Haraguchi Seiki, Kikuchi Kazuhiro, Nakai Michiko, Tokunaga Tomoyuki
Animal Breeding and Reproduction Division, NARO Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, Ibaraki 305-0901, Japan.
J Reprod Dev. 2012;58(6):707-16. doi: 10.1262/jrd.2012-008. Epub 2012 Sep 7.
Although the establishment of putative porcine embryonic stem cells (ESCs) has been reported, such cell lines quickly lose their self-renewal ability, as they easily differentiate or become extinct after only a limited number of passages in culture. ESC-like cells exhibiting self-renewal rather than pluripotency are considered to be a valuable resource in applications such as drug screening and toxicology testing in humans, livestock and veterinary medicine. Here, we report the generation of unique cell lines established from the inner cell mass (ICM) of porcine embryos by using inhibitors of glycogen synthase kinase 3β and mitogen-activated protein kinase kinase 1. These ICM-derived cell lines were initially cultured and passaged in conventional ES medium for human ESCs and showed porcine ESC-like morphology with alkaline phosphatase (AP) activity. After transfer to culture in ES medium containing inhibitors, the morphology of the colonies was dramatically changed, i.e., they were closely packed smooth-edged colonies with close cell-cell boundaries and showed the expression of undifferentiated markers including OCT4 (POU5F1) and NANOG. Notably, the self-renewal capacity and morphology of the cells were LIF-dependent, consistent with the expression of LIF receptors and phosphorylation of signal transducer and activator of transcription 3. To date, our established cell lines have been cultured continuously for over 100 passages without any overt morphological changes. Thus, the established cell lines reported here provide a new ESC-like cell culture system for use not only in the fields of veterinary medicine and livestock but also human medical research, since porcine physiology closely resembles that of humans.
尽管已有报道称建立了假定的猪胚胎干细胞（ESC），但此类细胞系很快就会失去自我更新能力，因为它们在培养中仅经过有限次数的传代后就容易分化或灭绝。表现出自我更新而非多能性的类ESC细胞被认为是人类、家畜和兽医学药物筛选和毒理学测试等应用中的宝贵资源。在此，我们报告了通过使用糖原合酶激酶3β和丝裂原活化蛋白激酶激酶1的抑制剂，从猪胚胎的内细胞团（ICM）建立独特细胞系的过程。这些源自ICM的细胞系最初在用于人类ESC的传统ES培养基中培养和传代，呈现出具有碱性磷酸酶（AP）活性的猪ESC样形态。在转移至含有抑制剂的ES培养基中培养后，集落的形态发生了显著变化，即它们是紧密堆积的边缘光滑的集落，细胞间边界紧密，并显示出包括OCT4（POU5F1）和NANOG在内的未分化标志物的表达。值得注意的是，细胞的自我更新能力和形态依赖于白血病抑制因子（LIF），这与LIF受体的表达以及信号转导子和转录激活子3的磷酸化一致。迄今为止，我们建立的细胞系已连续培养超过100代，没有任何明显的形态变化。因此，本文报道的已建立细胞系不仅为兽医学和家畜领域，也为人类医学研究提供了一种新的类ESC细胞培养系统，因为猪的生理学与人类非常相似。
