Isotopic Tracing of Nucleotide Sugar Metabolism in Human Pluripotent Stem Cells

• Published in: Cells (Basel, Switzerland) Switzerland), 2023-07, Vol.12 (13), p.1765
• Main Authors: Conte, Federica, Noga, Marek J, van Scherpenzeel, Monique, Veizaj, Raisa, Scharn, Rik, Sam, Juda-El, Palumbo, Chiara, van den Brandt, Frans C A, Freund, Christian, Soares, Eduardo, Zhou, Huiqing, Lefeber, Dirk J
• Format: Article
• Language: English
• Subjects: Analysis; Biosynthesis; Cell culture; Cell self-renewal; Chromatography, Liquid; Culture media; Embryo cells; Embryonic stem cells; Energy metabolism; Enzymes; Experiments; Fibroblasts; Galactose; Gene expression; Glucose; Glucose - metabolism; Glycolysis; Glycosylation; Health aspects; Humans; induced pluripotent stem cells; Isotopes; Liquid chromatography; Mass spectrometry; mass spectrometry-based isotopic tracing; Metabolic pathways; Metabolism; Metabolites; Metabolomics; nucleotide sugar metabolism; Nucleotides; O-GlcNAcylation; Penicillin; Pentose phosphate pathway; PGM1 deficiency; Phosphoglucomutase; Pluripotency; Pluripotent Stem Cells - metabolism; Protein turnover; Respiration; Scientific imaging; Stem cells; Sugars; Tandem Mass Spectrometry; Uridine Diphosphate
• ISSN: 2073-4409; 2073-4409
• 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.