Isotopic Tracing of Nucleotide Sugar Metabolism in Human Pluripotent Stem Cells

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-13(6)-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.

Automated summary

Metabolism plays a crucial role in cell functions and stem cell regulation. Nucleotide sugars (NSs) link glucose metabolism with cellular functions. Studying the synthesis of NSs is important for manipulating stem cell functions. Researchers developed a customizable medium and a mass-spectrometry method to trace NS biosynthesis using stable isotopes. They successfully traced carbon incorporation into NSs via glucose and other pathways in induced pluripotent stem cells (hiPSCs), and used these tools to study NS biosynthesis in hiPSCs from patients with deficiency of phosphoglucomutase 1 (PGM1), an enzyme regulating NS synthesis.