Functional neuronal circuitry and oscillatory dynamics in human brain organoids

<h3>Abstract</h3> Human brain organoids replicate much of the cellular diversity and developmental anatomy of the human brain. However, the physiological behavior of neuronal circuits within organoids remains relatively under-explored. With high-density CMOS microelectrode arrays and shank electrodes, we probed broadband and three-dimensional spontaneous activity of human brain organoids. These recordings simultaneously captured local field potentials (LFPs) and single unit activity. From spiking activity, we estimated a directed functional connectivity graph of synchronous neural network activity which showed a large number of weak functional connections enmeshed within a network skeleton of significantly fewer strong connections. Increasing the intrinsic inhibitory tone with a benzodiazepine altered the functional network graph of the organoid by suppressing the network skeleton. Simultaneously examining the spontaneous LFPs and their phase alignment to spiking showed that spike bursts were coherent with theta oscillations in the LFPs. An ensemble of spikes phase-locked to theta frequency oscillations were strongly interconnected as a sub-network within the larger network in which they were embedded. Our results demonstrate that human brain organoids have self-organized neuronal assemblies of sufficient size, cellular orientation, and functional connectivity to co-activate and generate field potentials from their collective transmembrane currents that phase-lock to spiking activity. These results point to the potential of brain organoids for the study of neuropsychiatric diseases, drug mechanisms, and the effects of external stimuli upon neuronal networks.

The advent of human induced pluripotent stem cell (hiPSC)-derived brain organoids represents a significant advance in environmental neurotoxicology, propelling the discipline toward human-relevant, mechanistic, and predictive in vitro paradigms. This review explores the utility of brain organoids in environmental neurotoxicology, which uniquely address critical limitations of traditional models by recapitulating key aspects of human brain development, including three-dimensional (3D) cytoarchitecture, multilineage cellular heterogeneity, and functional network activity. This review systematically elaborates on their construction principles, unique advantages in neurotoxicological research, and the significant progress made in elucidating mechanisms of toxicity. Notably, brain organoids exhibit enhanced sensitivity in identifying the subtle adverse outcomes of chronic, low-dose exposures to environmental contaminants, often eluding conventional approaches. Their key advantage lies in the greater capacity to deconstruct complex toxicological pathways, enabling precise tracing of adverse outcome pathways (AOPs). Future development requires enhancing model complexity through vascularization, promoting automation and standardization, and integrating artificial intelligence (AI) for data analysis. Concurrently, establishing sustained ethical oversight and standardized frameworks is essential to ultimately advance the field toward more precise and efficient hazard identification and risk characterization.Graphical abstractHighlights1. Human brain organoids bridge species gaps for more human-relevant neurotoxicity assessment.2. Brain organoids effectively recapitulate chronic, low-dose and mixture environmental risks.3. Integrating brain organoid data into the AOP frameworks enhances mechanistic understanding.4. Coupling brain organoids with organ-on-a-chip and AI advances next-generation risk assessment.

How well do brain organoids capture your brain?

Microcephaly-associated mutations disrupt microtubule-dependent WDR62 translocation from the Golgi complex to the mitotic spindle poles, impair mitotic progression, and alter neurogenic trajectories in patient induced Pluripotent Stem Cell-derived 2D and 3D models of human neurodevelopment.

Differentiation of human pluripotent stem cells to small brain-like structures known as brain organoids offers an unprecedented opportunity to model human brain development and disease. To provide a vascularized and functional in vivo model of brain organoids, we established a method for transplanting human brain organoids into the adult mouse brain. Organoid grafts showed progressive neuronal differentiation and maturation, gliogenesis, integration of microglia, and growth of axons to multiple regions of the host brain. In vivo two-photon imaging demonstrated functional neuronal networks and blood vessels in the grafts. Finally, in vivo extracellular recording combined with optogenetics revealed intragraft neuronal activity and suggested graft-to-host functional synaptic connectivity. This combination of human neural organoids and an in vivo physiological environment in the animal brain may facilitate disease modeling under physiological conditions.

