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Mutations in ARID1B, a member of the mSWI/SNF complex, cause severe neurodevelopmental phenotypes with elusive mechanisms in humans. The most common structural abnormality in the brain of ARID1B patients is agenesis of the corpus callosum (ACC), characterized by the absence of an interhemispheric white matter tract that connects distant cortical regions. Here, we find that neurons expressing SATB2, a determinant of callosal projection neuron (CPN) identity, show impaired maturation in ARID1B +/- neural organoids. Molecularly, a reduction in chromatin accessibility of genomic regions targeted by TCF-like, NFI-like, and ARID-like transcription factors drives the differential expression of genes required for corpus callosum (CC) development. Through an in vitro model of the CC tract, we demonstrate that this transcriptional dysregulation impairs the formation of long-range axonal projections, causing structural underconnectivity. Our study uncovers new functions of the mSWI/SNF during human corticogenesis, identifying cell-autonomous axonogenesis defects in SATB2 + neurons as a cause of ACC in ARID1B patients., Competing Interests: Declaration of interests J.A.K. is an inventor on patents describing the cerebral organoid technology (patent numbers: US10407664B2, EP12196954.7A) and co-founder and scientific advisory board member of a:head bio AG., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

During brain development, neural progenitors expand through symmetric divisions before giving rise to differentiating cell types via asymmetric divisions. Transition between those modes varies among individual neural stem cells, resulting in clones of different sizes. Imaging-based lineage tracing allows for lineage analysis at high cellular resolution but systematic approaches to analyse clonal behaviour of entire tissues are currently lacking. Here we implement whole-tissue lineage tracing by genomic DNA barcoding in 3D human cerebral organoids, to show that individual stem cell clones produce progeny on a vastly variable scale. By using stochastic modelling we find that variable lineage sizes arise because a subpopulation of lineages retains symmetrically dividing cells. We show that lineage sizes can adjust to tissue demands after growth perturbation via chemical ablation or genetic restriction of a subset of cells in chimeric organoids. Our data suggest that adaptive plasticity of stem cell populations ensures robustness of development in human brain organoids., (© 2024. The Author(s).)

Neural organoids model the development of the human brain and are an indispensable tool for studying neurodevelopment. Whole-organoid lineage tracing has revealed the number of progenies arising from each initial stem cell to be highly diverse, with lineage sizes ranging from one to more than 20,000 cells. This high variability exceeds what can be explained by existing stochastic models of corticogenesis and indicates the existence of an additional source of stochasticity. To explain this variability, we introduce the SAN model which distinguishes Symmetrically diving, Asymmetrically dividing, and Non-proliferating cells. In the SAN model, the additional source of stochasticity is the survival time of a lineage's pool of symmetrically dividing cells. These survival times result from neutral competition within the sub-population of all symmetrically dividing cells. We demonstrate that our model explains the experimentally observed variability of lineage sizes and derive the quantitative relationship between survival time and lineage size. We also show that our model implies the existence of a regulatory mechanism which keeps the size of the symmetrically dividing cell population constant. Our results provide quantitative insight into the clonal composition of neural organoids and how it arises. This is relevant for many applications of neural organoids, and similar processes may occur in other developing tissues both in vitro and in vivo., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: J.A.K. is on the supervisory and scientific advisory board of a:head bio AG (https://aheadbio.com) and is an inventor on several patents relating to cerebral organoids., (Copyright: © 2024 Pflug et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The establishment and maintenance of apical-basal polarity is a fundamental step in brain development, instructing the organization of neural progenitor cells (NPCs) and the developing cerebral cortex. Particularly, basally located extracellular matrix (ECM) is crucial for this process. In vitro, epithelial polarization can be achieved via endogenous ECM production, or exogenous ECM supplementation. While neuroepithelial development is recapitulated in neural organoids, the effects of different ECM sources in tissue morphogenesis remain underexplored. Here, we show that exposure to a solubilized basement membrane matrix substrate, Matrigel, at early neuroepithelial stages causes rapid tissue polarization and rearrangement of neuroepithelial architecture. In cultures exposed to pure ECM components or unexposed to any exogenous ECM, polarity acquisition is slower and driven by endogenous ECM production. After the onset of neurogenesis, tissue architecture and neuronal differentiation are largely independent of the initial ECM source, but Matrigel exposure has long-lasting effects on tissue patterning. These results advance the knowledge on mechanisms of exogenously and endogenously guided morphogenesis, demonstrating the self-sustainability of neuroepithelial cultures by endogenous processes., (© 2023 Institute of Molecular Biotechnology GmbH. Published under the terms of the CC BY 4.0 license.)

