Em Sadeesh, Kataria Meena, Shah Fozia, Yadav P S
Division of Animal Physiology and Reproduction, ICAR-Central Institute for Research on Buffaloes, Hisar, 125001, India.
Division of Biochemistry, ICAR-Indian Veterinary Research Institute, Bareilly, 243122, India.
Cytotechnology. 2016 Aug;68(4):593-608. doi: 10.1007/s10616-014-9805-1. Epub 2014 Dec 13.
The efficiency of two cell types, namely adult fibroblasts, and amniotic fluid stem (AFS) cells as nuclear donor cells for somatic cell nuclear transfer by hand-made cloning in buffalo (Bubalus bubalis) was compared. The in vitro expanded buffalo adult fibroblast cells showed a typical "S" shape growth curve with a doubling time of 40.8 h and stained positive for vimentin. The in vitro cultured undifferentiated AFS cells showed a doubling time of 33.2 h and stained positive for alkaline phosphatase, these cells were also found positive for undifferentiated embryonic stem cell markers like OCT-4, NANOG and SOX-2, which accentuate their pluripotent property. Further, when AFS cells were exposed to corresponding induction conditions, these cells differentiated into osteogenic, adipogenic and chondrogenic lineages which was confirmed through alizaran, oil red O and alcian blue staining, respectively. Cultured adult fibroblasts and AFS cells of passages 10-15 and 8-12, respectively, were used as nuclear donors. A total of 94 embryos were reconstructed using adult fibroblast as donor cells with cleavage and blastocyst production rate of 62.8 ± 1.8 and 19.1 ± 1.5, respectively. An overall cleavage and blastocyst formation rate of 71.1 ± 1.2 and 29.9 ± 2.2 was obtained when 97 embryos were reconstructed using AFS cells as donor cells. There were no significant differences (P > 0.05) in reconstructed efficiency between the cloned embryos derived from two donor cells, whereas the results showed that there were significant differences (P < 0.05) in cleavage and blastocyst rates between the cloned embryos derived from two donor cell groups. Average total cell numbers for blastocyst generated using AFS cells (172.4 ± 5.8) was significantly (P < 0.05) higher than from adult fibroblasts (148.2 ± 6.1). This study suggests that the in vitro developmental potential of the cloned embryos derived from AFS cells were higher than that of the cloned embryos derived from adult fibroblasts in buffalo hand-made cloning.
比较了两种细胞类型，即成年成纤维细胞和羊水干细胞作为核供体细胞，用于水牛（Bubalus bubalis）手工克隆体细胞核移植的效率。体外扩增的水牛成年成纤维细胞呈现典型的“S”形生长曲线，倍增时间为40.8小时，波形蛋白染色呈阳性。体外培养的未分化羊水干细胞倍增时间为33.2小时，碱性磷酸酶染色呈阳性，这些细胞对于未分化胚胎干细胞标志物如OCT-4、NANOG和SOX-2也呈阳性，这突出了它们的多能特性。此外，当羊水干细胞暴露于相应的诱导条件下时，这些细胞分别分化为成骨、成脂和成软骨谱系，这分别通过茜素红、油红O和阿尔辛蓝染色得到证实。分别使用第10 - 15代的成年成纤维细胞和第8 - 12代的羊水干细胞作为核供体。以成年成纤维细胞作为供体细胞共构建了94个胚胎，卵裂率和囊胚产生率分别为62.8±1.8和19.1±1.5。当以羊水干细胞作为供体细胞构建97个胚胎时，总体卵裂率和囊胚形成率分别为71.1±1.2和29.9±2.2。来自两种供体细胞的克隆胚胎在重构效率上无显著差异（P>0.05），而结果表明来自两个供体细胞组的克隆胚胎在卵裂率和囊胚率上存在显著差异（P<0.05）。使用羊水干细胞产生的囊胚平均总细胞数（172.4±5.8）显著高于成年成纤维细胞产生的囊胚（148.2±6.1）（P<0.05）。本研究表明，在水牛手工克隆中，来自羊水干细胞的克隆胚胎的体外发育潜力高于来自成年成纤维细胞的克隆胚胎。
