Vascular-Powered Mini-Brains Could Revolutionize Brain Drug Trials

Scientists have created a new mini-brain technology in the laboratory that better mimics the developing human brain. These "multiregional organoids" combine different parts of the brain with complex blood vessels, allowing for more realistic studies of how the brain forms and how diseases like autism and schizophrenia arise. This innovation could help better understand these disorders and test new treatments.

Neurological diseases represent a major challenge in the modern world, affecting more than a billion people. A particularly sensitive group is children with neurodevelopmental disorders. In 2019, approximately 317 million children worldwide had some form of neurodevelopmental disability, which has profound impacts on families and places a strain on healthcare and education systems.

To better understand how the brain develops and how these disorders arise, scientists have invested in the use of cerebral organoids, structures grown in the laboratory from stem cells that organize and grow in a manner similar to the developing human brain.

Recently, techniques for creating these organoids have improved. It is now possible to produce structures that combine different parts of the brain, such as the cortex (the outermost and most complex part), the midbrain, and the hindbrain (the innermost and most primitive regions). This allows for a more complete view of how the brain develops as a whole.

However, there were still important limitations to these approaches. The first is that, until now, it was not possible to integrate functional blood vessels into all of these regions simultaneously. The second is that, when attempts were made to include blood vessels, isolated human umbilical vein cells (called HUVECs) were used, which are useful but do not represent the true complexity of the cerebral vascular system.

In practice, the developing brain does not grow in isolation. It develops in conjunction with the blood vessel system (the endothelial system), which not only supplies nutrients and oxygen but also emits signals that directly influence the growth and specialization of brain cells. Therefore, ignoring this interaction can significantly limit the usefulness of laboratory-created models.

Some studies have attempted to generate blood vessels in organoids using bioreactors or microfluidic systems (which apply controlled pressure to cells), but these attempts focused only on one part of the brain and did not utilize complex vascular systems.

To overcome these limitations, researchers have developed a new technology called Multiregional Brain Organoids, or MRBOs. This platform combines, in a single structure, organoids representing different parts of the brain (cortex, midbrain, and hindbrain) along with complex endothelial organoids, mini blood vessel-shaped organoids containing various types of vascular cells: vascular stem cells, pericytes (which support the vessels), cells that promote new vessel formation, mature cells, and supporting cells.

This system more realistically mimics the neurovascular environment of the developing human brain.

Annie Kathuria and team. Credit: Will Kirk / Johns Hopkins University

The process of creating MRBOs involves two main steps. First, each organoid, whether cerebral or vascular, is formed individually through the activation of specific genetic signals that guide the fate of stem cells. Then, these organoids are joined under carefully adjusted conditions so that they fuse without losing their identities.

This means that the cortex, midbrain, hindbrain, and blood vessels maintain their unique characteristics while communicating and organizing as they would in the fetal brain.

To test the effectiveness of this new platform, the scientists used a technique called single-nucleus RNA sequencing. It allows them to analyze, cell by cell, which genes are active, identifying which cell types are present in each region.

The results showed that MRBOs form populations of brain cells specific to each region, as well as highly specialized vascular cells. Furthermore, the analyses showed that MRBOs can reproduce approximately 80% of the cell types found in the human fetal brain in the early stages of development.

The scientists also used a system called CellChat, which maps how cells "talk" to each other through signaling molecules. They discovered 13 interactions between brain cells and blood vessels that had never been described before.

e) Immunohistochemistry of MRBOs at day 60 showing PHOX2B, CD34, SV2A, Cux1, HOXB3, Nestin, SOX10, β-tubulin, ZO-1, and CD31. Scale bars: 50 µm (overall images), 25 µm (ZO-1, CD31). This panel presents images obtained by immunohistochemistry (a type of staining that highlights specific proteins in tissues) of MRBOs at day 60 of development. Each marker visualized serves to identify different cell types or structures: PHOX2B and HOXB3 indicate brainstem regions, CD34 and CD31 are used to mark blood vessels, SV2A is a marker of synapses (communication between neurons), Cux1 and β-tubulin indicate neurons, Nestin and SOX10 indicate neural and glial progenitor cells, ZO-1 is linked to the blood-brain barrier. Scale bars help visualize size.

