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Variance in iris color is related to the incidence of several important ocular diseases, including uveal melanoma and age-related macular degeneration. The purposes of this study were to determine the quantity and the types of melanin in cultured human uveal melanocytes in relation to the iris color. Sixty-one cell cultures of pure uveal melanocytes were isolated from donor eyes with various iris colors. The amount of eumelanin (EM) and pheomelanin (PM) of these cells was measured by chemical degradation and microanalytical high-performance liquid chromatography (HPLC) methods. The total amount of melanin was measured by both microanalytical methods and spectrophotometry. Total melanin content, measured by HPLC and spectrophotometry, correlated well with r = 0.872 (P < 0.0001). The quantity and type of melanin in iridal and choroidal melanocytes showed no significant difference (P > 0.05). When cells became senescent, the levels of EM, PM and total melanin were significantly increased. In both growing and senescent melanocytes, the quantity and type of melanin were closely correlated to the iris color. In cells from eyes with dark-colored irides (dark brown and brown), the amount of EM, the ratio of EM/PM and total melanin were significantly greater than that from eyes with light-colored irides (hazel, green, yellow-brown and blue) (P < 0.0001). The quantity of PM in uveal melanocytes from eyes with light-colored irides was slightly greater than that from dark-colored irides, although not statistically significant (P > 0.05). The present study shows that iris color is determined by both the quantity and the type of melanin in uveal melanocytes. These results suggest a possibility that uveal melanin in eyes with dark-colored irides is eumelanic at the surface and acts as an antioxidant while that in eyes with light-colored irides exposes pheomelanic core and behaves as a pro-oxidant.

The effects of various prostanoids on the growth, melanogenesis and dendrification of cultured iridal melanocytes were studied. Iridal melanocytes were isolated and cultured with medium supplemented with cAMP elevating agents and basic fibroblast growth factor (bFGF) (complete medium). The iridal melanocytes were plated into multiple well plates and cultured with complete medium or various deleted media with or without various prostanoids at different concentrations. After 6 days, the numbers of cells and dendrites were counted and melanin content was measured and compared with controls. Prostaglandin E(2), an EP(2)receptor agonist (AH 13205) and AGN 192093 (thromboxane mimetic) stimulated growth, melanogenesis and dendrification of cultured iridal melanocytes in cAMP-deleted medium. A mixed EP(1)and EP(3)receptor agonist (sulprostone), a EP(4)receptor agonist (ONO-AE1-329), IP receptor agonists (cicaprost or iloprost) and a TP receptor agonist (U-46619) showed no effect. Prostaglandin D(2)showed stimulating effects. However, these stimulating effects could not be blocked by the addition of a DP receptor antagonist (BW A868C). Furthermore, a DP receptor agonist (BW 245C) showed no effects, indicating that the effect of prostaglandin D(2)may involve receptors other than the DP receptor subtype. The present study indicates that: (1) among various EP receptor agonists, only an EP(2)receptor agonist has stimulating effects on iridal melanocytes; (2) DP, IP and TP receptor agonists do not have stimulating effects; and (3) the mechanisms of action of prostaglandin D(2)and AGN 192093 need further study., (Copyright 2001 Academic Press.)

The effect of various prostaglandins on the growth, melanogenesis and dendrification of cultured iridal melanocytes were studied. Iridal melanocytes were isolated and cultured with medium supplemented with cAMP elevating agents and basic fibroblast growth factor (complete medium). The iridal melanocytes were plated into multiple well plates and cultured with complete medium, cAMP-deleted medium with or without various prostaglandins at different concentrations. After 5-7 days, the numbers of cells and dendrites were counted and melanin content was measured and compared to controls. Prostaglandin A(2)and E(1)stimulated growth, melanogenesis and dendrification of cultured iridal melanocytes in cAMP-deleted medium. Prostaglandin F(2 alpha), PhXA85 and latanoprost showed no effect under similar experimental conditions. These results indicate that prostaglandin A(2)and E(1)stimulate growth and differentiation of iridal melanocytes through the activation of cAMP system. Failure of stimulation of melanogenesis of iridal melanocytes by prostaglandin F(2 alpha)and PhXA85 in vitro may be related to a different second messenger system activated by these prostaglandins and the selective stimulating effect of these prostaglandins on iridal melanocytes from mixed-colored irides., (Copyright 2000 Academic Press.)

