however, presynaptic sites become relocated along filopodia towards dendritic branches. This indicates the interactive growth of both pre- and postsynaptic partner towards one another during synaptogenesis, using filopodia as guides.     

The quest for the mechanism of melanin transfer

Skin pigmentation is accomplished by production of melanin in specialized membrane-bound organelles termed melanosomes and by transfer of these organelles from melanocytes to surrounding keratinocytes. The mechanism by which these cells transfer melanin is yet unknown. A central role has been established for the protease-activated receptor-2 of the keratinocyte which effectuates melanin transfer via phagocytosis. What exactly is being phagocytosed - naked melanin, melanosomes or melanocytic cell parts - remains to be defined. Analogy of melanocytes to neuronal cells and cells of the haemopoietic lineage suggests exocytosis of melanosomes and subsequent phagocytosis of naked melanin. Otherwise, microscopy studies demonstrate cytophagocytosis of melanocytic dendrites. Other plausible mechanisms are transfer via melanosome-containing vesicles shed by the melanocyte or transfer via fusion of keratinocyte and melanocyte plasma membranes with formation of tunnelling nanotubes. Molecules involved in transfer are being identified. Transfer is influenced by the interactions of lectins and glycoproteins and, probably, by the action of E-cadherin, SNAREs, Rab and Rho GTPases. Further clues as to what mechanism and molecular machinery will arise with the identification of the function of specific genes which are mutated in diseases that affect transfer.     

Growth characteristics of human epidermal melanocytes in pure culture with special reference to genetic differences

Human melanocyte cultures were established using disaggregated epidermal cell suspensions derived from foreskins and plated onto culture dishes in medium containing 2% fetal bovine serum, growth factors, hormones, and melanocyte growth factor (MGF) extracted from bovine hypothalamus (Wilkins et al., J.Cell. Physiol., 122:350, 1985). After 2 days in culture the cells were transferred to serum-free medium to eliminate keratinocyte and fibroblast growth. Melanocytes grew preferentially and pure melanocyte populations could be harvested after 12-16 days in vitro. Melanocytes were later subcultured in the presence of 1% FBS. Pure melanocyte cultures were characterized by light and electron microscopic criteria, as well as by cytochemical demonstration of the melanocyte-specific enzyme, tyrosinase. At the ultrastructural level, cultured melanocytes derived from black (negroid) neonatal skin (B-M) had numerous mature rod-shaped stage IV melanosomes, while white (caucasoid) skin-derived melanocytes (W-M) in culture contained no mature melanosomes. Growth rate, cell yield, and in vitro lifespan for B-M were more than twice that for W-M in pure melanocyte cultures in the presence of MGF. Our results suggest that MGF-dependent growth of B-M differs from that of W-M.     

Rab7 and Rab27a control two motor protein activities involved in melanosomal transport

Melanosomes are lysosome-related organelles that synthesize, store and transport melanin. In epidermal melanocytes, melanosomes mature and are transferred to surrounding keratinocytes, which is essential for skin and coat colour. Mouse coat colour mutants reveal a critical role for the small GTPase Rab27a, which recruits myosin Va through its effector protein melanophilin/Slac2a. Here we have studied how two different Rab GTPases control two motor proteins during subsequent phases in transport of melanosomes. We show that the small GTPase Rab7 mainly associates with early and intermediate stage melanosomes and Rab27a to intermediate and mature melanosomes. Rab27a is found in an active state on mature melanosomes in the tips of the dendrites. The Rab7-Rab7-interacting lysosomal protein-dynein pathway only controls early and intermediate stage melanosomes because the mature melanosomes lack Rab7 and associate with the actin network through Rab27a recruited MyoVa. Thus two Rab proteins regulate two different motor proteins, thereby controlling complementary phases in melanosome biogenesis: Rab7 controls microtubule-mediated transport of early and Rab27a the subsequent actin-dependent transport of mature melanosomes.     

A quantitative and morphological study of the pigmentary system of the chimpanzee with the light and electron microscope.

The epidermal melanocyte system of the chimpaneze was studied by the combined skin-splitting DOPA, and electron microscopic techniques. It is very similar to man. There are DOPA-positive epidermal melanocytes in all body regions regradless of the degree of macroscopic skin pigmentation or hirsutism. Furthermore, as in man, but in contrast to rodents, chimpanzee skin contains a very high level of melanocytes in the epidermis; approximately 3,320+/-350 per square millimeter skin. Chimpanzee melanosomes are long, wide, and fully melanized. In keratinocytes, these organelles are individually dispersed in all body regions, regardless of the degree of skin color, as is true for other mammalian species with large melanosomes.     

The slaty mutation affects the morphology and maturation of melanosomes in the mouse melanocytes

The slaty (Dct(slt)) mutation is known to reduce the activity of dopachrome tautomerase in melanocytes and to reduce the melanin content in skin, hairs and eyes. Although the melanosomes in slaty melanocytes are reported to be eumelanosome-like, detailed melanosome biogenesis is not well studied. To address this point, melanosomes in neonatal epidermal melanocytes from wild-type (Dct+/Dct+) mice at the slaty locus as well as its congenic mouse mutant (Dct(slt)/Dct(slt)) in serum-free primary culture were observed under the electron microscope. Wild-type melanocytes possessed exclusively elliptical melanosomes with internal longitudinal structures, whereas in mutant melanocytes, numerous spherical melanosomes with globular depositions of pigment and elliptical melanosomes as well as mixed type of the two melanosomes were observed. Mature stage IV melanosomes were greatly decreased in mutant melanocytes, whereas immature stage III melanosomes were more numerous than in wild-type melanocytes. These results suggest that the slaty mutation affects the morphology and maturation of melanosomes in mouse melanocytes.