SummaryDual inhibition (2i) of Ras–MEK–ERK and GSK3β pathways enables the derivation of embryo stem cells (ESCs) from refractory mouse strains and, for permissive strains, allows ESC derivation with no external protein factor stimuli involvement. In addition, blocking of ERK signalling in 8-cell-stage mouse embryos leads to ablation of GATA4/6 expression in hypoblasts, suggesting fibroblast growth factor (FGF) dependence of hypoblast formation in the mouse. In human, bovine or porcine embryos, the hypoblast remains unaffected or displays slight-to-moderate reduction in cell number. In this study, we demonstrated that segregation of the hypoblast and the epiblast in rabbit embryos is FGF independent and 2i treatment elicits only a limited reinforcement in favour of OCT4-positive epiblast populations against the GATA4-/6-positive hypoblast population. It has been previously shown that TGFβ/Activin A inhibition overcomes the pervasive differentiation and inhomogeneity of rat iPSCs, rat ESCs and human iPSCs while prompting them to acquire naïve properties. However, TGFβ/Activin A inhibition, alone or together with Rho-associated, coiled-coil containing protein kinase (ROCK) inhibition, was not compatible with the viability of rabbit embryos according to the ultrastructural analysis of preimplantation rabbit embryos by electron microscopy. In rabbit models ovulation upon mating allows the precise timing of progression of the pregnancy. It produces several embryos of the desired stage in one pregnancy and a relatively short gestation period, making the rabbit embryo a suitable model to discover the cellular functions and mechanisms of maintenance of pluripotency in embryonic cells and the embryo-derived stem cells of other mammals.

中文翻译：

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Ras-MEK-ERK和GSK3β通路双重抑制对体外培养兔胚胎发育的影响

总结 Ras-MEK-ERK 和 GSK3β 通路的双重抑制 (2i) 使得能够从难治小鼠品系中衍生胚胎干细胞 (ESC)，并且对于许可品系，允许衍生 ESC 而不涉及外部蛋白质因子刺激。此外，在 8 细胞期小鼠胚胎中阻断 ERK 信号会导致下胚层中 GATA4/6 表达的消融，这表明成纤维细胞生长因子 (FGF) 对小鼠下胚层形成的依赖性。在人、牛或猪胚胎中，下胚层不受影响或细胞数量略有减少。在这项研究中，我们证明了兔胚胎中下胚层和外胚层的分离与 FGF 无关，并且 2i 处理仅引起有限的增强，有利于 OCT4 阳性外胚层群体对抗 GATA4-/6 阳性下胚层群体。先前已经表明，TGFβ/激活素 A 抑制克服了大鼠 iPSC、大鼠 ESC 和人类 iPSC 的普遍分化和不均匀性，同时促使它们获得幼稚特性。然而，根据电子显微镜对植入前兔胚胎的超微结构分析，单独或与 Rho 相关的含有蛋白激酶 (ROCK) 的卷曲螺旋抑制一起抑制 TGFβ/激活素 A 与兔胚胎的活力不相容。在兔子模型中，交配时的排卵允许精确的妊娠进展时间。