Brain organoids: building higher-order complexity and neural circuitry models

Abstract

Brain organoids are 3D tissue models of the human brain that are derived from pluripotent stem cells (PSCs). They have enabled studies that were previously stymied by the inaccessibility of human brain tissue or the limitations of mouse models of some brain diseases. Despite their enormous potential, brain organoids have had significant limitations that prevented them from recapitulating the full complexity of the human brain and reduced their utility in disease studies. We describe recent progress in addressing these limitations, especially building complex organoids that recapitulate the interactions between multiple brain regions, and reconstructing in vitro the neural circuitry present in vivo. These major advances in the human brain organoid technology will remarkably facilitate brain disease modeling and neuroscience research.

Keywords: assembloids; brain development; brain disease; brain organoids; neural circuitry.

Figure 1.. Organoids recapitulating specific brain regions.

(A) Signaling pathways during neural tube development. Along the dorsal–ventral axis, SHH signaling drives ventral fate whereas WNT and BMP pathway activation favors dorsalization. In the anterior–posterior axis, low WNT concentration promotes forebrain specification whereas a higher WNT concentration leads to caudal fate. FGFs and retinoic acid (RA) also participate in the caudal fate induction. (B) Generation of region-specific brain organoids and the necessary patterning cues. Forebrain, midbrain, hindbrain, and spinal cord organoids can be induced by modifying the signaling pathways, as indicated in the corresponding frames. In the frames, ‘,’ indicates ‘and’, whereas ‘/’ indicates ‘or’. Abbreviations: BMPi, bone morphogenetic protein inhibitor; FGF-β, fibroblast growth factor β; LGE, lateral ganglionic eminence; MEKi, mitogen-activated protein kinase inhibitor; RXR, retinoid X receptor; SDF1, stromal cell-derived factor 1; SHHa, sonic hedgehog pathway activator; TGF-βi, transforming growth factor β inhibitor; WNTa, wingless/integrated pathway activator; WNTi, wingless/integrated pathway inhibitor. Figure created with BioRender.

Figure 2.. Methods to generate assembloids that recapitulate inter-brain-region interactions and neural circuitry.

Brain organoid assembloids include multi-region assembloids and multi-lineage assembloids. Multi-region assembloids can be used to recapitulate in vitro interactions between different brain regions and multi-lineage assembloids can integrate vascular structures and microglia cells into brain organoids. Multi-region assembloids are generated by fusing individually formed brain region-specific organoids using different methods such as (1) coculturing in tubes, (2) coembedding in Matrigel, (3) coculturing in multiwell plates, (4) acoustic devices, (5) 3D bioprinting, (6,9) 3D printed embedding molds, (7) transwell plates, and (8) biohybrid robot-on-a-chip. Multi-lineage assembloids refer to brain organoids with endothelial cells, pericytes, and microglial cells. These nonneuronal lineage-originated cells were incorporated into brain organoids by (2) coembedding in Matrigel, (3) coculturing in multiwell plates, (5) 3D bioprinting, or (10) microfluidic devices. Abbreviations: MGE, medial ganglionic eminence; MSN, medium spiny neuron; SVZ, subventricular zone; VZ, ventricular zone. Figure created with BioRender.

Figure 3.. Methods for measuring electrophysiological activity in brain organoids.

The techniques for obtaining electrophysiological recordings from brain organoids have been improved to enable long-term recording without interfering with organoid structures. These approaches include patch-clamp, multielectrode arrays (MEAs), 3D MEAs, 3D multifunctional mesoscale framework (MMF), shell MEAs, ‘pocket’-like MEAs, cyborg organoids, and kirigami electronics. Figure based on data from [,,,–,–59,62]. Figure created with BioRender.

Figure 4.. Methods for long-term culture of brain organoids.

Various approaches have been devised, including orbital shaker, spinning bioreactor, spinΩ mini bioreactor, vertical bioreactor, rotating-wall vessel bioreactor, and air–liquid interphase culture for sliced and whole organoid and organoid-on-a-chip models to improve the long-term culturing of brain organoids. Abbreviation: RWV, rotating wall vessel. Figure created with BioRender.