Human cortical organoids (hCOs), derived from human induced pluripotent stem cells (iPSCs), provide a platform to interrogate mechanisms of human brain development and diseases in complex three- dimensional tissues. However, current hCO development methods lack important non-neural tissues, such as the surrounding meningeal layer, that have been shown to be essential for normal corticogenesis and brain development. Here, we first generated hCOs from a single rosette to create more homogenous organoids with consistent size around 250 µm by day 5. We then took advantage of a 3D co-culture system to encapsulate brain organoids with a thin layer of meningeal cells from the very early stages of cortical development. Immunostaining analysis was performed to display different cortical layer markers during different stages of development. Real-time monitoring of organoid development using IncuCyte displayed enhanced morphology and increased growth rate over time. We found that meningeal-encapsulated organoids illustrated better laminar organization by exhibiting higher expression of REELIN by Cajal–Retzius neurons. Presence of meningeal cells resulted in a greater expansion of TBR2 intermediate progenitor cells (IPCs), the deep cortical layer (CTIP2) and upper cortical layer (BRN2). Finally, meningeal-encapsulated organoids enhanced outer radial glial and astrocyte formation illustrated by stronger expression of HOPX and GFAP markers, respectively. This study presents a novel 3D co-culture platform to more closely mimic the in vivo cortical brain structure and enable us to better investigating mechanisms underlying the neurodevelopmental disorders during embryonic development.

Human brain organoids provide a remarkable opportunity to model prenatal human brain biology in vitro by recapitulating features of in utero molecular, cellular and systems biology. An ethical concern peculiar to human brain organoids is whether they are or could become capable of supporting sentience through the experience of pain or pleasure and/or consciousness, including higher cognitive abilities such as self-awareness. Identifying the presence of these traits is complicated by several factors, beginning with consciousness—which is a highly contested concept among neuroscientists, cognitive scientists, and philosophers and so there is no agreed definition. Secondly, given human brain organoids are disembodied, there is no practical way to identify evidence of consciousness as we might in humans or animals. What would count as evidence of organoid consciousness is an emerging area of research. To address concerns about consciousness and human brain organoids, in this paper we clarify the morally relevant aspects of human consciousness, phenomenal experience and embodied development and explore the empirical basis of consciousness to develop a defensible framework for informed decision-making on the moral significance and utility of brain organoids, which can also guide regulation and future research of these novel biological systems.

The reduced pathogenicity of the omicron BA.1 sub-lineage compared to earlier variants is well described, although whether such attenuation is retained for later variants like BA.5 and XBB remains controversial. We show that BA.5 and XBB isolates were significantly more pathogenic in K18-hACE2 mice than a BA.1 isolate, showing increased neurotropic potential, resulting in fulminant brain infection and mortality, similar to that seen for original ancestral isolates. BA.5 also infected human cortical brain organoids to a greater extent than the BA.1 and original ancestral isolates. In the brains of mice, neurons were the main target of infection, and in human organoids neuronal progenitor cells and immature neurons were infected. The results herein suggest that evolving omicron variants may have increasing neurotropic potential.

The human brain organoid is a miniature three-dimensional tissue culture that can simulate the structure and function of the brain in an in vitro culture environment. Although we consider that human brain organoids could be used to understand brain development and diseases, experimental models of human brain organoids are so highly variable that we apply artificial intelligence (AI) techniques to investigate the development mechanism of the human brain. Therefore, this study briefly reviewed commonly used AI applications for human brain organoid-magnetic resonance imaging, electroencephalography, and gene editing techniques, as well as related AI algorithms. Finally, we discussed the limitations, challenges, and future study direction of AI-based technology for human brain organoids.

As deep‐space missions become increasingly feasible, understanding the health risks posed by galactic cosmic rays (GCR) is critical. This study investigates the effects of a simplified 5‐ion GCR simulation (simGCRsim) on human brain organoids derived from 12 human induced pluripotent stem cell (hiPSC) lines. Male organoids exhibit increased vulnerability, characterized by reduced SOX2‐positive neural progenitor populations, downregulation of outer kinetochore genes including components of the NDC80 complex, centromere loss, and elevated nuclear p53 accumulation. Female organoids also exhibit radiation‐induced neuronal damage but show comparatively lower levels of centromere disruption and p53 activation than males. To counteract these effects, an optogenetically controlled FGF2 signaling system is applied, leveraging plant‐derived light‐sensitive domains. Activation of this system enhances extracellular matrix (ECM) expression, resulting in significant neuroprotection against simGCRsim‐induced damage. Furthermore, exogenous ECM supplementation alone effectively mitigated radiation‐induced neural injury in both male and female brain organoids. These findings highlight the central role of ECM in neuroprotection under cosmic radiation exposure and suggest a potential strategy for safeguarding astronaut brain health during long‐duration spaceflight.

