Revolutionizing developmental neurotoxicity testing - a journey from animal models to advanced in vitro systems.

Developmental neurotoxicity (DNT) testing has seen enormous progress over the last two decades. Preceding even the publication of the animal-based OECD test guideline for DNT testing in 2007, a series of non-animal technology workshops and conferences (starting in 2005) shaped a community that has delivered a comprehensive battery of in vitro test methods (IVB). Its data interpretation is covered by a very recent OECD test guidance (No. 377). Here, we aim to overview the progress in the field, focusing on the evolution of testing strategies, the role of emerging technologies, and the impact of OECD test guidelines on DNT testing. In particular, this is an example of a targeted development of an animal-free testing approach for one of the most complex hazards of chemicals to human health. These developments started literally from a blank slate, with no proposed alternative methods available. Over two decades, cutting-edge science enabled the design of a testing approach that spares animals and enables throughput for this challenging hazard. While it is evident that the field needs guidance and regulation, the massive economic impact of decreased human cognitive capacity caused by chemical exposure should be prioritized more highly. Beyond this, the claim to fame of DNT in vitro testing is the enormous scientific progress it has brought for understanding the human brain, its development, and how it can be perturbed.

Developmental neurotoxicity (DNT) testing predicts the hazard of exposure to chemicals to human brain development. Comprehensive advanced non-animal testing strategies using cutting-edge technology can now replace animal-based approaches to assess this complex hazard. These strategies can assess large numbers of chemicals more accurately and efficiently than the animal-based approach. Recent OECD test guidance has formalized this battery of in vitro test methods for DNT, marking a pivotal achievement in the field. The shift towards non-animal testing reflects both a commitment to animal welfare and a growing recognition of the economic and public health impacts associated with impaired cognitive function caused by chemical exposures. These innovations ultimately contribute to safer chemical management and better protection of human health, especially during the vulnerable stages of brain development.

The Promise and Potential of Brain Organoids.

Brain organoids are 3D in vitro culture systems derived from human pluripotent stem cells that self-organize to model features of the (developing) human brain. This review examines the techniques behind organoid generation, their current and potential applications, and future directions for the field. Brain organoids possess complex architecture containing various neural cell types, synapses, and myelination. They have been utilized for toxicology testing, disease modeling, infection studies, personalized medicine, and gene-environment interaction studies. An emerging concept termed Organoid Intelligence (OI) combines organoids with artificial intelligence systems to generate learning and memory, with the goals of modeling cognition and enabling biological computing applications. Brain organoids allow neuroscience studies not previously achievable with traditional techniques, and have the potential to transform disease modeling, drug development, and the understanding of human brain development and disorders. The aspirational vision of OI parallels the origins of artificial intelligence, and efforts are underway to map a roadmap toward its realization. In summary, brain organoids constitute a disruptive technology that is rapidly advancing and gaining traction across multiple disciplines.

Blood extracellular vesicles carrying brain-specific mRNAs are potential biomarkers for detecting gene expression changes in the female brain.

The absence of non-invasive tests that can monitor the status of the brain is a major obstacle for psychiatric care. In order to address this need, we assessed the feasibility of using tissue-specific gene expression to determine the origin of extracellular vesicle (EV) mRNAs in peripheral blood. Using the placenta as a model, we discovered that 26 messenger RNAs that are specifically expressed in the placenta are present in EVs circulating in maternal blood. Twenty-three of these transcripts were either exclusively or highly expressed in maternal blood during pregnancy only and not in the postpartum period, verifying the feasibility of using tissue-specific gene expression to infer the tissue of origin for EV mRNAs. Using the same bioinformatic approach, which provides better specificity than isolating L1 cell-adhesion molecule containing EVs, we discovered that 181 mRNAs that are specifically expressed in the female brain are also present in EVs circulating in maternal blood. Gene set enrichment analysis revealed that these transcripts, which are involved in synaptic functions and myelination, are enriched for genes implicated in mood disorders, schizophrenia, and substance use disorders. The EV mRNA levels of 13 of these female brain-specific transcripts are associated with postpartum depression (adjusted p-vals = 3 × 10-5 to 0.08), raising the possibility that they can be used to infer the state of the brain. In order to determine the extent to which EV mRNAs reflect transcription in the brain, we compared mRNAs isolated from cells and EVs in an iPSC-derived brain microphysiological system differentiated for 3 and 9 weeks. We discovered that, although cellular and extracellular mRNA levels are not identical, they do correlate, and it is possible to extrapolate cellular RNA expression changes in the brain via EV mRNA levels. Our findings bring EV mRNAs to the forefront of peripheral biomarker development efforts in psychiatric diseases by demonstrating the feasibility of inferring transcriptional changes in the brain via blood EV mRNA levels.

A Novel Approach to Increase Glial Cell Populations in Brain Microphysiological Systems.

Brain microphysiological systems (bMPS) recapitulate human brain cellular architecture and functionality more closely than traditional monolayer cultures and have become increasingly relevant for the study of neurological function in health and disease. Existing 3D brain models vary in reflecting the relative populations of different cell types present in the human brain. Most models consist mainly of neurons, while glial cells represent a smaller portion of the cell populations. Here, by means of a chemically defined glial-enriched medium (GEM), an improved method to expand the population of astrocytes and oligodendrocytes without compromising neuronal differentiation in bMPS, is presented. An important finding is that astrocytes also change in morphology when cultured in GEM, more closely recapitulating primary culture astrocytes. GEM bMPS are electro-chemically active and show different patterns of calcium staining and flux. Synaptic vesicles and terminals observed by electron microscopy are also present. No significant changes in neuronal differentiation are observed by gene expression, however, GEM enhanced neurite outgrowth and cell migration, and differentially modulated neuronal maturation in two different cell lines. These results have the potential to significantly improve functionality of bMPS for the study of neurological diseases and drug discovery, contributing to the unmet need for safe human models.

