Glucose levels impact the morphology and cell-type composition of human cerebral organoids

Human cerebral organoids, derived from pluripotent stem cells, are powerful models for studying human brain development. The understanding of how morphogens can be used to guide patterning and differentiation has matured rapidly, however, the influence of basal media components on organoid development remains unclear. Standard organoid media frequently contain non- physiological concentrations of nutrients, including glucose, a central regulator of cellular metabolism and signaling. Here, we examine how glucose availability shapes cerebral organoid growth, morphology, and cell-type composition by comparing conventional hyperglycemic media to media with glucose levels more closely resembling physiological conditions. We find that organoids derived from multiple human pluripotent stem cell lines can grow in physiological glucose, but exhibit altered growth rates, structural features, and lineage distributions. In H9 embryonic stem cell-derived organoids, inhibition of the mTOR pathway under physiological glucose restores neurodevelopmental cell types otherwise diminished in these conditions. These findings highlight glucose as a key determinant of organoid lineage specification and cellular signaling. Importantly, however, glucose modulation does not reduce variability across organoids or cell lines, underscoring the need to better understand and control sources of heterogeneity to improve organoid models.