| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1999: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1998: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
Stem cell factor (SCF) and endothelin-3 (ET3) are both necessary for melanocyte development. In order to obtain immortal cell populations of melanoblasts which can survive without feeder cells, we first obtained an immortal cell population of neural crest cells form S1/+ and +/+ mice of strain WB by incubating with a culture medium supplemented with SCF and ET3, and designated them as NCC-SE3 cells. NCC-SE3 cells were bipolar, polygonal or round in shape and possessed stage I-III melanosomes (mainly stage I). They were positive to dihydroxyphenylalanine (DOPA) reaction and expressed KIT (a receptor tyrosin kinase), tyrosinase, tyrosinase related potein-1 (TRP1), tyrosinase related potein-2 (TRP2), and endothelin B receptor (ETRB) as determined by immunostaining. We next cultured NCC-SE3 cells by changing culture medium from the one supplemented with SCF+ET3 to the one supplemented with SCT of ET3. NCC-SE3 cells cultured with ET3 alone, designated as NCC-E3 cells, were bipolar in shape and had mainly stage II melanosomes and expressed the same proteins as did NCC-SE3 cells. However, NCC-SE3 cells cultured with SCF alone, designated as NCC-S4.1 cells, were polygonal in shape and had mainly stage I melanosomes. They are thought to be more immature, because they were positive to KIT, TRP1, and TRP2 but not to ETRB, tyrosinase, and DOPA reaction. When 12-o-tetradecanoy1-13-acetate (TPA) and cholera toxin were added to the culture medium, NCC-S4.1 cells changed the shape from polygonal to bipolar and became DOPA positive. This suggests that NCC-S4.1 cells are melanoblasts that have the potential to differentiate into melanocytes. These cell populations will be extremely useful to study factors which affect melanocyte development and melanogenesis.
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