An in vivo model of functional and vascularized human brain organoids.

How well do brain organoids capture your brain?

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.

Human brain organoids replicate much of the cellular diversity and developmental anatomy of the human brain. However, the physiology of neuronal circuits within organoids remains under-explored. With high-density CMOS microelectrode arrays and shank electrodes, we captured spontaneous extracellular activity from brain organoids derived from human induced pluripotent stem cells. We inferred functional connectivity from spike timing, revealing a large number of weak connections within a skeleton of significantly fewer strong connections. A benzodiazepine increased the uniformity of firing patterns and decreased the relative fraction of weakly connected edges. Our analysis of the local field potential demonstrate that brain organoids contain neuronal assemblies of sufficient size 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 action, and the effects of external stimuli upon neuronal networks.

In recent years, there has been much ethical debate about human brain organoid research. However, the possibility that human brain organoids might be human individuals has not been examined. This is an important issue because if human brain organoids are human individuals, then the creation of human brain organoids could be a form of human reproductive cloning. We examine the implications of two mainstream bioethical views on the nature of human individuals for human brain organoids: (1) Functional Integration Theory, the view that the capacity to integrate vital functions is the criterion for human individuality, and (2) Mental State Theory, the view that some mental capacity is the criterion for human individuality. We find that under either theory of human individuality, human brain organoids could be human individuals in the future. According to a version of Functional Integration Theory that locates the integrative function in the human brainstem (Brainstem Theory), a well-developed human brainstem organoid can be a human individual. Further, on Mental State Theory, a conscious human brain organoid can be a human individual. Our results, combined with the ban on human reproductive cloning, may provide some reasons to be more cautious in human brain organoid research. Alternatively, in light of the progress in human brain organoid research, it may be necessary to reconsider the ethics and laws surrounding human reproductive cloning. This research paves the way for the first time to examine the question of great ethical importance whether human brain organoids can be human individuals, and encourages further consideration.

<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.

Studying the Brain in a Dish: 3D Cell Culture Models of Human Brain Development and Disease.

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.

A Matter of Timing: Rudy Tanzi on Alzheimer's Disease

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental defects whose underlying mechanisms remain to be elucidated. A major roadblock is the lack of effective models recapitulating the early-onset neuronal impairment seen in the patients. Advances in the technology of induced pluripotent stem cells (iPSCs) enable the generation of three-dimensional (3D) brain organoids that can be used to investigate the impact of diseases on the development and organization of the nervous system. Researchers, including these authors, have recently introduced human brain organoids to model mitochondrial disorders. This paper reports a detailed protocol for the robust generation of human iPSC-derived brain organoids and their use in mitochondrial bioenergetic profiling and imaging analyses. These experiments will allow the use of brain organoids to investigate metabolic and developmental dysfunctions and may provide crucial information to dissect the neuronal pathology of mitochondrial diseases.

Recent developments in 3D cultures exploiting the self-organization ability of pluripotent stem cells have enabled the generation of powerful in vitro systems termed brain organoids. These 3D tissues recapitulate many aspects of human brain development and disorders occurring in vivo. When combined with improved differentiation methods, these in vitro systems allow the generation of more complex "assembloids," which are able to reveal cell diversities, microcircuits, and cell-cell interactions within their 3D organization. Here, the ways in which human brain organoids have contributed to demystifying the complexities of brain development and modeling of developmental disorders is reviewed and discussed. Furthermore, challenging questions that are yet to be addressed by emerging brain organoid research are discussed.

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.

Because our beliefs regarding our individuality, autonomy, and personhood are intimately bound up with our brains, there is a public fascination with cerebral organoids, the “mini-brain,” the “brain in a dish”. At the same time, the ethical issues around organoids are only now being explored. What are the prospects of using human cerebral organoids to better understand, treat, or prevent dementia? Will human organoids represent an improvement on the current, less-than-satisfactory, animal models? When considering these questions, two major issues arise. One is the general challenge associated with using any stem cell–generated preparation for in vitro modelling (challenges amplified when using organoids compared with simpler cell culture systems). The other relates to complexities associated with defining and understanding what we mean by the term “dementia.” We discuss 10 puzzles, issues, and stumbling blocks to watch for in the quest to model “dementia in a dish.”

Human cerebral organoids (COs) are three-dimensional self-organizing cultures of cerebral brain tissue differentiated from induced pluripotent stem cells. We have recently shown that COs are susceptible to infection with different subtypes of Creutzfeldt–Jakob disease (CJD) prions, which in humans cause different manifestations of the disease. The ability to study live human brain tissue infected with different CJD subtypes opens a wide array of possibilities from differentiating mechanisms of cell death and identifying neuronal selective vulnerabilities to testing therapeutics. However, the question remained as to whether the prions generated in the CO model truly represent those in the infecting inoculum. Mouse models expressing human prion protein are commonly used to characterize human prion disease as they reproduce many of the molecular and clinical phenotypes associated with CJD subtypes. We therefore inoculated these mice with COs that had been infected with two CJD subtypes (MV1 and MV2) to see if the original subtype characteristics (referred to as strains once transmitted into a model organism) of the infecting prions were maintained in the COs when compared with the original human brain inocula. We found that disease characteristics caused by the molecular subtype of the disease associated prion protein were similar in mice inoculated with either CO derived material or human brain material, demonstrating that the disease associated prions generated in COs shared strain characteristics with those in humans. As the first and only in vitro model of human neurodegenerative disease that can faithfully reproduce different subtypes of prion disease, these findings support the use of the CO model for investigating human prion diseases and their subtypes.