Dong Xiao, Tsung Hsiaochien, Mu Yulian, Liu Lixin, Chen Hongping, Zhang Li, Wang Hongjun, Feng Shutang
Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China; College of Life Science, Qingdao Agricultural University, Qingdao, China.
Xenotransplantation. 2014 Mar-Apr;21(2):140-8. doi: 10.1111/xen.12077. Epub 2013 Dec 16.
Human embryonic stem/germ (ES/EG) cell research poses ethical dilemma, it is therefore critical to establish alternative sources of cells for relevant studies. Considering the similarities between the inbred miniature Wuzhishan pigs (WZSP) and humans, ES/EG from these pigs can serve as potential substitutes in human research. In this study, we reported our results that successfully established stable EG cell lines from the WZSP.
Primordial germ cells (PGCs) were isolated from the genital ridges of pig fetuses at 25 to 28 days of pregnancy. To obtain stable EG cell line, PGCs were maintained on STO cells in DMEM containing multiple essential growth factors.
Two EG cell lines were established and characterized by positive alkaline phosphatase staining (AKP), expressions of Oct-4, SSEA-1, SSEA-3, SSEA-4, ability to differentiate into cells of all three germ layers in vitro, and generation of chimeric offsprings after microinjection and embryo transfer. Transmission electron microscopy demonstrated that the cytoplasmic structure of pig EG cells were rather simple and had a higher nuclear-to-cytoplasm ratio. Scanning electron microscopy showed the sizes of pig EG cells were similar to mouse EG cells. Both EG cell lines showed normal karyotypes. The EG cells were propagated for more than 20 passages and underwent multiple cycles of freezing and thawing, without losing their pluripotency (as distinguished by AKP staining).
Both in vitro and in vivo evidence strongly demonstrated that EG cells harvested from the inbred miniature WZSP were pluripotent and can be used for relevant pig or human studies.
人类胚胎干/生殖（ES/EG）细胞研究引发了伦理困境，因此为相关研究建立细胞替代来源至关重要。考虑到近交系五指山小型猪（WZSP）与人类的相似性，这些猪的ES/EG细胞可作为人类研究的潜在替代品。在本研究中，我们报告了成功从WZSP建立稳定EG细胞系的结果。
从妊娠25至28天的猪胎儿生殖嵴中分离原始生殖细胞（PGC）。为获得稳定的EG细胞系，将PGC维持在含有多种必需生长因子的DMEM中的STO细胞上。
建立了两个EG细胞系，其特征为碱性磷酸酶染色（AKP）阳性、Oct-4、SSEA-1、SSEA-3、SSEA-4表达阳性、体外分化为所有三个胚层细胞的能力以及显微注射和胚胎移植后产生嵌合后代。透射电子显微镜显示猪EG细胞的细胞质结构相当简单，核质比更高。扫描电子显微镜显示猪EG细胞的大小与小鼠EG细胞相似。两个EG细胞系均显示正常核型。EG细胞传代超过20次，并经历多次冻融循环，而不丧失其多能性（通过AKP染色区分）。
体外和体内证据均有力表明，从近交系五指山小型猪收获的EG细胞具有多能性，可用于相关的猪或人类研究。
