Engineering Brain Organoids: Toward Mature Neural Circuitry with an Intact Cytoarchitecture

Abstract

The emergence of brain organoids as a model system has been a tremendously exciting development in the field of neuroscience. Brain organoids are a gateway to exploring the intricacies of human-specific neurogenesis that have so far eluded the neuroscience community. Regardless, current culture methods have a long way to go in terms of accuracy and reproducibility. To perfectly mimic the human brain, we need to recapitulate the complex in vivo context of the human fetal brain and achieve mature neural circuitry with an intact cytoarchitecture. In this review, we explore the major challenges facing the current brain organoid systems, potential technical breakthroughs to advance brain organoid techniques up to levels similar to an in vivo human developing brain, and the future prospects of this technology.

Keywords: 3-dimensional culture; Bioengineering; Brain organoid; Multi-regional identity; Pluripotent stem cell.

Fig. 1

Signaling dynamics in the embryonic brain development. The schematic illustration of signaling factors in mouse embryonic brain development. The combinatory effects of morphogens determine the regionalization of the brain. Tel: telencephalon, Di: diencephalon, Mes: mesencephalon, Rhom: rhombencephalon, MHB: midbrain-hindbrain boundary, ChP: choroid plexus, FP: floor plate, MGE: medial ganglionic eminence, LGE: lateral ganglionic eminence, SHH: sonic hedgehog, FGF: fibroblast growth factor, BMP: bone morphogenic protein.

Fig. 2

Generation of brain organoids. The process of brain organoid generation consists first of aggregation of hPSCs into EBs and then further differentiation to organoids. EBs can undergo guided differentiation with small molecules into region-specific organoids or unguided differentiation to generate cerebral organoids. Though cerebral brain organoids successfully replicate many aspects of the human fetal brain, they are still limited with regard to many factors such as the depth and complexity of the cortical lamination.

Fig. 3

Engineering advances to overcome major hurdles of brain organoid technology. (A) Efforts to overcome the “batch effect” include using standardized microwells to generate homogenous EBs and generating organoids with single neural rosettes. (B) The tradeoff between the multi-regional complexity but low reproducibility of unguided cerebral organoids with high fidelity but low complexity of region-specific organoids may be overcome by generating mutli-regional organoids. Multi-regional organoids can be generated through assembloid production, morphogen gradients created by artificial signaling centers, and chemical and/or light inducible systems. (C) Mimicking the neural ECM in brain organoid culture systems is a challenge that has been addressed by modulating the properties of both naturally-derived and synthetic biomaterials. (D) Various approaches may be used to overcome the “diffusion limit” to enhance the long-term culture and mature of brain organoids. These include the use of bioreactors, organoid slice culture at the air-liquid interface, and vascularization through in vivo transplantation. (E) Organoids lack many important connections among various cellular subtypes within the human brain. These missing cell types may be accounted for through microglia integration (neuro-immune), CFS producing ChP organoid generation (meninges-brain), and neuromuscular organoid generation (PNS-CNS).

Fig. 4

Advanced tools to analyze the functional properties of the brain organoid system. (A∼C) Lineage tracing tools to elucidate spatiotemporal dynamics of developing organoids, including dynamic lineage tracers and cell tracking. (D, E) Electrophysiological methods to monitor electrical activity in brain organoids, including voltage imaging and bioelectronic interfaces.