NARIC, Agricultural Biotechnology Institute, Animal Biotechnology Department, 2100Gödöllő, Szent-Györgyi A. str. 4., Hungary.
Department of Laboratory Medicine, Semmelweis University, 1088Budapest, Szentkiralyi str. 46, Hungary.
Zygote. 2020 Jun;28(3):183-190. doi: 10.1017/S0967199419000753. Epub 2020 Mar 20.
Dual 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.
双重抑制 Ras-MEK-ERK 和 GSK3β 通路可使胚胎干细胞（ESCs）从难治性小鼠品系中分离出来，对于允许的品系，允许在没有外部蛋白因子刺激参与的情况下分离 ESC。此外，在 8 细胞期小鼠胚胎中阻断 ERK 信号通路会导致下胚层中 GATA4/6 表达的消融，这表明在小鼠中，成纤维细胞生长因子（FGF）依赖于下胚层的形成。在人类、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胚层和上胚层的分离是独立于 FGF 的，2i 处理仅对 OCT4 阳性上胚层群体相对于 GATA4-/6 阳性下胚层群体产生有限的强化。先前已经表明，TGFβ/Activin A 抑制克服了大鼠 iPSCs、大鼠 ESCs 和人 iPSCs 的普遍分化和异质性，同时促使它们获得原始特性。然而，TGFβ/Activin A 抑制，单独或与 Rho 相关卷曲螺旋蛋白激酶（ROCK）抑制一起，根据电子显微镜对植入前兔胚胎的超微结构分析，与兔胚胎的存活不兼容。在兔模型中，交配后的排卵允许妊娠进展的时间精确控制。它在一次妊娠中产生了几个所需阶段的胚胎，并且妊娠期相对较短，使兔胚胎成为发现胚胎细胞和其他哺乳动物胚胎来源干细胞中维持多能性的细胞功能和机制的合适模型。
