1H NMR metabolite monitoring during the differentiation of human induced pluripotent stem cells provides new insights into the molecular events that regulate embryonic chondrogenesis

The integration of cell metabolism with signalling pathways, transcription factor networks and epigenetic mediators is critical in coordinating molecular and cellular events during embryogenesis. Induced pluripotent stem cells (IPSCs) are an established model for embryogenesis, germ layer specification and cell lineage differentiation, advancing the study of human embryonic development and the translation of innovations in drug discovery, disease modelling and cell-based therapies. The metabolic regulation of IPSC pluripotency is mediated by balancing glycolysis and oxidative phosphorylation, but there is a paucity of data regarding the influence of individual metabolite changes during cell lineage differentiation. We used 1H NMR metabolite fingerprinting and footprinting to monitor metabolite levels as IPSCs are directed in a three-stage protocol through primitive streak/mesendoderm, mesoderm and chondrogenic populations. Metabolite changes were associated with central metabolism, with aerobic glycolysis predominant in IPSC, elevated oxidative phosphorylation during differentiation and fatty acid oxidation and ketone body use in chondrogenic cells. Metabolites were also implicated in the epigenetic regulation of pluripotency, cell signalling and biosynthetic pathways. Our results show that 1H NMR metabolomics is an effective tool for monitoring metabolite changes during the differentiation of pluripotent cells with implications on optimising media and environmental parameters for the study of embryogenesis and translational applications.

细胞代谢与信号通路、转录因子网络及表观遗传调控介质的协同整合，对于协调胚胎发生过程中的分子与细胞事件至关重要。诱导多能干细胞（induced pluripotent stem cells，IPSCs）是研究胚胎发生、胚层特化及细胞谱系分化的经典模型，推动了人类胚胎发育研究，以及药物研发、疾病建模与细胞治疗领域创新成果的转化应用。诱导多能干细胞多能性的代谢调控依赖糖酵解与氧化磷酸化的动态平衡，但目前关于细胞谱系分化过程中单一代谢物变化的调控作用仍缺乏相关数据。本研究采用氢核磁共振（1H NMR）代谢物指纹图谱与代谢足迹分析技术，在诱导多能干细胞经三阶段定向分化为原条/中内胚层、中胚层及软骨细胞群的过程中，对其代谢物水平进行监测。代谢物变化与核心代谢通路密切相关：诱导多能干细胞中以有氧糖酵解为主，分化过程中氧化磷酸化水平升高，而软骨细胞群则依赖脂肪酸氧化与酮体利用。代谢物亦参与多能性调控、细胞信号转导及生物合成通路的表观遗传调控环节。本研究结果表明，氢核磁共振代谢组学是监测多能干细胞分化过程中代谢物变化的有效工具，可为优化胚胎发生研究及转化应用所需的培养体系与环境参数提供参考。