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. 2025 Nov;56(11):3294-3305.
doi: 10.1161/STROKEAHA.125.051788. Epub 2025 Sep 4.

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

Carina Seah #  1   2 , Braxton R Schuldt #  2 , James M Vicari  1 , Connie S Lebakken  3 , William D Richards  3 , Kaylie Greuel  3 , Kailyn T Parham  3 , Grace Rabinowitz  2 , Christopher P Kellner  2
Affiliations

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

Carina Seah et al. Stroke. 2025 Nov.

Abstract

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.

Keywords: astrocytes; cerebral hemorrhage; hemorrhagic stroke; neurons; organoids.

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Conflict of interest statement

Dr Kellner directs research at Mount Sinai, supported by research grants from Integra, Penumbra, Viz.AI, Siemens, Medtronic, Minnetronix, Longeviti, Irras, ICE Neurosystems, CVAID, Endostream, and Microtransponder; and has equity in Precision Recovery, Borealis, E8, Borvo, and Metis Innovative. Metis Innovative is an investment group that has coordinated investments in Synchron, Proprio, Fluid Biomed, Von Vascular, Precision Recovery, and Phantom Neuro. Dr Lebakken, W.D. Richards, K. Gruel, and K.T. Parham are employees of Stem Pharm and have equity and options in Stem Pharm. Dr Lebakken reports stock options in Stem Pharm, Incorporated; a patent issued for US20220017873A1 Human Pluripotent Stem Cell–Based Models for Predictive Developmental Neural Toxici licensed to Stem Pharm, Incorporated; stock holdings in Stem Pharm, Incorporated; grants from National Institutes of Health to other; a provisional patent for 63/847677 CENTRAL NERVOUS SYSTEM ORGANOIDS; service as Chief Operating Officer for Stem Pharm, Incorporated; employment by Stem Pharm, Incorporated; and securities holdings in Stem Pharm, Incorporated. W.D. Richards reports a patent issued for US20220017873A1 Human Pluripotent Stem Cell-based models for predictive neural toxicity licensed to Stem Pharm, Inc.; a provisional patent for 63/847677 Central Nervous System Organoids; grants from National Institutes of Health to other; and stock options in Stem Pharm, Inc. K. Greuel reports a provisional patent for 63/847677 Central Nervous System Organoids; employment by Stem Pharm, Inc; and a patent issued for US20220017873A1 Human Pluripotent Stem Cell-based models for neural toxicity licensed to Stem Pharm, Inc. K.T. Parham reports a provisional patent for 63/847677 Central Nervous System Organoids; employment by Stem Pharm, Inc; a patent issued for US20220017873A1 Human Pluripotent Stem Cell - Based Models for Predictive Developmental Neurotoxicit licensed to Stem Pharm, Inc; and stock options in Stem Pharm, Inc. The other authors report no conflicts.

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