Metabolic Assessment in Human Pluripotent Stem Cell-Derived Cerebral Organoids Using HR-MAS NMR Spectroscopy

Brain metabolism is vital to healthy brain function and is often altered in disease; yet direct investigation in patients is challenging. Although animal models are commonly used for studying brain metabolism, their use is under increasing scrutiny due to concerns of animal welfare and model validity. Human pluripotent stem cell (hPSC)-derived cerebral organoids (COs) present a unique opportunity to model human brain developmental and neuropathological processes, allowing for detailed metabolic characterization via multiple approaches. Here, we applied high-resolution magic-angle spinning (HR-MAS) proton nuclear magnetic resonance (¹H-NMR) spectroscopy to analyze metabolite levels in hPSC-derived COs, establishing a pipeline to study neurometabolic pathways in these engineered human brain tissues. We identified and quantified 17 metabolites in hPSC-derived COs at different stages of maturity. The high spectral quality (linewidth < 4 Hz, SNR > 65) allowed detection of metabolite levels in 85- to 312-day-old hPSC-derived COs, which exhibited a metabolic profile similar to human fetal brain, with key distinguishing features relative to human adult brain, including: elevated lactate levels; approximately equimolar glutamate and glutamine levels; low N-acetylaspartate levels; and an abundance of hypotaurine. In summary, this study presents direct metabolic assessment in intact COs via HR-MAS ¹H-NMR spectroscopy. Our approach provides a platform for investigating human brain metabolism and its alteration in human brain models of neurodegeneration.