Anand T, Kumar D, Singh M K, Shah R A, Chauhan M S, Manik R S, Singla S K, Palta P
Animal Biotechnology Center, National Dairy Research Institute, Karnal-132001, India.
Reprod Domest Anim. 2011 Feb;46(1):50-8. doi: 10.1111/j.1439-0531.2009.01564.x.
In this study, inner cell mass (ICM) cells were isolated from in vitro produced buffalo blastocysts and were cultured on mitomycin-C treated buffalo foetal fibroblast feeder layer for producing embryonic stem (ES) cells. Among different sources (hatched vs expanded blastocysts) or methods (enzymatic vs mechanical), mechanical isolation of ICM from hatched blastocysts resulted in the highest primary colony formation rate and the maximum passage number up to which ES cells survived. Putative ES cells expressed alkaline phosphatase and exhibited a normal karyotype up to passage 7. Putative ES cells and embryos at 2- to 4-cell, 8- to 16-cell, morula and blastocyst stages strongly expressed stage-specific embryonic antigen (SSEA)-4 but lacked expressions of SSEA-1 and SSEA-3. Putative ES cells also expressed tumour rejection antigen (TRA)-1-60, TRA-1-81 and Oct4. Whereas in all early embryonic stages, TRA-1-60 was observed only in the periplasmic space, and TRA-1-81 expression was observed as small spots at a few places inside the embryos, both these markers were expressed by ICM. Oct4 expression, which was observed at all the embryonic stages and also in the trophectoderm, was the strongest in the ICM. Buffalo putative ES cells possess a unique pluripotency-related surface antigen phenotype, which resembles that of the ICM.
在本研究中，从体外生产的水牛囊胚中分离出内细胞团（ICM）细胞，并将其培养在经丝裂霉素-C处理的水牛胎儿成纤维细胞饲养层上以生产胚胎干细胞（ES细胞）。在不同来源（孵化囊胚与扩张囊胚）或方法（酶解法与机械法）中，从孵化囊胚中机械分离ICM导致最高的原代集落形成率和ES细胞存活的最大传代数。推定的ES细胞表达碱性磷酸酶，并且在传代7之前表现出正常的核型。推定的ES细胞以及处于2至4细胞、8至16细胞、桑葚胚和囊胚阶段的胚胎强烈表达阶段特异性胚胎抗原（SSEA）-4，但缺乏SSEA-1和SSEA-3的表达。推定的ES细胞还表达肿瘤排斥抗原（TRA）-1-60、TRA-1-81和Oct4。而在所有早期胚胎阶段，TRA-1-60仅在周质空间中观察到，TRA-·1-81的表达在胚胎内部的一些地方表现为小点，这两种标记物均由ICM表达。在所有胚胎阶段以及滋养外胚层中均观察到的Oct4表达，在ICM中最强。水牛推定的ES细胞具有独特的多能性相关表面抗原表型，类似于ICM的表型。