f) Immunostaining of brain organoids for MBP, Nestin, Cux1, CTIP2, GFAP, and MAP2. Scale bars: 50 μm (overall DAPI), 25 μm (individual markers). Here, scientists demonstrated the staining of specific organoids to analyze different cell types. The markers used help identify specific brain functions: MBP shows myelin cells (the covering of neurons), Nestin indicates neural stem cells, Cux1 and CTIP2 identify different layers of the cerebral cortex, GFAP marks astrocytes (supporting cells), and MAP2 reveals neuron dendrites. The image also uses DAPI, a stain that marks cell nuclei.

g) MHO whole-mount immunostaining for DAPI, Gephyrin, Sox10, MAP2, TH, PHOX2B, β-tubulin, and VAChT. Scale bar: 50 μm. This panel used a technique called "whole-mount immunostaining," which allows visualization of the entire organoid at once, rather than cutting slices. The markers used reveal varied neurological functions: DAPI for cell nuclei, Gephyrin for inhibitory synapses, Sox10 for glial cells, MAP2 for neurons, TH (tyrosine hydroxylase) and VAChT for dopaminergic and cholinergic neurons, respectively, PHOX2B for brainstem neurons, β-tubulin for the neuronal cytoskeleton.

h) Endothelial organoids showing uptake of CD31, PDGFβ, VEGFR2, and Dil-Ac-LDL. This panel shows vascular organoids (which mimic blood vessels) with foci of proteins important for vessel development. CD31 is a classic marker of endothelial cells, PDGFβ and VEGFR2 are molecules involved in vessel formation and cell growth. Dil-Ac-LDL uptake is a technique that demonstrates the cells' ability to absorb blood-like particles, decreasing the amount of LDL that is functionally similar to actual vascular cells.

This all confirms that these cells behave like human blood vessels.

One of the most interesting discoveries was that certain chemical signals emitted by blood vessels are essential for the development of the hindbrain, but not for the cortex. This indicates that blood vessels actively participate in the regional formation of the brain, helping to shape its different parts in unique ways.

This new platform represents a huge advance in science, as it allows us to study the human brain much more closely, considering multiple regions simultaneously and incorporating the role of blood vessels. This opens up new possibilities for investigating the causes of neurodevelopmental disorders such as autism, schizophrenia, and bipolar disorder, all characterized by communication failures between brain regions.

MRBO technology therefore offers a powerful tool for understanding these diseases, testing treatments, and studying how environmental and genetic factors influence the development of the still-forming brain.

READ MORE:

Multi-Region Brain Organoids Integrating Cerebral, Mid-Hindbrain, and Endothelial Systems

Anannya Kshirsagar, Hayk Mnatsakanyan, Sai Kulkarni, John Guo, Kai Cheng, Luke Daniel Ofria, Oce Bohra, Ram Sagar, Vasiliki Mahairaki, Christian E Badr, and Annie Kathuria

Advanced Science, e03768, 08 July 2025

https://doi.org/10.1002/advs.202503768

Abstract: 

Brain organoid technology has revolutionized the ability to model human neurodevelopment in vitro. However, current techniques remain limited by their reliance on simplified endothelial cell populations. Multi-Region Brain Organoids (MRBOs) are engineered that integrate cerebral, mid/hindbrain, and complex endothelial organoids into one structure. Unlike earlier approaches based on isolated Human Umbilical Vein Endothelial Cells, the endothelial organoids contain diverse vascular cell types, including progenitors, mature endothelial cells, pericytes, proliferating angiogenic cells, and stromal cells. The strategy employs sequential modulation of key developmental pathways to generate individual organoids, followed by optimized fusion conditions that maintain regional identities while supporting cellular integration. Single-nucleus RNA sequencing reveals that MRBOs develop discrete neural populations specific to each brain region alongside specialized endothelial populations that establish paracrine signaling networks. Integration analysis with human fetal brain data shows that MRBOs contribute to 80% of cellular clusters found in human fetal brain tissue (Carnegie stages 12–16). CellChat analysis identifies 13 previously uncharacterized endothelial-neural signaling interactions. Endothelial-derived factors are uncovered that support intermediate progenitor populations during hindbrain development, but not cerebral development, revealing a role for endothelial populations in regional brain patterning. This platform enables matching of multiple developmental regions while incorporating endothelial components, providing opportunities for studying neurodevelopmental disorders with disrupted neural-endothelial interactions.