Dysregulation of BRD4 Function Underlies the Functional Abnormalities of MeCP2 Mutant Neurons.

Rett syndrome (RTT), mainly caused by mutations in methyl-CpG binding protein 2 (MeCP2), is one of the most prevalent intellectual disorders without effective therapies. Here, we used 2D and 3D human brain cultures to investigate MeCP2 function. We found that MeCP2 mutations cause severe abnormalities in human interneurons (INs). Surprisingly, treatment with a BET inhibitor, JQ1, rescued the molecular and functional phenotypes of MeCP2 mutant INs. We uncovered that abnormal increases in chromatin binding of BRD4 and enhancer-promoter interactions underlie the abnormal transcription in MeCP2 mutant INs, which were recovered to normal levels by JQ1. We revealed cell-type-specific transcriptome impairment in MeCP2 mutant region-specific human brain organoids that were rescued by JQ1. Finally, JQ1 ameliorated RTT-like phenotypes in mice. These data demonstrate that BRD4 dysregulation is a critical driver for RTT etiology and suggest that targeting BRD4 could be a potential therapeutic opportunity for RTT.

Imaging three-dimensional brain organoid architecture from meso- to nanoscale across development.

Organoids are stem cell-derived three-dimensional cultures offering a new avenue to model human development and disease. Brain organoids allow the study of various aspects of human brain development in the finest details in vitro in a tissue-like context. However, spatial relationships of subcellular structures, such as synaptic contacts between distant neurons, are hardly accessible by conventional light microscopy. This limitation can be overcome by systems that quickly image the entire organoid in three dimensions and in super-resolution. To that end we have developed a system combining tissue expansion and light-sheet fluorescence microscopy for imaging and quantifying diverse spatial parameters during organoid development. This technique enables zooming from a mesoscopic perspective into super-resolution within a single imaging session, thus revealing cellular and subcellular structural details in three spatial dimensions, including unequivocal delineation of mitotic cleavage planes as well as the alignment of pre- and postsynaptic proteins. We expect light-sheet fluorescence expansion microscopy to facilitate qualitative and quantitative assessment of organoids in developmental and disease-related studies.

Vascularized Brain Assembloids With Enhanced Cellular Complexity Provide Insights Into the Cellular Deficits of Tauopathy.

Advanced technologies have enabled the engineering of self-organized 3-dimensional (3D) cellular structures from human induced pluripotent stem cells (hiPSCs), namely organoids, which recapitulate some key features of tissue development and functions of the human central nervous system (CNS). While hiPSC-derived 3D CNS organoids hold promise in providing a human-specific platform for studying CNS development and diseases, most of them do not incorporate the full range of implicated cell types, including vascular cell components and microglia, limiting their ability to accurately recreate the CNS environment and their utility in the study of certain aspects of the disease. Here we have developed a novel approach, called vascularized brain assembloids, for constructing hiPSC-derived 3D CNS structures with a higher level of cellular complexity. This is achieved by integrating forebrain organoids with common myeloid progenitors and phenotypically stabilized human umbilical vein endothelial cells (VeraVecs), which can be cultured and expanded in serum-free conditions. Compared with organoids, these assembloids exhibited enhanced neuroepithelial proliferation, advanced astrocytic maturation, and increased synapse numbers. Strikingly, the assembloids derived from hiPSCs harboring the tauP301S mutation exhibited increased levels of total tau and phosphorylated tau, along with a higher proportion of rod-like microglia-like cells and enhanced astrocytic activation, when compared to the assembloids derived from isogenic hiPSCs. Additionally, the tauP301S assembloids showed an altered profile of neuroinflammatory cytokines. This innovative assembloid technology serves as a compelling proof-of-concept model, opening new avenues for unraveling the intricate complexities of the human brain and accelerating progress in the development of effective treatments for neurological disorders.

Biallelic null variants in PNPLA8 cause microcephaly by reducing the number of basal radial glia.

