Brain Organoids / Neural Organoids

The cerebrum accounts for more than half of the brain's volume and serves as the command center underlying complex phenomena such as perception, thought, language, attention, episodic memory, and voluntary movement. Cerebral organoids are emerging as a promising model for studying aspects of human brain development that remain poorly understood. A key example is the diversification of human cortical cell populations—such as outer radial glia (oRG) in the upper cortical layers and callosal projection neurons (CPN)—which underwent marked, species-specific expansion during human evolution.

Development and Structure of the Cerebral Cortex in vivo

The cerebral cortex is formed through the following developmental process:

• Early proliferative phase (neural epithelium proliferation)
  After neural tube closure, neural epithelium cells (NECs) repeatedly self-renew through symmetric division, expanding the neuroepithelial cell pool.
• Neurogenesis (neuron supply)
  With the initiation of neurogenesis, NECs transition into radial glial cells (RGCs). RGCs whose cell bodies lie on the ventricular side, with processes extending toward the basal surface, are specifically referred to as apical radial glial cells (aRGCs). These cells undergo asymmetric division, producing one aRGC and either an intermediate progenitor cell (IPC) or a neuron.
• Formation of the six-layer structure
  Neurons generated by aRGCs are sequentially arranged from the deep layers (layers V/VI) to the superficial layers (layers II/III/IV), giving rise to the distinct six-layered structure of the cerebral cortex.
• Regulation by the marginal zone (MZ)
  Cajal–Retzius cells, which originate from regions outside the cerebral cortical primordium, migrate tangentially to form the marginal zone (MZ), where they regulate the proper laminar positioning of neurons.

1. Pre-patterning

This method involves the addition of exogenous signals (small-molecule compounds or growth factors), to differentiation induction toward specific target brain regions.

Conventional directed neural differentiation from mouse ES cells mainly relied on embryoid body (EB) formation under serum-containing conditions. However, its low neural differentiation efficiency remained a persistent bottleneck. To address this, Sasai, Watanabe and colleagues developed the SFEB method, a floating culture technique using serum-free medium. They succeeded in inducing Foxg1, a transcription factor specifically expressed in the telencephalon, ventricular zone, subventricular zone, and intermediate zone, confirming induction of telencephalic progenitor cells. Further, introducing a ROCK inhibitor into the culture system greatly improved cell survival, making it possible to reproduce these results in human cells.

The SFEB method, however, had reproducibility issues, including variability in the size of the resulting cell aggregates and induction efficiencies for Foxg1-positive cells that fell below 50%. Eiraku and colleagues resolved this issue with the SFEBq method. By using low-adhesion 96-well plates to promote rapid cell aggregation, they succeeded in producing uniform aggregates. As a result, the efficiency of Foxg1 expression was increased to a maximum of 75%.

2. Self-patterning

This approach maximally exploits the stem cells' inherent capacity for self-organization, without relying on exogenous patterning factors.

The protocol developed by Lancaster and colleagues promotes the stem cells' intrinsic differentiation potential (self-patterning) without using exogenous signaling factors. Instead of controlling differentiation with compounds, EBs are embedded in Matrigel droplets and cultured in a spinning bioreactor, which enhances nutrient absorption and three-dimensional cell growth.

In this culture system, a neuroepithelial sheet exhibiting apical N-cadherin polarity forms around ventricle-like cavities. Long-term culture of more than 10 months is possible, and structures resembling choroid tissue and retina form alongside cerebral cortical tissue. Forebrain, midbrain, and hindbrain markers are initially detected, but forebrain marker expression becomes predominant and is maintained as culture progresses.

Despite the absence of exogenous patterning factors, expression of the forebrain marker Foxg1 and the cortical marker Emx1 is ultimately detected. Furthermore, layered structures resembling the ventricular zone, subventricular zone, and cortical plate, as well as structures and marker expression characteristic of bRGCs—primate-specific neural stem cells—have been observed within the tissue.

Human stem cell-derived neurons provide an important foundation for drug discovery and the development of treatments for serious neurological diseases. They are also widely studied to understand neural development, function, and dysfunction. In recent years, appropriate multicellular tissue and microenvironments have proven essential for physiological modeling. As a result, in vitro three-dimensional organoid culture systems, which more accurately recapitulate multicellular structures, are increasingly being adopted.

Regionalized Organoids

Regionalized organoids refer to the technology—and the resulting tissue—for creating specific functional and structural regions (patterning) within a single organoid by recapitulating in vivo developmental processes when generating three-dimensional organs or tissues from pluripotent stem cells (such as iPS cells).

Examples of Regionalized Organoids

In addition to the organoids introduced above, many other methods for generating organoids have been reported, such as those described below.

1. Choroid Plexus Organoids

This organoid originates from the roof plate, located at the most dorsal part of the dorsomedial telencephalic region, and forms in a region where high concentrations of BMP and Wnt signals are present during development. Based on cerebral cortex induction conditions, a canonical Wnt signaling activator (CHIR99021) and BMP4 protein are continuously added from culture day 18 onward, once neural regionalization is complete. This enables the efficient three-dimensional induction of choroid plexus epithelial tissue that is positive for LMX1A, OTX2, and transthyretin.

2. Hippocampus Organoids

This organoid originates from the medial pallium of the medial telencephalon and is positioned between the cerebral cortex and the choroid plexus. As with choroid plexus induction, CHIR99021 and BMP4 are used, but only for three days, from culture day 18 to 21. This induces the dorsomedial telencephalon. Long-term culture (60 days or more) leads to the expression of the hippocampal marker ZBTB20 and the dentate gyrus neuron marker PROX1.

3. Spinal Cord Organoids

During spinal cord development, concentration gradients of factors secreted by organizers (dorsal BMP/Wnt and ventral Shh signaling) pattern the dorsal region into six progenitor domains and the ventral region into five.
Deliberately removing the BMP inhibitor (LDN-193189) and Shh agonist (SAG) from the conventional motor neuron induction protocol induces a broader, continuous neuroepithelium spanning from the dorsal to the intermediate regions. Concentration-dependent addition of exogenous SAG then enables precise control of patterning toward the intermediate or ventral spinal cord.

4. Ventral Telencephalon Organoids

This region primarily generates inhibitory interneurons and comprises the lateral ganglionic eminence (LGE), medial ganglionic eminence (MGE), and caudal ganglionic eminence (CGE).
Shh signaling drives patterning into the LGE (GSH2-positive), MGE (NKX2.1-positive), and CGE (CoupTFII-positive). Under cerebral cortex differentiation conditions, adding a low concentration (30 nM) of SAG during culture days 15–21 gives rise to PAX6-positive (cerebral cortex) and GSH2-positive (LGE) regions within a continuous epithelium. In contrast, adding a high concentration (500 nM) of SAG suppresses PAX6 and GSH2 expression, efficiently inducing an NKX2.1-positive neuroepithelium and achieving complete ventralization.