Tosolini Matteo, Jouneau Alice
INRA, UMR1198 Biologie du Développement et Reproduction, F-78350, Jouy-en-Josas, France.
Methods Mol Biol. 2016;1341:41-8. doi: 10.1007/7651_2015_207.
Mouse embryonic stem cells (ESCs) derive from the inner cell mass (ICM) of a blastocyst. These cells are pluripotent and thus able to generate both somatic and germinal lineages. It is possible to maintain ESCs in different pluripotent states depending on the in vitro culture conditions. Classically, ESCs are cultured in the presence of serum and LIF, which sustain the naive state of pluripotency but in this metastable state cells exhibit a large degree of heterogeneity. In the last few years, it has been discovered that when ESCs are cultured in a chemically defined medium (without serum), in the presence of LIF and with the addition of two small molecules (in particular the inhibitors of MAPK and Gsk-3 pathways), they reach a ground state of pluripotency where cells are more homogeneous and more "ICM-like." In this protocol, we describe how we culture mouse ESCs and the way we switch them from naive to ground state.
小鼠胚胎干细胞（ESCs）源自囊胚的内细胞团（ICM）。这些细胞具有多能性，因此能够产生体细胞和生殖细胞谱系。根据体外培养条件，可以将ESCs维持在不同的多能状态。传统上，ESCs在血清和白血病抑制因子（LIF）存在的情况下进行培养，这维持了多能性的原始状态，但在这种亚稳态下，细胞表现出很大程度的异质性。在过去几年中，人们发现，当ESCs在化学成分明确的培养基（无血清）中培养，在LIF存在且添加两种小分子（特别是丝裂原活化蛋白激酶（MAPK）和糖原合成酶激酶-3（Gsk-3）途径的抑制剂）时，可以达到多能性的基态，此时细胞更加均匀，更“类似ICM”。在本方案中，我们描述了如何培养小鼠ESCs以及将它们从原始状态转换为基态的方法。
