Isotopic Tracing of Nucleotide Sugar Metabolism in Human Pluripotent Stem Cells
Federica Conte,
Marek J. Noga,
Monique van Scherpenzeel,
Raisa Veizaj,
Rik Scharn,
Juda-El Sam,
Chiara Palumbo,
Frans C. A. van den Brandt,
Christian Freund,
Eduardo Soares,
Huiqing Zhou,
Dirk J. Lefeber
Affiliations
Federica Conte
Department of Neurology, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands
Marek J. Noga
Department of Clinical Genetics, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands
Monique van Scherpenzeel
GlycoMScan B.V., 5349 AB Oss, The Netherlands
Raisa Veizaj
Department of Neurology, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands
Rik Scharn
Department of Neurology, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands
Juda-El Sam
Department of Neurology, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands
Chiara Palumbo
Department of Neurology, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands
Frans C. A. van den Brandt
GlycoMScan B.V., 5349 AB Oss, The Netherlands
Christian Freund
hiPSC Hotel, LUMC, 2333 ZC Leiden, The Netherlands
Eduardo Soares
Department of Molecular Developmental Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University, 6525 GA Nijmegen, The Netherlands
Huiqing Zhou
Department of Neurology, Amsterdam University Medical Centres, Location Academic Medical Center, Amsterdam Neuroscience, University of Amsterdam, 1098 XH Amsterdam, The Netherlands
Dirk J. Lefeber
Department of Neurology, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands
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 13C6-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.
