Handyside Alan H, O'Neill Gerard T, Jones Mary, Hooper Martin L
MRC Experimental Embryology and Teratology Unit, MRC Laboratories, Woodmansterne Road, SM5 4EF, Carshalton, Surrey, England.
Department of Pathology, University of Edinburgh, Teviot Place, EH8 9AG, Edinburgh, England.
Rouxs Arch Dev Biol. 1989 May;198(1):48-56. doi: 10.1007/BF00376370.
The derivation of a karyotypically normal embryonal stem (ES) cell line, E14, from inner cell masses (ICMs) isolated by immunosurgery from 129/Ola late mouse blastocysts is described. Disaggregated ICMs were cultured on mitotically-arrested fibroblast feeder layers in droplets of medium conditioned with Buffalo rat liver (BRL) cells under oil. BRL-conditioned medium inhibits the differentiation of established embryonal carcinoma (EC) and ES cell lines which can be maintained indefinitely in the complete absence of feeder cells (Smith and Hooper 1987). At clonal densities, however, a combination of BRL-conditioned medium and a feeder layer was most effective in preventing the differentiation of E14 cells. This effect was less pronounced at higher passage suggesting it may be particularly important to use a combination in the early stages of isolation. Once established, E14 has been maintained in BRL-conditioned medium alone. In non-conditioned medium on agarose, E14 cells formed embryoid bodies which when allowed to reattach differentiated into a wide variety of tissues. An HPRT-deficient sub line of E14, E14TG2a, has been demonstrated to form germline chimaeras with high efficiency after injection into blastocysts (Hooper et al. 1987). The modifications to the ES cell isolation procedure described here may improve the efficiency with which karyotypically normal lines can be derived.
本文描述了从129/Ola晚期小鼠囊胚中通过免疫手术分离的内细胞团（ICM）中获得核型正常的胚胎干细胞（ES）系E14的过程。将分散的ICM接种在经丝裂霉素处理的成纤维细胞饲养层上，置于油下的水牛大鼠肝（BRL）细胞条件培养基液滴中培养。BRL条件培养基可抑制已建立的胚胎癌细胞（EC）和ES细胞系的分化，这些细胞系在完全没有饲养细胞的情况下可无限期维持（Smith和Hooper，1987）。然而，在克隆密度下，BRL条件培养基和饲养层的组合在防止E14细胞分化方面最为有效。在传代次数较高时，这种效果不太明显，这表明在分离的早期阶段使用这种组合可能特别重要。一旦建立，E14已单独在BRL条件培养基中维持培养。在琼脂糖上的非条件培养基中，E14细胞形成胚状体，当允许其重新附着时可分化为多种组织。已证明E14的一个次黄嘌呤磷酸核糖转移酶（HPRT）缺陷亚系E14TG2a在注入囊胚后能高效形成种系嵌合体（Hooper等人，1987）。本文所述的ES细胞分离程序的改进可能会提高获得核型正常细胞系的效率。
