Continuous suppression of Mek1/2 impairs the developmental potential of mouse embryonic stem cells. Mus musculus

Simultaneous inhibition of Gsk3α/β and Mek1/2 activity in the presence of LIF (2i/L) induces a naïve state in mouse embryonic stem cells (ESCs) that resembles the inner cell mass (ICM) of the pre-implantation embryo. Since the ICM exists only transiently in vivo, it remains unclear how continuous propagation of naïve ESCs in vitro affects their stability and functionality. Here we show that extended culture of male ESCs in 2i/L results in the progressive erosion of genomic imprints, loss of H2A.X binding, and accumulation of chromosomal aberrations. Consistent with these observations, we find that the developmental potential of ESCs cultured in 2i/L is impaired. Mechanistically, we demonstrate that Mek1/2 inhibition is predominantly responsible for these effects, in part through downregulation of DNA methyltransferases. Additionally, we demonstrate that female ESCs cultured in conventional serum/LIF media phenocopy male ESCs cultured in 2i/L, including the aforementioned epigenetic and developmental abnormalities. Finally, we show that replacement of the Mek1/2 inhibitor with a Src inhibitor preserves the epigenetic and genomic integrity as well as developmental potential of ESCs. Taken together, our data suggest that, while suppression of Mek1/2 in ESCs maintains an ICM-like epigenetic state, continuous suppression results in irreversible changes that compromise their developmental potential. Overall design: Profiling allelic expressions of imprinted genes in iPSCs cultured in serum/LIF and 2i/LIF.

在白血病抑制因子（leukemia inhibitory factor, LIF）存在的条件下，同时抑制Gsk3α/β与Mek1/2的活性（即2i/L培养体系），可诱导小鼠胚胎干细胞（embryonic stem cells, ESCs）进入类似植入前胚胎内细胞团（inner cell mass, ICM）的初始态。由于内细胞团在体内仅瞬时存在，目前尚不清楚体外持续传代培养初始态ESCs会对其稳定性与功能产生何种影响。本研究发现，雄性ESCs在2i/L体系中长时间培养时，会逐渐出现基因组印记丢失、H2A.X结合丧失以及染色体畸变累积的现象。与上述观察结果一致，我们发现经2i/L培养的ESCs其发育潜能受到损伤。从机制层面而言，我们证实Mek1/2抑制是引发上述效应的主要诱因，其作用部分通过下调DNA甲基转移酶（DNA methyltransferases）实现。此外，我们发现使用常规血清-LIF培养基培养的雌性ESCs，其表观遗传与发育异常表型与雄性ESCs在2i/L体系中培养的情况完全一致。最后，我们证实将Mek1/2抑制剂替换为Src抑制剂，可保留ESCs的表观遗传与基因组完整性，同时维持其发育潜能。综上，本研究数据表明：尽管在ESCs中抑制Mek1/2可维持类似ICM的表观遗传状态，但持续抑制会引发不可逆的改变，进而损害其发育潜能。整体实验设计：对在血清-LIF与2i/LIF培养基中培养的诱导多能干细胞（induced pluripotent stem cells, iPSCs）进行印记基因的等位基因表达谱分析。