Multi-region brain organoids for unprecedented insights into human neurodevelopmental disorders

08 July 2025

Despite extensive biomedical research, disease-modifying treatments for human neurodevelopmental disorders such as autism spectrum disorder and schizophrenia are still lacking. Neurology and psychiatry are consistently the lowest success categories in drug development, with a 95-97% failure rate in clinical trials. Owing to inter-species differences in brain anatomy and physiology, animal models do not recapitulate human-specific molecular and cellular pathways responsible for the onset of human neurological disorders, nor do they capture high heterogeneity of patient populations.

By contrast, human brain organoids have the ability to capture human-specific brain physiology and model disease-specific pathophysiology, allowing to obtain human-relevant insights into disease mechanisms and develop effective therapies. Through the use of patient-derived cells, brain organoids can model patient-specific features, opening the door to personalized medicine.

Current approaches to generation of human brain organoids for preclinical research are typically limited to single-region models and simplified endothelial cell populations, such as isolated human umbilical vein endothelial cells. To enhance the physiological relevance of brain organoids, Anannya Kshirsagar et al. from Johns Hopkins University have employed human iPSCs to engineer multi-region brain organoids (MRBO) that integrate cerebral, mid-hindbrain, and endothelial organoids into one structure. The obtained endothelial organoids better capture the diversity of vascular cell types, including progenitors, mature endothelial cells, pericytes, proliferating angiogenic cells, and stromal cells.

These MRBO were formed by fusion of organoids using the hanging-drop method, a low-cost and reproducible technique that leverages gravity to bring pre-formed organoids into close contact within a suspended droplet of culture medium. Its contact-free or low-shear environment supports natural cell-cell interactions, adherence, merger and integration dynamics.

As confirmed by snRNA-seq analysis, the resulting MRBO maintained the key cell populations from their constituent organoids until day 60. Dimensional reduction and clustering of the integrated dataset showed that MRBO contribute to 80% of cellular clusters found in human fetal brain at Carnegie stage of 12–16, corresponding roughly to 29–42 days post‑fertilization, indicating that the fused organoids have the capacity to recapitulate major neurodevelopmental events with unprecedented fidelity. Electrophysiological recordings of MRBOs using microelectrode arrays revealed significant increases in spike frequency, burst rates, and network synchronization compared to individual region organoids, creating novel opportunities for studying circuit development and dysfunction. 

Incorporation of endothelial system with multiple brain regions makes it possible to study the effects of endothelial-derived factors on the development of several brain regions, investigate the impact of disrupted neural-endothelial interactions on neuronal migration, synaptogenesis, metabolic support, oxidative stress and inflammation, and develop targeted therapies for neurovascular dysfunction.

In future iterations, the Johns Hopkins University team will focus on incorporating additional cell types, particularly microglia and other immune cells, to further enhance physiological relevance of the multi-region brain organoid model and expand preclinical research applications.

What Puts It on the Frontier

• Engineering of multi-region human brain organoids (MRBO) by fusing cerebral, mid-hindbrain, and endothelial organoids into one structure

• Engineering of endothelial organoids that better capture the diversity of vascular cell types (progenitors, pericytes, angiogenic cells, stromal cells)

Impact Snapshot

• Enhanced physiological relevance of human brain organoids

• Improved ability to recapitulate disease- and patient-specific pathophysiology in vitro

• Elucidation of mechanisms that underly human neurodevelopmental disorders

• Development of targeted therapies. Personalized drug response testing. Formulation of a therapeutic strategy tailored to individual patients

Reference 

Kshirsagar A, Mnatsakanyan H, Kulkarni S, Guo J, Cheng K, Ofria LD, Bohra O, Sagar R, Mahairaki V, Badr CE, et al. Multi‐Region brain organoids integrating cerebral, Mid‐Hindbrain, and endothelial systems. Advanced Science. Jul 2025; 12(33):e03768. https://doi.org/10.1002/advs.202503768