Human pluripotent stem cell (hPSC)-derived GABAergic neurons offer potential for treating neurological disorders by restoring disrupted inhibitory circuits, yet current differentiation methods show limited efficiency, purity, and subtype specificity. We present an approach for generating lateral/caudal ganglionic eminence (LGE/CGE) organoids (LCOs) from hPSC-derived brain organoids (BOs) without external signal induction. LCOs bud from the BO surface and are predominantly composed of LGE/CGE-type GABAergic neurons that mature into functional inhibitory neurons. LCOs and BOs exhibit distinct subtype compositions: LCOs contain both LGE-type neurons, with the capacity to form striatal medium spiny neurons, and abundant CGE-type neurons, whereas BOs contain fewer CGE-type neurons. Single-cell transcriptomic analysis reveals that LCOs closely resemble human embryonic LGE/CGE neurons at gestational weeks 12-13. We also developed a method to selectively enrich CXCR4+ CGE-type neurons from LCOs. This platform enables efficient generation of human LGE/CGE-type GABAergic neurons for disease modeling and cell therapy development.

Arboviruses, transmitted by blood-sucking arthropods, are responsible for significant human and animal diseases, including fever, hemorrhagic fever, and encephalitis, posing a serious threat to global public health. Nevertheless, research on the mechanisms of arbovirus infection and the development of therapeutic interventions has been impeded. This delay is primarily due to the limitations inherent in current in vitro research models, including cell cultures and animal models. The simplicity of cell types and interspecies differences present significant obstacles to advancing our understanding of arbovirus infection mechanisms and the development of effective drugs. Human brain organoids, derived from human pluripotent stem cells or human embryonic stem cells and cultured in three-dimensional systems, more accurately replicate the extensive neuronal cellular diversity and key characteristics of human neurodevelopment. These organoids serve as an ideal model for investigating the intricate interactions between viruses and human hosts, and providing a novel platform for the development of antiviral drugs. In this review, we summarize how brain organoid models complement classical approaches to accelerate research into the infection mechanisms of arboviruses, with a particular focus on the types of neural cells, key factors, and cellular signaling pathways involved in the arbovirus infection of brain organoids that have been reported. Furthermore, we examine the development of brain organoids, address their current limitations, and propose future directions to enhance the application of brain organoids in the study of arboviral infectious diseases.

The increasing need for ethical, human-relevant, and efficient alternatives to animal testing is driving the development of New Approach Methodologies (NAMs) in safety assessment and drug development. However, the inherent complexity of neurological diseases presents a significant challenge to fully replace animal models in this field. In neuroscience, a range of NAMs, from traditional 2D cell cultures to advanced brain organoids and alternative vertebrate models like zebrafish, demonstrate complementary strengths and limitations. Together, these models support translational research, including the investigation of neurodevelopment, disease, and neurotoxicity. While human and mouse brain organoids that mimic the structural and functional properties of mammalian brain tissue hold great promise, their applicability for high-throughput screening is hindered by their cost- and time-intensive nature. Complementary approaches such as embryonic and larval zebrafish models and the emerging zebrafish brain organoids provide faster, cost-effective, and scalable yet biologically relevant platforms for early-phase screening, thanks to the zebrafish’s rapid development, conserved vertebrate neuroanatomy, and proven value in toxicology. This review maps the current landscape of NAMs in neuroscience, examining approaches ranging from 2D and 3D in vitro systems to zebrafish models. It highlights the advantages and challenges of the different models, including a comparison of human, mouse, and zebrafish brain organoids, and outlines the future directions for integrating these complementary systems into robust, efficient, and ethically responsible pipelines for both early-phase toxicity testing and drug discovery.

Enhanced Radiation-Sparing Effects of Ultra-High Dose Rate Proton Radiation (FLASH-RT) in a Human Induced Pluripotent Stem Cell-Derived Cerebral Organoid Model

We present a custom recording platform that enables electrophysiological recordings with Neuropixels 2.0 from human brain and heart organoids by maintaining a stable, physiological environment of 5% CO2, 37 °C, and 95% relative humidity. The platform combines a modular positioning system with real-time camera guidance for precise probe alignment. We validated the system by recording consistent bursting activity in brain organoids over a 110-minperiod and in heart organoids for up to 11h.