Brain organoids: Human 3D models to investigate neuronal circuits assembly, function and dysfunction

•Advances in brain organoid technologies offer novel approaches for modeling human brain development and diseases.•Assembloids and organ-on-chip are emerging as models to increase circuit complexity and allow multi-organs interactions.•Single-cell multi-omic maps are needed to benchmark brain organoids against human brain tissue.•Long-term brain organoid cultures have the potential to model human brain connectivity.•Vascularization of brain organoids and/or xenografts enable further neuronal maturation. The human brain is characterized by an extraordinary complexity of neuronal and nonneuronal cell types, wired together into patterned neuronal circuits, which represent the anatomical substrates for the execution of high-order cognitive functions. Brain circuits' development and function is metabolically supported by an intricate network of selectively permeable blood vessels and finely tuned by short-range interactions with immune factors and immune cells. The coordinated cellular and molecular events governing the assembly of this unique and complex structure are at the core of intense investigation and pose legitimate questions about the best modeling strategies. Unceasing advancements in stem cell technologies coupled with recent demonstration of cell self-assembly capacity have enabled the exponential growth of brain organoid protocols in the past decade. This provides a compelling solution to investigate human brain development, a quest often halted by the inaccessibility of brain tissues and the lack of suitable models. We review the current state-of-the-art on the generation of brain organoids, describing the latest progresses in unguided, guided, and assembloids protocols, as well as organoid-on-a-chip strategies and xenograft approaches. High resolution genome wide sequencing technologies, both at the transcriptional and epigenomic level, enable the molecular comparative analysis of multiple brain organoid protocols, as well as to benchmark them against the human fetal brain. Coupling the molecular profiling with increasingly detailed analyses of the electrophysiological properties of several of these systems now allows a more accurate estimation of the protocol of choice for a given biological question. Thus, we summarize strengths and weaknesses of several brain organoid protocols and further speculate on some potential future endeavors to model human brain development, evolution and neurodevelopmental and neuropsychiatric diseases.