Analysis of co-expression of OCT4, NANOG and SOX2 in pluripotent cells of the porcine embryo, in vivo and in vitro
Analysis of co-expression of OCT4, NANOG and SOX2 in pluripotent cells of the porcine embryo, in vivo and in vitro
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DOI：
10.1016/j.theriogenology.2010.09.019
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发表时间：
2011-02-01
期刊：
影响因子：
2.8
通讯作者：
Roelen, Bernard A. J.
中科院分区：
文献类型：
--
作者：
du Puy, Leonie;Lopes, Susana M. Chuva de Sousa;Roelen, Bernard A. J.
为了获得猪胚胎干 (ES) 细胞系，必须确定猪胚胎含有易在体外进行未分化自我更新的多能细胞的时间窗口。因此，我们首先使用整体原位杂交、定量逆转录（RT）-PCR和整体免疫荧光研究了胚胎期（E）6.5至E10.5之间囊胚期猪胚胎多能性和谱系分离关键因子的空间和时间表达模式。在 ICM 和外胚层中均检测到 NANOG 和 SOX2 的表达，而 OCT4 表达在 E9.5 时仅限于外胚层。令人惊讶的是 ICM 和外胚层细胞也表达 CK18。因此，在体外测试了维持人 ES 细胞和小鼠外胚层干细胞 (EpiSC) 未分化生长的生长因子维持猪 ICM 和外胚层细胞未分化自我更新的能力。与源自外胚层细胞的原代培养物相比，ICM 细胞的培养物产生了更高比例的具有 ES 样形态的原代集落。这些未分化的集落持续表达 OCT4、NANOG、SOX2 和 CK18。 CK18 的表达表明这些细胞与人类 ES 细胞和小鼠 EpiSC 比与小鼠 ES 细胞更相似。尽管未分化的培养物维持有限的传代，但如 RT-PCR 所表征，ICM 和外胚层培养物迅速分化为中胚层、外胚层和内胚层来源的细胞类型。这些结果表明，猪ICM和外胚层细胞不能用目前使用的人ES细胞培养条件进行体外培养。然而，重要的是，OCT4、NANOG 和 SOX2 这三者已知在其他系统中形成多能性的自动调节网络，它们也由猪外胚层和培养中的未分化原代集落共同表达。 (C) 2011 Elsevier Inc. 保留所有权利。
To derive porcine embryonic stem (ES) cell lines, the time window during which porcine embryos contain pluripotent cells that are predisposed to undifferentiated self-renewal in vitro must be identified. Therefore we first studied the spatial and temporal expression pattern of key factors in pluripotency and lineage segregation of blastocyst-stage porcine embryos between embryonic days (E) 6.5 and E10.5 using whole mount in situ hybridization, quantitative reverse transcription (RT)-PCR and whole mount immunofluorescence. Expression of NANOG and SOX2 was detected in both the ICM and epiblast, while OCT4 expression became restricted to the epiblast at E9.5. Surprisingly ICM and epiblast cells also expressed CK18. Consequently, growth factors which sustain the undifferentiated growth of human ES cells and mouse epiblast stem cells (EpiSCs) were tested for their ability to sustain undifferentiated self-renewal of porcine ICM and epiblast cells in vitro. Cultures of ICM cells resulted in a higher percentage of primary colonies with an ES-like morphology compared to primary cultures derived from epiblast cells. These undifferentiated colonies sustained expression of OCT4, NANOG, SOX2 and CK18. The expression of CK18 suggests that these cells are more similar to human ES cells and mouse EpiSCs than to mouse ES cells. Although undifferentiated cultures were maintained for limited passages, ICM and epiblast cultures rapidly differentiated into cell types of mesodermal, ectodermal, and endodermal origin, as characterized by RT-PCR. These results demonstrate that porcine ICM and epiblast cells can not be cultured in vitro with currently used human ES cell culture conditions. Importantly however, the trio of OCT4, NANOG and SOX2, which are known to form an autoregulatory network for pluripotency in other systems, are co-expressed also by porcine epiblasts, and by undifferentiated primary colonies in culture. (C) 2011 Elsevier Inc. All rights reserved.