Effects of clinostat-based microgravity simulation on the growth and differentiation of cerebral organoids using a 3D-clinostat

ABSTRACT The human brain is highly sensitive to altered gravitational environments, yet the molecular and cellular consequences of microgravity remain poorly understood due to the limited accessibility of astronaut neural tissue. Here, we employ human cerebral organoids cultured in a 3D clinostat to clinostat-based simulation and investigate neurodevelopmental responses. Organoids were exposed to continuous clinostat-based simulation for 30 days (C30), 15 days followed by 15 days of normal gravity (C15-N15), or standard gravity controls (N30). Neurochemical profiling revealed marked reductions in acetylcholine, dopamine, norepinephrine, and glutamate under microgravity, with partial recovery after re-adaptation; by contrast, GABA levels increased persistently, suggesting an excitatory–inhibitory imbalance. Transcriptomic analysis demonstrated widespread downregulation of pluripotency markers (OCT4, NANOG, SOX2), regional identity genes (FOXG1, DLX2, NKX2.1, SIX6), and neuronal differentiation markers (MAP2, DCX, VGLUT1), whereas astrocytic GFAP was robustly elevated and cortical identity marker SATB2 recovered upon re-exposure to normal gravity. Despite extensive transcriptional changes, protein levels of the neuronal marker MAP2 and the pluripotency marker SOX2 remained unchanged. Together, these findings indicate that clinostat-based simulation disrupts stemness, regional specification, and neurotransmitter balance, while promoting astrocytic lineage bias and partial recovery upon gravitational re-exposure. This study establishes human cerebral organoids as a tractable ground-based simulation model for probing brain vulnerability to space environments and for identifying candidate biomarkers and countermeasures to safeguard astronaut neurocognitive health during long-duration missions.