Reduced MEK inhibition preserves genomic stability in naive human embryonic stem cells.
|核心内容：
人类胚胎干细胞(HESCs)的两种状态，类似于着床后的上胚层细胞的primed状态和类似于植入前的上胚层的naive状态，都可以用技术捕获。
幼稚（naive）细胞特有的培养条件允许体外研究植入前发育，但据报道会导致染色体异常，这损害了它们在研究和潜在治疗应用中的效用。
虽然MEK抑制对于幼稚状态是必不可少的，但我们在这里表明，MEK抑制的减少促进了幼稚hESCs的建立和维持，这些细胞保留了幼稚细胞特有的特征，包括全局DNA低甲基化、HERVK表达和两条活跃的X染色体。
hESCs cultured in m5i/LAF are hypomethylated and have two active X chromosomes；（mimic function of 2i）
我们进一步表明，在这些改良条件下培养的人胚胎干细胞增殖更快，染色体异常较少，并且表现出MAPK组分的磷酸化水平，DNA损伤/修复调节因子和细胞周期的变化。
Naive hESCs cultured in m5i/LAF retain a more stable karyotype.(but avoid the side-effect of 2i)
因此，我们对目前的方法进行了简单的修改(m5i/LAF)，可以在幼稚的人类胚胎干细胞中实现旺盛的生长和减少基因组的不稳定性。原文摘要：
Human embryonic stem cells (hESCs) can be captured in a primed state in which they resemble the postimplantation epiblast, or in a naive state where they resemble the preimplantation epiblast.
Naive-cell-specific culture conditions allow the study of preimplantation development ex vivo but reportedly lead to chromosomal abnormalities, which compromises their utility in research and potential therapeutic applications.
Although MEK inhibition is essential for the naive state, here we show that reduced MEK inhibition facilitated the establishment and maintenance of naive hESCs that retained naive-cell-specific features, including global DNA hypomethylation, HERVK expression, and two active X chromosomes.
We further show that hESCs cultured under these modified conditions proliferated more rapidly; accrued fewer chromosomal abnormalities; and displayed changes in the phosphorylation levels of MAPK components, regulators of DNA damage/repair, and cell cycle.
We thus provide a simple modification to current methods that can enable robust growth and reduced genomic instability in naive hESCs.
Through systematic characterization of primed and naive hESCs maintained under different culture conditions, we uncovered an unexpected sensitivity of naive pluripotent stem cells to MAPK signal inhibition, which affects their proliferation rate, survival, and genome integrity.
We propose that minimal MAPK activity is beneficial for the preservation of both robust growth potential and genomic stability in naive human pluripotent stem cells maintained in 5i/LAF (model in Supplementary Fig. 9f).
Our data also suggest that different hESC lines respond differently to MEK inhibition.
For instance, we found that the concentration of PD03 could be reduced to 0.5 μM in WIBR3 hESCs and to 0.25 μM in UCLA4 hESCs without adverse effects (data not shown).
We suggest that MEK inhibitors should ideally be titrated in a cell-line-dependent fashion.
The observation that attenuated MEK inhibition affects the phosphorylation levels of proteins that regulate DNA damage and repair and leads to a reduced number of γ-H2AX foci provides a possible molecular explanation for the observed phenotypes.
Our finding that naive hESCs cultured in 5i/LAF and t2iLGöY exhibited differences in MAPK activity despite equivalent concentrations of MEKi suggests that the BRAF and SRC inhibitors in 5i/LAF media further dampen the MAPK pathway compared with t2iLGöY media.
dampen[ˈdæmpən]: To dampen something such as someone's enthusiasm or excitement means to make it less lively or intense.
The notion that MAPK signaling is less repressed in t2iLGöY-cultured hESCs than in 5i/LAF-cultured hESCs parallels our observations in m5i/LAF-cultured hESCs and is consistent with the following.
First, t2iLGöY-cultured cells, like m5i/LAF-cultured cells, divide more rapidly and are karyotypically more stable than 5i/LAFcultured hESCs10,27.
Second, we found that we could not reduce the concentration of PD03 to 0.5 μM in hESCs continually cultured in t2iLGöY without inducing differentiation (Supplementary Fig. 2c,d).
MEK1 inhibition is sufficient to sustain naive pluripotency in 5i/LAF
Third, UCLA lines that are refractory to naive conversion with bulk passaging in 0.5 μM PD03 could not be transferred from 5i/LAF to t2iLGöY, which suggests a requirement for stronger suppression of the MAPK pathway in certain hESC lines.
Fourth, the majority of cells cultured in t2iLGöY media have two active X chromosomes but lack XIST transcription similar to cells expanded in m5i/LAF medium.
Finally, hESCs maintained in t2iLGöY had levels of global methylation that were higher than those in naive cells expanded in 5i/LAF medium but similar to those in m5i/LAF-cultured hESCs (Fig. 4b, Supplementary Fig. 6b, and ref. 27).
The transcriptional, proteomic, phosphoproteomic, and genomic methylation data we have generated here will provide a useful resource for future studies aimed at dissecting the mechanisms underlying naive and primed pluripotency.
Our results may also have a bearing on potential therapeutic applications of naive pluripotent stem cells, as chromosomal stability is of the utmost importance in cell and gene therapy applications.参考文献：https://sci-hub.ren/10.1038/s41592-018-0104-1
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m5i/LAF 减少MEK的抑制（不是完全去抑制，因为MEK的抑制对于维持多能性很重要），并可以使幼稚的人类胚胎干细胞实现旺盛的生长和减少基因组的不稳定性。
