Sun L, Bradford C S, Barnes D W
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331-7305, USA.
Mol Mar Biol Biotechnol. 1995 Mar;4(1):43-50.
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.
使用源自小鼠胚胎的成纤维细胞作为饲养层是在培养中建立多能性小鼠胚胎干细胞（ES细胞）的主要步骤之一。为了从斑马鱼获得多能性ES细胞培养物，从斑马鱼原肠胚早期胚胎建立了一种成纤维样细胞培养物，命名为斑马鱼胚胎成纤维细胞（ZEF），用作饲养层。在没有饲养层的情况下从斑马鱼早期胚胎开始的原代培养中，培养第二天出现了黑素细胞。相比之下，在含有汇合单层ZEF或水牛大鼠肝（BRL）细胞的共培养物中，黑素生成明显受到抑制。BRL细胞是常用于小鼠ES细胞的饲养层细胞。在含有重组人分化抑制活性（DIA）的培养基中起始的原代培养物或在BRL饲养层细胞培养物条件培养基中未观察到黑素生成的抑制。斑马鱼胚胎细胞与任何一种饲养层共培养时增殖显著增强。在ZEF和BRL饲养层上短期共培养的斑马鱼胚胎细胞，当从饲养层移除并悬浮培养时会产生黑素细胞并形成类胚体样结构，这表明细胞在培养中仍保持多能性。
