Intelliwaste: NMR of 13 C-labeled Spent Media Enables Non-Invasive Metabolic Fingerprinting of Pluripotent Stem Cells and LIS1-Associated Neuropathology

Abstract Metabolic dysregulation is increasingly recognized as a key contributor to neurodevelopmental disorders. Here, we present Intelliwaste , a non-invasive, cost-effective method for profiling carbon metabolism in pluripotent stem cells and brain organoids using 13 C-labeled metabolites and 1 H and 13 C NMR spectroscopy. This approach enables longitudinal analysis of extracellular fluxes without disrupting cell viability. We apply Intelliwaste to human embryonic stem cells (hESCs) cultured in a defined media enriched with >95% 13 C 1 -Glucose. Under these conditions, 13 C 3 -lactate emerged as the most abundant labeled product, with 20–50-fold lower fluxes to 13 C 3 -alanine, 13 C 2 -acetate, 13 C 3 -serine, and 13 C 3 -pyruvate, and 100–300-fold lower fluxes to 13 C 1 -formate and multiple 13 C-labeled glutamate species. These profiles allow for precise quantification of fractional metabolic isotopic labeling and glucose-derived carbon flow. To demonstrate biological utility, we first examine the effect of L-glutamine omission , which selectively reduces 13 C 3 -alanine/ 13 C 3 -lactate and 13 C 4 -glutamate/ 13 C 3 -lactate flux ratios, while the 13 C 3 -Glutamate/ 13 C 3 -Lactate and 13 C 2 -Glutamate/ 13 C 3 -Lactate flux ratios remained unchanged. These findings suggest a specific role for extracellular glutamine in modulating the activity of alanine aminotransferase and pyruvate carboxylase. We then characterized LIS1 mutant hESCs —a model of lissencephaly—and observed significantly increased flux ratios involving 13 C 4 -, 13 C 3 -, and 13 C 2 -glutamate relative to 13 C 3 -lactate, indicating enhanced glutamate production via the TCA cycle. These findings establish Intelliwaste as a powerful tool for metabolic profiling in the study of human neurodevelopment and disease. Its non-destructive nature makes it particularly well-suited for tracking metabolic changes during differentiation and in patient-derived organoid models of neurological disorders.