Pigs share anatomical and physiological traits with humans and can serve as a large-animal model for translational medicine. Bona fide porcine pluripotent stem cells (PSCs) could facilitate testing cell and drug therapies. Agriculture and biotechnology may benefit from the ability to produce immune cells for studying animal infectious diseases and to readily edit the porcine genome in stem cells. Isolating porcine PSCs from preimplantation embryos has been intensively attempted over the past decades. We previously reported the derivation of expanded potential stem cells (EPSCs) from preimplantation embryos and by reprogramming somatic cells of multiple mammalian species, including pigs. Porcine EPSCs (pEPSCs) self-renew indefinitely, differentiate into embryonic and extra-embryonic lineages, and permit precision genome editing. Here we present a highly reproducible experimental procedure and data of an optimized and robust porcine EPSC culture system and its use in deriving new pEPSC lines from preimplantation embryos and reprogrammed somatic cells. No particular expertise is required for the protocols, which take ~4–6 weeks to complete. Importantly, we successfully established pEPSC lines from both in vitro fertilized and somatic cell nuclear transfer-derived embryos. These new pEPSC lines proliferated robustly over long-term passaging and were amenable to both simple indels and precision genome editing, with up to 100% targeting efficiency. The pEPSCs differentiated into embryonic cell lineages in vitro and teratomas in vivo, and into porcine trophoblast stem cells in human trophoblast stem cell medium. We show here that pEPSCs have unique epigenetic features, particularly H3K27me3 levels substantially lower than fibroblasts.

猪与人类具有相同的解剖学和生理学特征，可以作为转化医学的大型动物模型。真正的猪多能干细胞（PSC）可以促进细胞和药物疗法的测试。农业和生物技术可能受益于生产用于研究动物传染病的免疫细胞以及在干细胞中轻松编辑猪基因组的能力。过去几十年来，人们广泛尝试从植入前胚胎中分离猪 PSC。我们之前报道过通过对包括猪在内的多种哺乳动物物种的体细胞进行重编程，从植入前胚胎中衍生出扩展的潜在干细胞（EPSC）。猪 EPSC (pEPSC) 可以无限地自我更新，分化为胚胎和胚胎外谱系，并允许精确的基因组编辑。在这里，我们提出了一个高度可重复的实验程序和数据，优化和强大的猪 EPSC 培养系统及其在从植入前胚胎和重编程体细胞衍生新 pEPSC 系中的用途。该协议不需要特殊的专业知识，大约需要 4-6 周才能完成。重要的是，我们成功地从体外受精和体细胞核移植衍生的胚胎中建立了 pEPSC 系。这些新的 pEPSC 系在长期传代中增殖强劲，并且适合简单的插入缺失和精确的基因组编辑，靶向效率高达 100%。pEPSC在体外分化为胚胎细胞谱系，在体内分化为畸胎瘤，并在人滋养层干细胞培养基中分化为猪滋养层干细胞。我们在此表明​​ pEPSC 具有独特的表观遗传特征，特别是 H3K27me3 水平大大低于成纤维细胞。