Department of Neurology, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.
Department of Clinical Genetics, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands.
Cells. 2023 Jul 3;12(13):1765. doi: 10.3390/cells12131765.
Metabolism not only produces energy necessary for the cell but is also a key regulator of several cellular functions, including pluripotency and self-renewal. Nucleotide sugars (NSs) are activated sugars that link glucose metabolism with cellular functions via protein N-glycosylation and O-GlcNAcylation. Thus, understanding how different metabolic pathways converge in the synthesis of NSs is critical to explore new opportunities for metabolic interference and modulation of stem cell functions. Tracer-based metabolomics is suited for this challenge, however chemically-defined, customizable media for stem cell culture in which nutrients can be replaced with isotopically labeled analogs are scarcely available. Here, we established a customizable flux-conditioned E8 (FC-E8) medium that enables stem cell culture with stable isotopes for metabolic tracing, and a dedicated liquid chromatography mass-spectrometry (LC-MS/MS) method targeting metabolic pathways converging in NS biosynthesis. By C-glucose feeding, we successfully traced the time-course of carbon incorporation into NSs directly via glucose, and indirectly via other pathways, such as glycolysis and pentose phosphate pathways, in induced pluripotent stem cells (hiPSCs) and embryonic stem cells. Then, we applied these tools to investigate the NS biosynthesis in hiPSC lines from a patient affected by deficiency of phosphoglucomutase 1 (PGM1), an enzyme regulating the synthesis of the two most abundant NSs, UDP-glucose and UDP-galactose.
代谢不仅产生细胞所需的能量，还是几种细胞功能（包括多能性和自我更新）的关键调节剂。核苷酸糖 (NSs) 是激活的糖，通过蛋白质 N-糖基化和 O-GlcNAcylation 将葡萄糖代谢与细胞功能联系起来。因此，了解不同代谢途径如何在 NSs 的合成中汇聚对于探索代谢干扰和调节干细胞功能的新机会至关重要。示踪代谢组学非常适合这项挑战，然而，用于干细胞培养的化学定义、可定制的培养基中，几乎没有可用于替代营养物的同位素标记类似物。在这里，我们建立了一种可定制的通量条件 E8 (FC-E8) 培养基，该培养基可用于稳定同位素标记的代谢追踪的干细胞培养，以及一种专门针对 NS 生物合成中汇聚的代谢途径的液相色谱-质谱 (LC-MS/MS) 方法。通过 C-葡萄糖喂养，我们成功地直接通过葡萄糖、间接地通过其他途径（如糖酵解和戊糖磷酸途径）追踪了诱导多能干细胞 (hiPSC) 和胚胎干细胞中碳掺入 NS 的时间过程。然后，我们应用这些工具来研究来自患有磷酸葡萄糖变位酶 1 (PGM1) 缺乏症患者的 hiPSC 系中的 NS 生物合成，PGM1 是调节两种最丰富的 NSs（UDP-葡萄糖和 UDP-半乳糖）合成的酶。
