Human Cerebral Organoids as an Organotypic 3D in-vitro Model for Neuroscience under Earth and Space Conditions

Kurzfassung

During long-duration human space exploration, astronauts are exposed to a combination of ionizing radiation and microgravity, while their effects on the central nervous system remain incompletely understood at the molecular level. In this thesis, human cerebral organoids were used as a three-dimensional, human-relevant invitro model to investigate the individual and combined effects of these space-relevant stressors on neural tissue and to evaluate the ketamine derivative HW774 as a potential neuroprotective countermeasure. Organoids were exposed to X-ray and ⁵⁶Fe-ion irradiation in combination with clinorotation-simulated microgravity, as well as to real altered-gravity conditions during a parabolic flight campaign and two MAPHEUS sounding rocket missions (MAPHEUS14 and MAPHEUS16). Cellular and transcriptional responses were assessed using LDH assays, immunofluorescence staining, RT-qPCR, and bulk RNA sequencing, followed by differential expression, gene set enrichment, and weighted gene co-expression network analyses. Under simulated space conditions, only limited radiation dose-dependent effects were detected across markers of cell death, astrocytic reactivity, and neuronal survival, whereas culture condition was identified as a major source of variation. Differences between X-ray and ⁵⁶Fe-ion irradiation were mainly transient. Clinorotation was associated with impaired organoid growth and maturation in a developmental stage-dependent manner, increased apoptotic signaling at later timepoints, and altered expression of mechanosensitive channel genes, whereas glial progenitor dynamics were influenced primarily by organoid age. In the parabolic flight experiment, a broad transcriptional response was induced by HW774, characterized by reduced neuronal programs, and increased mitochondrial and cellular stress-associated transcription. This response was attenuated under real altered-gravity conditions, while gravity-associated changes were detected in mechanotransduction- and cell adhesion-related pathways. Across MAPHEUS14 and MAPHEUS16, reduced expression of mechanosensitive channel genes, protocadherinmediated cell adhesion genes, and selected synaptic markers was consistently observed in HyperG and MicroG samples, together with increased translational and mitochondrial metabolic activity. These patterns were reproduced across both missions and organoid ages, although their interpretation was limited by chamberassociated effects and batch variability. Overall, human cerebral organoids tolerated the applied experimental conditions, supporting their use as a platform for space biology research. Although definitive mechanistic conclusions cannot yet be drawn, a reproducible experimental framework was established for investigating the effects of altered gravity and ionizing radiation on human neural tissue. Future studies with larger sample sizes, improved controls, and optimized exposure conditions will be required to resolve gravity- and radiation-specific mechanisms and to further evaluate HW774 as a potential neuroprotective countermeasure