Patatin-like phospholipase domain-containing lipase 8 (PNPLA8), one of the calcium-independent phospholipase A2 enzymes, is involved in various physiological processes through the maintenance of membrane phospholipids. Biallelic variants in PNPLA8 have been associated with a range of paediatric neurodegenerative disorders. However, the phenotypic spectrum, genotype-phenotype correlations and the underlying mechanisms are poorly understood. Here, we newly identified 14 individuals from 12 unrelated families with biallelic ultra-rare variants in PNPLA8 presenting with a wide phenotypic spectrum of clinical features. Analysis of the clinical features of current and previously reported individuals (25 affected individuals across 20 families) showed that PNPLA8-related neurological diseases manifest as a continuum ranging from variable developmental and/or degenerative epileptic-dyskinetic encephalopathy to childhood-onset neurodegeneration. We found that complete loss of PNPLA8 was associated with the more profound end of the spectrum, with congenital microcephaly. Using cerebral organoids generated from human induced pluripotent stem cells, we found that loss of PNPLA8 led to developmental defects by reducing the number of basal radial glial cells and upper-layer neurons. Spatial transcriptomics revealed that loss of PNPLA8 altered the fate specification of apical radial glial cells, as reflected by the enrichment of gene sets related to the cell cycle, basal radial glial cells and neural differentiation. Neural progenitor cells lacking PNPLA8 showed a reduced amount of lysophosphatidic acid, lysophosphatidylethanolamine and phosphatidic acid. The reduced number of basal radial glial cells in patient-derived cerebral organoids was rescued, in part, by the addition of lysophosphatidic acid. Our data suggest that PNPLA8 is crucial to meet phospholipid synthetic needs and to produce abundant basal radial glial cells in human brain development.

Low-intensity ultrasound ameliorates brain organoid integration and rescues microcephaly deficits.

Human brain organoids represent a remarkable platform for modelling neurological disorders and a promising brain repair approach. However, the effects of physical stimulation on their development and integration remain unclear. Here, we report that low-intensity ultrasound significantly increases neural progenitor cell proliferation and neuronal maturation in cortical organoids. Histological assays and single-cell gene expression analyses revealed that low-intensity ultrasound improves the neural development in cortical organoids. Following organoid grafts transplantation into the injured somatosensory cortices of adult mice, longitudinal electrophysiological recordings and histological assays revealed that ultrasound-treated organoid grafts undergo advanced maturation. They also exhibit enhanced pain-related gamma-band activity and more disseminated projections into the host brain than the untreated groups. Finally, low-intensity ultrasound ameliorates neuropathological deficits in a microcephaly brain organoid model. Hence, low-intensity ultrasound stimulation advances the development and integration of brain organoids, providing a strategy for treating neurodevelopmental disorders and repairing cortical damage.

Weighing the moral status of brain organoids and research animals.

Recent advances in human brain organoid systems have raised serious worries about the possibility that these in vitro 'mini-brains' could develop sentience, and thus, moral status. This article considers the relative moral status of sentient human brain organoids and research animals, examining whether we have moral reasons to prefer using one over the other. It argues that, contrary to common intuitions, the wellbeing of sentient human brain organoids should not be granted greater moral consideration than the wellbeing of nonhuman research animals. It does so not by denying that typical humans have higher moral status than animals, but instead by arguing that none of the leading justifications for granting humans higher moral status than nonhuman animals apply to brain organoids. Additionally, it argues that there are no good reasons to be more concerned about the well-being of human brain organoids compared to those generated from other species.

Modeling neurological disorders using brain organoids.

Neurological disorders are challenging to study given the complexity and species-specific features of the organ system. Brain organoids are three dimensional structured aggregates of neural tissue that are generated by self-organization and differentiation from pluripotent stem cells under optimized culture conditions. These brain organoids exhibit similar features of structural organization and cell type diversity as the developing human brain, creating opportunities to recapitulate disease phenotypes that are not otherwise accessible. Here we review the initial attempt in the field to apply brain organoid models for the study of many different types of human neurological disorders across a wide range of etiologies and pathophysiologies. Forthcoming advancements in both brain organoid technology as well as analytical methods have significant potentials to advance the understanding of neurological disorders and to uncover opportunities for meaningful therapeutic intervention.

Deconstructing and reconstructing the human brain with regionally specified brain organoids.

Brain organoids, three-dimensional neural cultures recapitulating the spatiotemporal organization and function of the brain in a dish, offer unique opportunities for investigating the human brain development and diseases. To model distinct parts of the brain, various region-specific human brain organoids have been developed. In this article, we review current approaches to produce human region-specific brain organoids, developed through the endeavor of many researchers. We highlight the applications of human region-specific brain organoids, especially in reconstructing regional interactions in the brain through organoid fusion. We also outline the existing challenges to drive forward further the brain organoid technology and its applications for future studies.

Microfluidic Brain-on-a-Chip: From Key Technology to System Integration and Application.

