Feeder cell cultures for zebrafish embryonal cells in vitro.

Use of fibroblast cells derived from mouse embryos as feeder layers was one of the major steps leading to the establishment of pluripotential mouse embryonal stem (ES) cells in culture. In attempts to obtain a culture of pluripotential ES cells from zebrafish, a culture of fibroblastoid cells, designated zebrafish embryo fibroblast (ZEF), was established from early gastrula stage zebrafish embryos for use as feeder layer. In primary cultures initiated from early embryos of zebrafish without feeder layers, melanocytes appeared on the second day of culture. In contrast, melanogenesis was markedly suppressed in cocultures containing confluent monolayers of ZEF or Buffalo rat liver (BRL) cells. BRL cells are commonly used feeder layer cells for mouse ES cells. Suppression of melanogenesis was not observed in primary cultures initiated in medium containing human recombinant differentiation-inhibiting activity (DIA) or in medium conditioned by cultures of BRL feeder cells. Proliferation of zebrafish embryonal cells was enhanced significantly in cocultures with either feeder layer. Zebrafish embryonal cells cocultured short-term on ZEF and BRL feeder layers gave rise to melanocytes and formed embryoid body-like structures when removed from feeder layers and cultured in suspension, suggesting that the cells remained pluripotent in culture.