Strojek R M, Reed M A, Hoover J L, Wagner T E
Edison Animal Biotechnology Center Ohio University, Wilson Hall - West Green Athens, OH 45701 USA.
Theriogenology. 1990 Apr;33(4):901-13. doi: 10.1016/0093-691x(90)90825-e.
Variable conditions were tested to determine an in-vitro cultivation method for the formation of morphologically undifferentiated embryonic stem cells from the inner cell mass (ICM) derived outgrowth of porcine blastocysts. Although all 16 Day-9 embryos failed to form colonies, 14 such colonies were obtained from a total of 69 Day-10 embryos when they were co-cultivated with porcine uterine fibroblast (PUF) cells over a 6-day period. The best results were obtained in Dulbecco's modified Eagle medium (DMEM) with 10% fetal calf serum and 10% porcine serum supplemented with bovine insulin and beta-mercaptoethanol, in which six out of seven embryos formed adequate ICM-derived colonies. Since murine fibroblasts were not found to be suitable feeder cells in this procedure, an endocrine synergistic interaction, which promotes embryonic attachment and colony formation, between porcine blastocysts and PUF cells is hypothesized. Continued propagation of the ICM-derived cells was not dependent on these factors; a total of seven cell lines were obtained after three to five subsequent passages on murine feeder-layers that resembled morphologically undifferentiated embryonic cells.
对各种不同条件进行了测试，以确定一种体外培养方法，用于从猪囊胚的内细胞团（ICM）衍生的外植体中形成形态上未分化的胚胎干细胞。尽管所有16个第9天的胚胎都未能形成集落，但当69个第10天的胚胎与猪子宫成纤维细胞（PUF）共同培养6天时，共获得了14个这样的集落。在含有10%胎牛血清和10%猪血清，并添加牛胰岛素和β-巯基乙醇的杜氏改良伊格尔培养基（DMEM）中获得了最佳结果，在这种培养基中，7个胚胎中有6个形成了足够的源自ICM的集落。由于在该过程中发现鼠成纤维细胞不是合适的饲养细胞，因此推测猪囊胚与PUF细胞之间存在促进胚胎附着和集落形成的内分泌协同相互作用。源自ICM的细胞的持续传代不依赖于这些因素；在鼠饲养层上经过三到五次后续传代后，共获得了七个细胞系，这些细胞系在形态上类似于未分化的胚胎细胞。