As an ideal in vitro model, brain-on-chip (BoC) is an important tool to comprehensively elucidate brain characteristics. However, the in vitro model for the definition scope of BoC has not been universally recognized. In this review, BoC is divided into brain cells-on-a- chip, brain slices-on-a-chip, and brain organoids-on-a-chip according to the type of culture on the chip. Although these three microfluidic BoCs are constructed in different ways, they all use microfluidic chips as carrier tools. This method can better meet the needs of maintaining high culture activity on a chip for a long time. Moreover, BoC has successfully integrated cell biology, the biological material platform technology of microenvironment on a chip, manufacturing technology, online detection technology on a chip, and so on, enabling the chip to present structural diversity and high compatibility to meet different experimental needs and expand the scope of applications. Here, the relevant core technologies, challenges, and future development trends of BoC are summarized.

Galangin Rescues Alzheimer's Amyloid-ß Induced Mitophagy and Brain Organoid Growth Impairment.

Dysfunctional mitochondria and mitophagy are hallmarks of Alzheimer's disease (AD). It is widely accepted that restoration of mitophagy helps to maintain cellular homeostasis and ameliorates the pathogenesis of AD. It is imperative to create appropriate preclinical models to study the role of mitophagy in AD and to assess potential mitophagy-targeting therapies. Here, by using a novel 3D human brain organoid culturing system, we found that amyloid-ß (Aß1-42,10 µM) decreased the growth level of organoids, indicating that the neurogenesis of organoids may be impaired. Moreover, Aß treatment inhibited neural progenitor cell (NPC) growth and induced mitochondrial dysfunction. Further analysis revealed that mitophagy levels were reduced in the brain organoids and NPCs. Notably, galangin (10 µM) treatment restored mitophagy and organoid growth, which was inhibited by Aß. The effect of galangin was blocked by the mitophagy inhibitor, suggesting that galangin possibly acted as a mitophagy enhancer to ameliorate Aß-induced pathology. Together, these results supported the important role of mitophagy in AD pathogenesis and suggested that galangin may be used as a novel mitophagy enhancer to treat AD.

Application of Human Brain Organoids-Opportunities and Challenges in Modeling Human Brain Development and Neurodevelopmental Diseases.

Brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that reflect early brain organization. These organoids contain different cell types, including neurons and glia, similar to those found in the human brain. Human brain organoids provide unique opportunities to model features of human brain development that are not well-reflected in animal models. Compared with traditional cell cultures and animal models, brain organoids offer a more accurate representation of human brain development and function, rendering them suitable models for neurodevelopmental diseases. In particular, brain organoids derived from patients' cells have enabled researchers to study diseases at different stages and gain a better understanding of disease mechanisms. Multi-brain regional assembloids allow for the investigation of interactions between distinct brain regions while achieving a higher level of consistency in molecular and functional characterization. Although organoids possess promising features, their usefulness is limited by several unresolved constraints, including cellular stress, hypoxia, necrosis, a lack of high-fidelity cell types, limited maturation, and circuit formation. In this review, we discuss studies to overcome the natural limitations of brain organoids, emphasizing the importance of combinations of all neural cell types, such as glia (astrocyte, oligodendrocytes, and microglia) and vascular cells. Additionally, considering the similarity of organoids to the developing brain, regionally patterned brain organoid-derived neural stem cells (NSCs) could serve as a scalable source for cell replacement therapy. We highlight the potential application of brain organoid-derived cells in disease cell therapy within this field.

Human Brain Organoids in Migraine Research: Pathogenesis and Drug Development.

Human organoids are small, self-organized, three-dimensional (3D) tissue cultures that have started to revolutionize medical science in terms of understanding disease, testing pharmacologically active compounds, and offering novel ways to treat disease. Organoids of the liver, kidney, intestine, lung, and brain have been developed in recent years. Human brain organoids are used for understanding pathogenesis and investigating therapeutic options for neurodevelopmental, neuropsychiatric, neurodegenerative, and neurological disorders. Theoretically, several brain disorders can be modeled with the aid of human brain organoids, and hence the potential exists for understanding migraine pathogenesis and its treatment with the aid of brain organoids. Migraine is considered a brain disorder with neurological and non-neurological abnormalities and symptoms. Both genetic and environmental factors play essential roles in migraine pathogenesis and its clinical manifestations. Several types of migraines are classified, for example, migraines with and without aura, and human brain organoids can be developed from patients with these types of migraines to study genetic factors (e.g., channelopathy in calcium channels) and environmental stressors (e.g., chemical and mechanical). In these models, drug candidates for therapeutic purposes can also be tested. Here, the potential and limitations of human brain organoids for studying migraine pathogenesis and its treatment are communicated to generate motivation and stimulate curiosity for further research. This must, however, be considered alongside the complexity of the concept of brain organoids and the neuroethical aspects of the topic. Interested researchers are invited to join the network for protocol development and testing the hypothesis presented here.

