Karlinski Zur, M. | Search bioRxiv

bioRxiv2026

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

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 13C-labeled metabolites and 1H and 13C 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% 13C1-Glucose. Under these conditions, 13C3-lactate emerged as the most abundant labeled product, with 20-50-fold lower fluxes to 13C3-alanine, 13C2-acetate, 13C3-serine, and 13C3-pyruvate, and 100-300-fold lower fluxes to 13C1-formate and multiple 13C-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 13C3-alanine/13C3-lactate and 13C4-glutamate/13C3-lactate flux ratios, while the 13C3-Glutamate/13C3-Lactate and 13C2-Glutamate/13C3-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 13C4-, 13C3-, and 13C2-glutamate relative to 13C3-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.

cell biology↗

bioRxiv2024

Altered Extracellular Matrix Structure and Elevated Stiffness in a Brain Organoid Model for Disease

The viscoelasticity of tissues impacts their shape, as well as the growth and differentiation of their cells. Nevertheless, little is known about changes in viscoelastic properties during brain malformations. Lissencephaly is a severe malformation of cortical development caused by LIS1 mutations, which results in a lack of cortical convolutions. Here, we show that human-derived brain organoids with LIS1 mutation are stiffer than control ones at multiple developmental times. This stiffening is accompanied by abnormal ECM expression and organization, as well as elevated water content, as measured by diffusion-weighted MRI. Proteolytic cleavage of ECM components by short-term treatment with the catalytic subunit of MMP9 reduced the stiffening and water diffusion levels of mutated organoids to control levels. Finally, based on the molecular and rheological properties obtained, we generated a computational microstructure mechanical model that can successfully predict mechanical changes that follow differential ECM localization and integrity in the developing brain. Overall, our study reveals that LIS1 is essential for the expression and organization of ECM proteins during brain development, and its mutation leads to a substantial viscoelastic change. To our knowledge, this is the first study to elucidate how tissue mechanics change in disease states using human brain organoids.

developmental biology↗