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Zika virus infections of human stem cell-derived cerebral organoids reveal viral lineage-specific pathogenesis responses.
UNLABELLED: Zika virus (ZIKV) has multiple lineages and strains that cause a range of disease severity, underscoring the need to elucidate differential neuropathogenesis mechanisms. Here, we performed systematic, side-by-side comparisons of African, Asian, and American ZIKV lineage infections using cerebral organoids derived from human embryonic stem cells, a relevant human model experimental system. African lineage ZIKV strains, as well as the ancestral Asian Malaysia strain, persistently infected neural progenitor cells, causing apoptosis and severe disruption of ventricular cytoarchitecture. In contrast, contemporary Asian and American lineage viruses were cleared from ventricles, coinciding with low apoptosis and reduced neuropathology. Single-cell RNA sequencing demonstrated upregulated cell-type-specific antiviral signaling during American lineage infections, coinciding with viral clearance from ventricular progenitor cells. Conversely, pathogenic African lineage infections were associated with apoptosis, reduced STAT2 and IFIT1 protein levels, and enhanced activation of stress pathways. African lineage and ancestral Malaysian strain infections induced mitochondrial oxidative stress. Scavenging the reactive oxygen species improved ventricular cytoarchitecture and progenitor survival, but without reducing viral titers. Together, these findings suggest that lineage- and strain-specific host stress responses, rather than viral burden alone, contribute to ZIKV-induced neurodevelopmental damage. An implication of this study is that host-directed therapeutic strategies, used to improve host tolerance to viral infection, may benefit clinical outcomes. IMPORTANCE: This study provides a systematic comparison of Zika virus (ZIKV) lineage infections in a relevant human model system to correlate mechanisms of host cellular responses with neuropathogenesis. By analyzing African, Asian, and American ZIKV infections side by side in cerebral organoids derived from human embryonic stem cells, we link persistent infection of neural progenitor cells to elevated cellular stress responses and structural disruption of organoid ventricles. Structural disruption was reduced by adding a hydroxyl radical scavenger, but without lowering viral titer. These data are important in strongly suggesting that host responses to viral infection can be of equal or greater importance than viral burden in determining pathogenesis. This study provides mechanistic insight into how closely related viral lineage infections result in divergent outcomes in the developing brain. The data have added importance for suggesting the potential value of host-directed therapeutics to improve ZIKV tolerance without addressing viral titer.
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Impaired neural stress resistance and loss of REST in bipolar disorder.
Neurodevelopmental changes and impaired stress resistance have been implicated in the pathogenesis of bipolar disorder (BD), but the underlying regulatory mechanisms are unresolved. Here we describe a human cerebral organoid model of BD that exhibits altered neural development, elevated neural network activity, and a major shift in the transcriptome. These phenotypic changes were reproduced in cerebral organoids generated from iPS cell lines derived in different laboratories. The BD cerebral organoid transcriptome showed highly significant enrichment for gene targets of the transcriptional repressor REST. This was associated with reduced nuclear REST and REST binding to target gene recognition sites. Reducing the oxygen concentration in organoid cultures to a physiological range ameliorated the developmental phenotype and restored REST expression. These effects were mimicked by treatment with lithium. Reduced nuclear REST and derepression of REST targets genes were also observed in the prefrontal cortex of BD patients. Thus, an impaired cellular stress response in BD cerebral organoids leads to altered neural development and transcriptional dysregulation associated with downregulation of REST. These findings provide a new model and conceptual framework for exploring the molecular basis of BD.
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Self-Organized Synchronous Calcium Transients in a Cultured Human Neural Network Derived from Cerebral Organoids.
The cerebrum is a major center for brain function, and its activity is derived from the assembly of activated cells in neural networks. It is currently difficult to study complex human cerebral neuronal network activity. Here, using cerebral organoids, we report self-organized and complex human neural network activities that include synchronized and non-synchronized patterns. Self-organized neuronal network formation was observed following a dissociation culture of human embryonic stem cell-derived cerebral organoids. The spontaneous individual and synchronized activity of the network was measured via calcium imaging, and subsequent analysis enabled the examination of detailed cell activity patterns, providing simultaneous raster plots, cluster analyses, and cell distribution data. Finally, we demonstrated the feasibility of our system to assess drug-inducible dynamic changes of the network activity. The comprehensive functional analysis of human neuronal networks using this system may offer a powerful tool to access human brain function.
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Cellular Characterization and Neural Electrophysiology in Cerebral Organoid Models of Neurological Disorders.
Human cerebral organoids (hCOs), generated in vitro from induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs), exhibit cellular compositions similar to those of specific human brain regions. These organoids simulate early stages of brain development and can serve as in vitro disease models, providing a unique platform for studying neurodevelopmental processes and investigating underlying disease mechanisms. This review systematically summarizes research progress on cellular characteristics and neural electrophysiology in hCO-based models of neurodevelopmental disorders, neurodegenerative diseases, psychiatric disorders, epilepsy, viral infections, and traumatic brain injuries. These studies have elucidated the mechanisms underlying neuroelectrophysiological dysfunction in related diseases and facilitated innovative therapeutic explorations. Current limitations include prolonged culture durations and high costs, insufficient standardization that compromises reproducibility, the absence of neurovascular units that restrict pathological fidelity, immature laminar architectures that hinder complex circuit modeling, and electrophysiological bottlenecks in network-level analysis. Future efforts should focus on optimizing hCO culture protocols, innovating electrophysiological detection technologies, and promoting their clinical translation.