Playing Brains: The Ethical Challenges Posed by Silicon Sentience and Hybrid Intelligence in DishBrain.

The convergence of human and artificial intelligence is currently receiving considerable scholarly attention. Much debate about the resulting Hybrid Minds focuses on the integration of artificial intelligence into the human brain through intelligent brain-computer interfaces as they enter clinical use. In this contribution we discuss a complementary development: the integration of a functional in vitro network of human neurons into an in silico computing environment.To do so, we draw on a recent experiment reporting the creation of silico-biological intelligence as a case study (Kagan et al., 2022b). In this experiment, multielectrode arrays were plated with stem cell-derived human neurons, creating a system which the authors call DishBrain. By embedding the system into a virtual game-world, neural clusters were able to receive electrical input signals from the game-world and to respond appropriately with output signals from pre-assigned motor regions. Using this design, the authors demonstrate how the DishBrain self-organises and successfully learns to play the computer game 'Pong', exhibiting 'sentient' and intelligent behaviour in its virtual environment.The creation of such hybrid, silico-biological intelligence raises numerous ethical challenges. Following the neuroscientific framework embraced by the authors themselves, we discuss the arising ethical challenges in the context of Karl Friston's Free Energy Principle, focusing on the risk of creating synthetic phenomenology. Following the DishBrain's creator's neuroscientific assumptions, we highlight how DishBrain's design may risk bringing about artificial suffering and argue for a congruently cautious approach to such synthetic biological intelligence.

Rotenone, an environmental toxin, causes abnormal methylation of the mouse brain organoid's genome and ferroptosis.

More and more reports have pointed out that rotenone, as an insecticide, has high neurotoxicity and reproductive toxicity to livestock and mammals. As a highly physiological correlation system of internal organs, quasi-organs have great potential in the fields of drug toxicity and efficacy test, toxicology research, developmental biology and so on. In this study, brain organs (mBOs) derived from mouse neural stem cells were used to investigate the effects of rotenone on the physiological activity and epigenetic modification of mBOs. At the same time, Rotenone could significantly stimulate the increase of the concentration of LPO, lactic acid and hydroxyl radical in mBOs, and inhibit the expression of neuronal marker Tuj1, CHAT, PAX6 and so on. Further analysis showed that Rotenonem could induce mitochondrial damage in mBOs. The results of qPCR and Western blot showed that Rotenone could up-regulate the expressions of ferroptosis promoting protein p53, Cox2 and so on, while inhibit the expressions of negative regulatory protein of ferroptosis GPX4, FTH1, SLC7A11. In addition, the results of RRBS-Seq sequencing showed that the methylation modification at DMR level in Rotenone-treated mBOs group was significantly higher than that in Ctrl group. The results of KEGG analysis showed that compared with Ctrl, the genes with hypermethylation of promoter and Genebody in Rotenone-treated mBOs were mainly located in the Neuro active ligand-receptor interaction signal transduction pathway. In summary, rotenone can significantly lead to abnormal methylation of mouse brain organs, and lead to the loss of normal physiological function of neurons by inducing ferroptosis.

Aberrant Cortical Layer Development of Brain Organoids Derived from Noonan Syndrome-iPSCs.

Noonan syndrome (NS) is a genetic disorder mainly caused by gain-of-function mutations in Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2). Although diverse neurological manifestations are commonly diagnosed in NS patients, the mechanisms as to how SHP2 mutations induce the neurodevelopmental defects associated with NS remain elusive. Here, we report that cortical organoids (NS-COs) derived from NS-induced pluripotent stem cells (iPSCs) exhibit developmental abnormalities, especially in excitatory neurons (ENs). Although NS-COs develop normally in their appearance, single-cell transcriptomic analysis revealed an increase in the EN population and overexpression of cortical layer markers in NS-COs. Surprisingly, the EN subpopulation co-expressing the upper layer marker SATB2 and the deep layer maker CTIP2 was enriched in NS-COs during cortical development. In parallel with the developmental disruptions, NS-COs also exhibited reduced synaptic connectivity. Collectively, our findings suggest that perturbed cortical layer identity and impeded neuronal connectivity contribute to the neurological manifestations of NS.

Brain Regional Identity and Cell Type Specificity Landscape of Human Cortical Organoid Models.