Self-organizing three-dimensional cellular models derived from human pluripotent stem cells or primary tissue have great potential to provide insights into how the human nervous system develops, what makes it unique and how disorders of the nervous system arise, progress and could be treated. Here, to facilitate progress and improve communication with the scientific community and the public, we clarify and provide a basic framework for the nomenclature of human multicellular models of nervous system development and disease, including organoids, assembloids and transplants., (© 2022. Springer Nature Limited.)

Understanding etiology of human neurological and psychiatric diseases is challenging. Genomic changes, protracted development, and histological features unique to human brain development limit the disease aspects that can be investigated using model organisms. Hence, in order to study phenotypes associated with human brain development, function, and disease, it is necessary to use alternative experimental systems that are accessible, ethically justified, and replicate human context. Human pluripotent stem cell (hPSC)-derived brain organoids offer such a system, which recapitulates features of early human neurodevelopment in vitro, including the generation, proliferation, and differentiation of neural progenitors into neurons and glial cells and the complex interactions among the diverse, emergent cell types of the developing brain in three-dimensions (3-D). In recent years, numerous brain organoid protocols and related techniques have been developed to recapitulate aspects of embryonic and fetal brain development in a reproducible and predictable manner. Altogether, these different organoid technologies provide distinct bioassays to unravel novel, disease-associated phenotypes and mechanisms. In this review, we summarize how the diverse brain organoid methods can be utilized to enhance our understanding of brain disorders. FACTS: Brain organoids offer an in vitro approach to study aspects of human brain development and disease. Diverse brain organoid techniques offer bioassays to investigate new phenotypes associated with human brain disorders that are difficult to study in monolayer cultures. Brain organoids have been particularly useful to study phenomena and diseases associated with neural progenitor morphology, survival, proliferation, and differentiation. OPEN QUESTION: Future brain organoid research needs to aim at later stages of neurodevelopment, linked with neuronal activity and connections, to unravel further disease-associated phenotypes. Continued improvement of existing organoid protocols is required to generate standardized methods that recapitulate in vivo-like spatial diversity and complexity.

Turbidity and opaqueness are inherent properties of tissues that limit the capacity to acquire microscopic images through large tissues. Creating a uniform refractive index, known as tissue clearing, overcomes most of these issues. These methods have enabled researchers to image large and complex 3D structures with unprecedented depth and resolution. However, tissue clearing has been adopted to a limited extent due to a combination of cost, time, complexity of existing methods and potential negative impact on fluorescence signal. Here, we describe 2Eci (2nd generation ethyl cinnamate-based clearing), which can be used to clear a wide range of tissues in several species, including human organoids, Drosophila melanogaster , zebrafish, axolotl and Xenopus laevis , in as little as 1-5 days, while preserving a broad range of fluorescent proteins, including GFP, mCherry, Brainbow and Alexa-conjugated fluorophores. Ethyl cinnamate is non-toxic and can easily be used in multi-user microscope facilities. This method opens up tissue clearing to a much broader group of researchers due to its ease of use, the non-toxic nature of ethyl cinnamate and broad applicability., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Brain tumors are among the most lethal and devastating cancers. Their study is limited by genetic heterogeneity and the incompleteness of available laboratory models. Three-dimensional organoid culture models offer innovative possibilities for the modeling of human disease. Here we establish a 3D in vitro model called a neoplastic cerebral organoid (neoCOR), in which we recapitulate brain tumorigenesis by introducing oncogenic mutations in cerebral organoids via transposon- and CRISPR-Cas9-mediated mutagenesis. By screening clinically relevant mutations identified in cancer genome projects, we defined mutation combinations that result in glioblastoma-like and central nervous system primitive neuroectodermal tumor (CNS-PNET)-like neoplasms. We demonstrate that neoCORs are suitable for use in investigations of aspects of tumor biology such as invasiveness, and for evaluation of drug effects in the context of specific DNA aberrations. NeoCORs will provide a valuable complement to the current basic and preclinical models used to study brain tumor biology.

Evolutionary development of the human brain is characterized by the expansion of various brain regions. Here, we show that developmental processes specific to humans are responsible for malformations of cortical development (MCDs), which result in developmental delay and epilepsy in children. We generated a human cerebral organoid model for tuberous sclerosis complex (TSC) and identified a specific neural stem cell type, caudal late interneuron progenitor (CLIP) cells. In TSC, CLIP cells over-proliferate, generating excessive interneurons, brain tumors, and cortical malformations. Epidermal growth factor receptor inhibition reduces tumor burden, identifying potential treatment options for TSC and related disorders. The identification of CLIP cells reveals the extended interneuron generation in the human brain as a vulnerability for disease. In addition, this work demonstrates that analyzing MCDs can reveal fundamental insights into human-specific aspects of brain development.