Zhao X E, Zheng Y M
College of Veterinary Medicine, Northwest A&F University and Key Laboratory of Animal Reproductive Physiology and Embryo Technology, Ministry of Agriculture, Yangling, Shaanxi 712100, China.
Animal. 2010 Jun;4(6):921-9. doi: 10.1017/S1751731110000121.
The aim of this study was to determine the developmental ability of cloned embryos derived from porcine neural stem (NS) cells, amniotic fluid-derived stem (AFS) cells, differentiated cells from NS and AFS cells, fetal fibroblast (FF) cells, adult fibroblast (AF) cells and mammary gland epithelial (MGE) cells. NS, AFS and FF cells were isolated from embryonic day 30 porcine fetus, AF and MGE cells were isolated from adult pig. NS and AFS cells were induced to differentiate into different cell types, respectively. Stem cells and their differentiated cells were harvested for analysis of the markers using reverse transcription PCR. NS and AFS cells, their differentiated cells, FF, AF and MGE cells were used for nuclear transfer, respectively. A total of 100 two-cell stage cloned embryos derived from each cell line were transferred into the oviducts of surrogate mothers. The results showed that the neurospheres were positive for the undifferentiated neural cell marker, Nestin and NS cells widely expressed NogoA, DCX, CyclinD2, CD133, Hes1, Oct4, Desmin, CD-90, Nanog and Sox2. AFS cells widely expressed NogoA, DCX, CyclinD2, CD133, Hes1, Nanog, Sox2, Oct4, Desmin and CD-90. Both NS and AFS cells were differentiated into astrocyte (GFAP+), oligodendrocyte (GalC+), neuron (NF+, NSE+ and MAP2+), adipocyte (LPL+ and PPARγ-D+), osteoblast (Osteonectin+ and Osteocalcin+), myocyte (myf-6+ and myoD+) and endothelium (CD31+, CD34+, CD144+ and eNOS+). Four cloned fetuses (28 and 32 days) derived from NS and AFS cells were obtained. The developmental potential of the cloned embryos derived from stem cells (NS and AFS cells) were higher (P < 0.05) than that of the cloned embryos derived from somatic cells (the differentiated cells from NS and AFS cells, FF cells, AF cells and MGE cells), which suggests that the undifferentiated state of the donor cells increases cloning efficiency.
本研究的目的是确定源自猪神经干细胞（NS）、羊水干细胞（AFS）、NS和AFS细胞的分化细胞、胎儿成纤维细胞（FF）、成年成纤维细胞（AF）以及乳腺上皮细胞（MGE）的克隆胚胎的发育能力。NS、AFS和FF细胞从妊娠30天的猪胚胎中分离得到，AF和MGE细胞从成年猪中分离得到。NS和AFS细胞分别被诱导分化为不同的细胞类型。收获干细胞及其分化细胞，使用逆转录PCR分析标志物。NS和AFS细胞、它们的分化细胞、FF、AF和MGE细胞分别用于核移植。从每个细胞系获得的总共100个二细胞期克隆胚胎被移植到代孕母猪的输卵管中。结果显示，神经球对未分化神经细胞标志物巢蛋白呈阳性，NS细胞广泛表达NogoA、双皮质素（DCX）、细胞周期蛋白D2、CD133、Hes1、八聚体结合转录因子4（Oct4）、结蛋白、CD-90、Nanog和性别决定区Y框蛋白2（Sox2）。AFS细胞广泛表达NogoA、DCX、细胞周期蛋白D2、CD133、Hes1、Nanog、Sox2、Oct4、结蛋白和CD-90。NS和AFS细胞均分化为星形胶质细胞（胶质纤维酸性蛋白阳性）、少突胶质细胞（半乳糖脑苷脂阳性）、神经元（神经丝蛋白阳性、神经元特异性烯醇化酶阳性和微管相关蛋白2阳性）、脂肪细胞（脂蛋白阳性和过氧化物酶体增殖物激活受体γ - D阳性）、成骨细胞（骨连接蛋白阳性和骨钙素阳性）、肌细胞（肌原性因子6阳性和肌分化抗原阳性）和内皮细胞（血小板内皮细胞黏附分子-1阳性、CD34阳性、血管内皮钙黏蛋白阳性和内皮型一氧化氮合酶阳性）。获得了4个源自NS和AFS细胞的克隆胎儿（28天和32天）。源自干细胞（NS和AFS细胞）的克隆胚胎的发育潜能高于源自体细胞（NS和AFS细胞的分化细胞、FF细胞、AF细胞和MGE细胞）的克隆胚胎（P < 0.05），这表明供体细胞的未分化状态提高了克隆效率。