In vitro models of corticogenesis from pluripotent stem cells (PSCs) have greatly improved our understanding of human brain development and disease. Among these, 3D cortical organoid systems are able to recapitulate some aspects of in vivo cytoarchitecture of the developing cortex. Here, we tested three cortical organoid protocols for brain regional identity, cell type specificity and neuronal maturation. Overall, all protocols gave rise to organoids that displayed a time-dependent expression of neuronal maturation genes such as those involved in the establishment of synapses and neuronal function. Comparatively, guided differentiation methods without WNT activation generated the highest degree of cortical regional identity, whereas default conditions produced the broadest range of cell types such as neurons, astrocytes and hematopoietic-lineage-derived microglia cells. These results suggest that cortical organoid models produce diverse outcomes of brain regional identity and cell type specificity and emphasize the importance of selecting the correct model for the right application.

Studying Human Neurodevelopment and Diseases Using 3D Brain Organoids.

In vitro differentiation of pluripotent stem cells provides a systematic platform to study development and disease. Recent advances in brain organoid technology have created new opportunities to investigate the formation and function of the human brain, under physiological and pathological conditions. Brain organoids can be generated to model the cellular and structural development of the human brain, and allow the investigation of the intricate interactions between resident neural and glial cell types. Combined with new advances in gene editing, imaging, and genomic analysis, brain organoid technology can be applied to address questions pertinent to human brain development, disease, and evolution. However, the current iterations of brain organoids also have limitations in faithfully recapitulating the in vivo processes. In this perspective, we evaluate the recent progress in brain organoid technology, and discuss the experimental considerations for its utilization.Dual Perspectives Companion Paper: Integrating CRISPR Engineering and hiPSC-Derived 2D Disease Modeling Systems, by Kristina Rehbach, Michael B. Fernando, and Kristen J. Brennand.

Applications of brain organoids in neurodevelopment and neurological diseases.

A brain organoid is a self-organizing three-dimensional tissue derived from human embryonic stem cells or pluripotent stem cells and is able to simulate the architecture and functionality of the human brain. Brain organoid generation methods are abundant and continue to improve, and now, an in vivo vascularized brain organoid has been encouragingly reported. The combination of brain organoids with immune-staining and single-cell sequencing technology facilitates our understanding of brain organoids, including the structural organization and the diversity of cell types. Recent publications have reported that brain organoids can mimic the dynamic spatiotemporal process of early brain development, model various human brain disorders, and serve as an effective preclinical platform to test and guide personalized treatment. In this review, we introduce the current state of brain organoid differentiation strategies, summarize current progress and applications in the medical domain, and discuss the challenges and prospects of this promising technology.

Modeling genetic epilepsies in a dish.

Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells, provide a powerful platform for mechanistic studies of disorders of neurodevelopment and neural networks. hPSC models of autism, epilepsy, and other neurological disorders are also advancing the path toward designing and testing precision therapies. The field is evolving rapidly with the addition of genome-editing approaches, expanding protocols for the two-dimensional (2D) differentiation of different neuronal subtypes, and three-dimensional (3D) human brain organoid cultures. However, the application of these techniques to study complex neurological disorders, including the epilepsies, remains a challenge. Here, we review previous work using both 2D and 3D hPSC models of genetic epilepsies, as well as recent advances in the field. We also describe new strategies for applying these technologies to disease modeling of genetic epilepsies, and discuss current challenges and future directions.

Ethical Aspects of Brain Organoid Research in News Reports: An Exploratory Descriptive Analysis.

Background and Objectives: Brain organoids are self-assembled, three-dimensional (3D) aggregates generated from pluripotent stem cells. These models are useful for experimental studies on human brain development and function and are therefore increasingly used for research worldwide. As their increasing use raises several ethical questions, we aimed to assess the current state of the press on brain organoid research using a cross-sectional database to understand the extent of discussion of this subject in the public. Materials and Methods: We conducted a descriptive analysis of news reports obtained from the Nexis Uni database, searched in April 2020. After extracting the news reports, the number of published reports in each year and the included terms were analyzed. Results: Up to April 2020, 332 news reports had been published, with over half of them published in the United States and the United Kingdom, with the numbers gradually increasing every year. In total, 113 (34.0%) news reports included ethics-related keywords, and the ratio of studies before and after the study-period midpoint was significantly increased (21.0% (2013-2016) vs. 38.2% (2017-2020); p = 0.0066, Chi-square test with Yates' continuity correction). Conclusions: Although news reports on the ethical aspects of brain organoid research have been increasing gradually, there was a bias in the region of publication. Additional studies focusing on the ethical aspects of brain organoid research should strive to assess the public perception on the subject in different parts of the world.