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Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids.
The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain. Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.
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Zika Virus Depletes Neural Progenitors in Human Cerebral Organoids through Activation of the Innate Immune Receptor TLR3.
Emerging evidence from the current outbreak of Zika virus (ZIKV) indicates a strong causal link between Zika and microcephaly. To investigate how ZIKV infection leads to microcephaly, we used human embryonic stem cell-derived cerebral organoids to recapitulate early stage, first trimester fetal brain development. Here we show that a prototype strain of ZIKV, MR766, efficiently infects organoids and causes a decrease in overall organoid size that correlates with the kinetics of viral copy number. The innate immune receptor Toll-like-Receptor 3 (TLR3) was upregulated after ZIKV infection of human organoids and mouse neurospheres and TLR3 inhibition reduced the phenotypic effects of ZIKV infection. Pathway analysis of gene expression changes during TLR3 activation highlighted 41 genes also related to neuronal development, suggesting a mechanistic connection to disrupted neurogenesis. Together, therefore, our findings identify a link between ZIKV-mediated TLR3 activation, perturbed cell fate, and a reduction in organoid volume reminiscent of microcephaly.
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Advances, challenges, and opportunities of human midbrain organoids for modelling of the dopaminergic system.
Dopaminergic neurons in the ventral midbrain are critical for regulating movement, cognition, and emotion. Ventral midbrain organoids can be used to model both development and diseases of the dopaminergic system, especially Parkinson's disease. Here, we summarize recent advances and remaining challenges in developing such three-dimensional organoids from human pluripotent stem cells. We outline how ventral midbrain organoid systems have progressed from early three-dimensional culture models to sophisticated, engineered, multiregional systems that more accurately replicate the complex network of dopaminergic neurons. Furthermore, we examine how the development of organoid models from other brain regions, particularly the forebrain, provides complementary insights that can accelerate progress also in the field of midbrain organoids, towards the generation of more advanced in vitro systems for midbrain dopaminergic neurons and their circuitry. Such cutting-edge human stem cell-based models offer powerful platforms for investigating dopaminergic neuron generation, function, and connectivity, thereby enhancing disease modelling, drug discovery, and the development of targeted cell-based therapies.
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NeuRoDev resolves lifelong temporal and cellular variation in human cortical gene expression.
Understanding how the human brain develops and functions requires direct analysis of human cells. Single-cell atlases open unprecedented opportunities to survey cell physiological molecular states as the brain develops. However, technical challenges limit their potential. We present NeuRoDev, a computational resource with highly curated transcriptomic data and novel analytical tools to investigate neuronal and glial development in the human cortex. NeuRoDev compresses ∼1M single-cell transcriptomes into integrative summary networks of reproducible cell clusters that capture temporal and cellular variation across all stages of human brain development. It provides a reference framework to directly interrogate cellular maturation dynamics, contextualize gene function, and interpret experimental organoid models. We use NeuRoDev to investigate developmental variation in cell physiology, reconstruct genesis and maturation dynamics in neuronal and glial cells, and interpret time-series data from human organoids. NeuRoDev is provided as a freely available software package and web applications for interactive data analysis.
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Single-cell genomic analysis of human cerebral organoids.
Investigating early brain development has previously relied on using primary developing brain tissue or two-dimensional cell culture models. Recently, stem cell-derived three-dimensional cell culture systems, collectively called brain organoids, have been developed that can faithfully recapitulate many aspects of early brain development. Together with the ability to reprogram fibroblast or blood cells into induced pluripotent stem cells from humans with neurodevelopmental disorders, this opens new inroads to study patient-specific brain development in a personalized cell culture model. Studying the transcriptomes and regulatory landscape of single cells within brain organoids presents a major advance to understand cell-type specific features and transient states during development, and to link these states to their underlying regulatory logic at high resolution. In this protocol, we describe how to generate single-cell RNA-seq and ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) data from the same suspension of organoid cells and focus on reducing batch effects by multiplexing multiple individuals in one experiment. Moreover, we outline basic data processing, analysis, and strategies to correct for batch effects, to account for variability in organoids and for integrating gene expression and open chromatin data.
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Modeling brain development and diseases with human cerebral organoids.
Understanding the mechanisms that underlie human brain development and neurological and neuropsychiatric disorders is one of the key topics of neurobiology. Because of the poor accessibility of human and non-human primate brain tissues, the current perception and understanding of human brain development have been mainly derived from studies of rodents. However, some human-specific features of neural development cannot be well characterized by these animal models. Thanks to the advances in stem cell technologies, brain organoids are being under rapid development, showing the promising applications in decoding the human brain development and uncovering the pathology of brain diseases. In this review, we mainly summarized the recent advances in the development of brain organoid technology and discussed the limitations, applications and future prospects of this promising field.