Establishment of melanoblast cell lines and analysis of the factor involving melanocyte development

干细胞因子(SCF)和内皮素-3(ET3)都是黑素细胞发育所必需的。为了获得在没有饲养层细胞的情况下仍能存活的黑素母细胞的永生化细胞群，我们首先用添加SCF和ET3的培养液从菌株WB的S1/和/小鼠中获得了神经脊细胞永生化细胞群，命名为NCC-SE3细胞。NCC-SE3细胞呈双极、多角形或圆形，具有I-III期黑素小体，主要为I期。免疫组化检测KIT、酪氨酸酶、酪氨酸酶相关蛋白-1(TRP1)、酪氨酸酶相关蛋白-2(Trp2)和内皮素B受体(ETRB)的表达。接下来，我们将NCC-SE3细胞的培养液从添加SCF ET3的培养液更换为添加ET3的SCT的培养液。单独用ET3培养的NCC-SE3细胞，命名为NCC-E3细胞，呈双极形态，主要为II期黑素小体，表达与NCC-SE3细胞相同的蛋白。而单独与SCF共同培养的NCC-SE3细胞，命名为NCC-S4.1细胞，形态呈多角形，以I期黑素小体为主。它们被认为更不成熟，因为它们对KIT、TRP1和Trp2呈阳性，而对ETRB、酪氨酸酶和DOPA反应不呈阳性。加入TPA和霍乱毒素后，NCC-S4.1细胞由多角形变为双极形，呈DOPA阳性。这表明NCC-S4.1细胞是具有分化为黑素细胞的潜能的黑素母细胞。这些细胞群体将对研究影响黑素细胞发育和黑素生成的因素非常有用。

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.