The presence of eumelanin and pheomelanin in irides from eyes of various colors was determined and quantified by a highly specific microanalytical procedure based on chemical degradation. Significant differences in the type of melanin were observed in the stroma and iris pigment epithelium (IPE) fractions obtained by micro-dissection of the iris specimens. Melanin from the IPE is essentially eumelanin, while the pigment in IPE-scraped iris (consisting mainly of stroma plus anterior IPE) proved to be both eumelanic and pheomelanic. A pheomelanic-type pigmentation was associated with green irides, while green-blue mixed-color irides were mostly eumelanic; by contrast, green-brown mixed-color and brown irides could not be placed into either of the two categories and probably feature a mixed pigment content. Blue irides invariably exhibited very low pigment content. Analysis of cultured iridial melanocytes in the growing stage showed a significant shift to pheomelanic pigmentation when compared with those in IPE-scraped tissues, providing evidence that growth of iridial melanocyte induce a marked change of melanin metabolism. After senescence, cultured melanocytes exhibited a marked increase in pigment content, most of the variation was associated with the eumelanin content. These results represent the first direct evidence for the presence of eumelanin and pheomelanin in human irides, and suggest that differences in stromal pigmentation are due not only to the quantity, but also the nature of the melanin pigment., (Copyright 1998 Academic Press.)

Objective: To culture iris pigment epithelium (IPE) from surgical iridectomy specimens of eyes with and without exfoliation syndrome., Methods: The IPE was treated to obtain a single cell suspension. Cells were cultured in Ham F12 nutrient mixture, which was supplemented with 30% fetal bovine serum, 50-micrograms/mL [corrected] gentamicin, and 2-mmol/L glutamine. After confluence, the cells were detached using a 0.125% trypsin-0.01% edetic acid solution, resuspended, diluted, and subcultured. The IPE from primary cultures and subcultures was studied by transmission electron microscopy. Immunocytochemical staining was performed., Results: In the primary cultures of IPE from patients with exfoliation syndrome, curved, cross-banded, fine fibrils (diameter, 10-15 nm; periodicity, 10-14 nm) were found on the cell surface. Thicker fibrils (diameter, 24-48 nm; periodicity, 24-36 nm) were found external to the fine fibrils. Subcultures contained mainly fine fibrils. The IPE cells stained positively with anticytokeratin, S100 protein, and vimentin antibodies., Conclusion: Iris pigment epithelium can be successfully cultured from eyes with exfoliation syndrome. Studying the production of exfoliation material in vitro should provide information about the pathogenesis of exfoliation syndrome and about the nature of the exfoliation material. The cultivation of normal IPE from surgical specimens provides a source for the study of the growth regulation and pharmacophysiology of IPE in vitro.

Purpose: To study melanogenesis by cultured human uveal melanocytes, and the relationship between melanin production by uveal melanocytes in vitro with the degree of iris pigmentation in vivo., Methods: Melanin content, melanin production, and tyrosinase activity of cultured uveal melanocytes derived from eyes of various iris color were measured at different stages of cultivation., Results: Cultured uveal melanocytes maintained a constant level of melanin content, expressed tyrosinase activity, and produced measurable amounts of melanin in vitro. Melanosomes in different stages were seen ultrastructurally. Melanin production correlated directly with the degree of iris pigmentation of the eyes from which the uveal melanocytes were isolated. Tyrosinase activity of cultured uveal melanocytes from black versus white donors was significantly different, but, among white donors, there was no correlation with iris pigmentation or with melanin production in vitro., Conclusion: Cultured uveal melanocytes can produce melanin in vitro. Cultured uveal melanocytes isolated from eyes of different iris color maintained their inherent capacity for melanogenesis. Therefore, cultured uveal melanocytes are an excellent model system for studying melanogenesis in uveal melanocytes in vitro.

There have been very few attempts to isolate and culture human iris pigment epithelium (IPE). Earlier efforts that used whole iris explant methods did not achieve pure cultures of IPE. We have developed methods for separating the IPE from the iris stroma of post-mortem eyes that avoid contamination by other cell types. Three different isolation methods were studied: direct dissection, enzyme digestion, and enzyme-assisted microdissection. The latter method yielded the best results. After treatment with enzyme solution, the IPE was easily separated from the stroma under the stereomicroscope and subsequently cultured with supplemented F12 medium. With this method, approximately 2.3 x 10(5) cells were isolated from each iris with an average viability of 90.2%. IPE cells isolated from 19 of 24 eyes grew to confluence in primary culture. The IPE could be maintained in pure culture for many generations over several months with up to 20 population doublings. Cultured IPE demonstrated cytokeratin and S-100 protein by immunocytochemistry. Some of these cells also displayed desmin, indicating origin from the anterior IPE. Cultured IPE cells retained most of the characteristics of IPE in vivo, such as apical/basal polarization, microvilli, and many cell junctions. Gradual dilution of pigment occurred in the dividing IPE cells, suggesting an inability to produce melanin in vitro. A subpopulation of the IPE cells contained myofilaments by electron microscopy, also indicating a anterior IPE origin. This method provides a source for large numbers of human IPE cells and could be useful in studies of the biology of IPE and the role of IPE in pathogenesis of several eye diseases, most notably exfoliation syndrome and its associated glaucomas.