Single-Cell Sequencing of Human Neural Organoids in a Model of Intracerebral Hemorrhage Reveals Temporally Dynamic Responses to Blood.

Background: Intracerebral hemorrhage leads to significant morbidity and mortality due to primary mechanical and secondary neurotoxic injury to brain parenchyma. Timing of surgical evacuation to ensure optimal outcomes is controversial, with recent evidence suggesting early intervention improves functional outcome. Here, we characterize the impact of blood-induced secondary injury on diverse brain cell types in a scalable organoid model of intracerebral hemorrhage.

Methods: Human neural organoids consisting of excitatory neurons, inhibitory neurons, neural progenitor cells, astrocytes, endothelial cells, and microglia were generated from induced pluripotent stem cell-derived cells and treated with 5% blood for either 6 or 24 hours. Organoids were dissociated and analyzed by single-cell RNA sequencing.

Results: Single-cell sequencing of 96 725 cells across 18 organoids resolved intermediate progenitors, neural progenitor cells, microglia, inhibitory neurons, endothelial cells, excitatory neurons, and astrocytes. Twenty-four-hour exposure to blood induced cellular reactivity, whereas 6-hour exposure did not. Intermediate progenitors, endothelial cells, and astrocytes were the most reactive to blood and exhibited gene expression patterns corresponding to astrocyte reactivity, angiogenesis, and progenitor cell ischemic excitotoxicity. Drug repurposing analysis identified neurotransmitter-modulating, vasculature-remodeling, and protein synthesis pathways as potential therapeutic targets, mitigating blood-induced neurotoxicity.

Conclusions: Blood exposure induces transcriptomic changes in a temporal and cell type-specific manner, particularly mediated by astrocyte reactivity, angiogenesis, and impairment of neurogenesis. Early and complete removal of blood after 6 hours of blood exposure mitigates secondary neurotoxicity seen in 24-hour exposure to blood. Transcriptomic signatures of blood-mediated neurotoxicity may potentially be reversed by antiadrenergic, dopamine agonist, and vasodilatory mechanisms.