Metabolic enhancement of developmental pausing (embryos)

Embryonic stem (ES) cells and embryos reversibly pause via chemical mTOR inhibition. In this study, we investigate the tissue-specific response to mTORi-induced pausing in ES and trophoblast stem (TS) cells. To resolve the sequential rewiring of the proteome, we conducted a time-series proteomics experiment at 1, 3, 6, 12, 24, and 48 hours upon induction of pausing, and at 1, 3, 6, 12, 24, and 48 hours upon release of pausing in ES and TS cells. We find that ES, but not TS cells pause reversibly. To optimise developmental pausing conditions, we reasoned that by understanding the difference in pausing response of ES and TS cells, we could identify which pathways are essential for pausing. We found that KEGG pathways related to amino acid degradation, fatty acid degradation, and DNA repair are upregulated in ES cells, but downregulated in TS cells during entry into pausing. Moreover, by targeted metabolomics, we found a depletion of short chain carnitines in the paused ES cells. To extend the length of developmental pausing, we supplemented paused embryos with L-carnitine. The L-carnitine supplementation facilitates lipid usage and prolongs the pausing length by 19 days through the establishment of a more dormant state.

胚胎干细胞（Embryonic Stem, ES）与胚胎可通过化学性哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）抑制作用实现可逆性细胞停滞。本研究针对ES细胞与滋养层干细胞（Trophoblast Stem, TS）在mTOR抑制剂（mTOR inhibitor, mTORi）诱导的细胞停滞过程中的组织特异性应答展开探究。为解析蛋白质组的时序重编程过程，本研究分别在细胞停滞诱导后的1、3、6、12、24及48小时，以及停滞解除后的1、3、6、12、24及48小时，对两类细胞开展了时序蛋白质组学实验。研究发现，ES细胞可实现可逆性停滞，而TS细胞则无此特性。为优化发育性细胞停滞的培养条件，我们推测：通过解析ES细胞与TS细胞在停滞应答上的差异，可明确对细胞停滞至关重要的信号通路。我们发现，在细胞停滞启动阶段，与氨基酸降解、脂肪酸降解及DNA修复相关的京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路在ES细胞中呈上调表达，而在TS细胞中则呈下调表达。此外，通过靶向代谢组学分析，我们发现处于停滞状态的ES细胞中短链肉碱出现耗竭。为延长发育性细胞停滞的持续时长，我们向停滞胚胎中添加了左旋肉碱（L-carnitine）。左旋肉碱的添加可促进脂质利用，并通过构建更为彻底的休眠状态，将停滞时长延长19天。