A novel approach to increase glial cell populations in brain microphysiological systems.

Brain microphysiological systems (bMPS), which recapitulate human brain cellular architecture and functionality more closely than traditional monolayer cultures, have become a practical, non-invasive, and increasingly relevant platform for the study of neurological function in health and disease. These models include 3D spheroids and organoids as well as organ-on-chip models. Currently, however, existing 3D brain models vary in reflecting the relative populations of the different cell types present in the human brain. Most of the models consist mainly of neurons, while glial cells represent a smaller portion of the cell populations. Here, by means of a chemically defined glial-enriched medium (GEM), we present an improved method to expand the population of astrocytes and oligodendrocytes without compromising neuronal differentiation in bMPS. An important finding is that astrocytes not only increased in number but also changed in morphology when cultured in GEM, more closely recapitulating primary culture astrocytes. We demonstrate oligodendrocyte and astrocyte enrichment in GEM bMPS using a variety of complementary methods. We found that GEM bMPS are electro-chemically active and showed different patterns of Ca +2 staining and flux. Synaptic vesicles and terminals observed by electron microscopy were also present. No significant changes in neuronal differentiation were observed by gene expression, however, GEM enhanced neurite outgrowth and cell migration, and differentially modulated neuronal maturation in two different iPSC lines. Our results have the potential to significantly improve in vivo-like functionality of bMPS for the study of neurological diseases and drug discovery, contributing to the unmet need for safe human models.

Deconvoluting gene and environment interactions to develop an "epigenetic score meter" of disease.

Human health is determined both by genetics (G) and environment (E). This is clearly illustrated in groups of individuals who are exposed to the same environmental factor showing differential responses. A quantitative measure of the gene-environment interactions (GxE) effects has not been developed and in some instances, a clear consensus on the concept has not even been reached; for example, whether cancer is predominantly emerging from "bad luck" or "bad lifestyle" is still debated. In this article, we provide a panel of examples of GxE interaction as drivers of pathogenesis. We highlight how epigenetic regulations can represent a common connecting aspect of the molecular bases. Our argument converges on the concept that the GxE is recorded in the cellular epigenome, which might represent the key to deconvolute these multidimensional intricated layers of regulation. Developing a key to decode this epigenetic information would provide quantitative measures of disease risk. Analogously to the epigenetic clock introduced to estimate biological age, we provocatively propose the theoretical concept of an "epigenetic score-meter" to estimate disease risk.

G × E interactions as a basis for toxicological uncertainty.

To transfer toxicological findings from model systems, e.g. animals, to humans, standardized safety factors are applied to account for intra-species and inter-species variabilities. An alternative approach would be to measure and model the actual compound-specific uncertainties. This biological concept assumes that all observed toxicities depend not only on the exposure situation (environment = E), but also on the genetic (G) background of the model (G × E). As a quantitative discipline, toxicology needs to move beyond merely qualitative G × E concepts. Research programs are required that determine the major biological variabilities affecting toxicity and categorize their relative weights and contributions. In a complementary approach, detailed case studies need to explore the role of genetic backgrounds in the adverse effects of defined chemicals. In addition, current understanding of the selection and propagation of adverse outcome pathways (AOP) in different biological environments is very limited. To improve understanding, a particular focus is required on modulatory and counter-regulatory steps. For quantitative approaches to address uncertainties, the concept of "genetic" influence needs a more precise definition. What is usually meant by this term in the context of G × E are the protein functions encoded by the genes. Besides the gene sequence, the regulation of the gene expression and function should also be accounted for. The widened concept of past and present "gene expression" influences is summarized here as Ge. Also, the concept of "environment" needs some re-consideration in situations where exposure timing (Et) is pivotal: prolonged or repeated exposure to the insult (chemical, physical, life style) affects Ge. This implies that it changes the model system. The interaction of Ge with Et might be denoted as Ge × Et. We provide here general explanations and specific examples for this concept and show how it could be applied in the context of New Approach Methodologies (NAM).

Organoid intelligence (OI) - The ultimate functionality of a brain microphysiological system.

Understanding brain function remains challenging as work with human and animal models is complicated by compensatory mechanisms, while in vitro models have been too simple until now. With the advent of human stem cells and the bioengineering of brain microphysiological systems (MPS), understanding how both cognition and long-term memory arise is now coming into reach. We suggest combining cutting-edge AI with MPS research to spearhead organoid intelligence (OI) as synthetic biological intelligence. The vision is to realize cognitive functions in brain MPS and scale them to achieve relevant short- and long-term memory capabilities and basic information processing as the ultimate functional experimental models for neurodevelopment and neurological function and as cell-based assays for drug and chemical testing. By advancing the frontiers of biological computing, we aim to (a) create models of intelligence-in-a-dish to study the basis of human cognitive functions, (b) provide models to advance the search for toxicants contributing to neurological diseases and identify remedies for neurological maladies, and (c) achieve relevant biological computational capacities to complement traditional computing. Increased understanding of brain functionality, in some respects still superior to today's supercomputers, may allow to imitate this in neuromorphic computer architectures or might even open up biological computing to complement silicon computers. At the same time, this raises ethical questions such as where sentience and consciousness start and what the relationship between a stem cell donor and the respective OI system is. Such ethical discussions will be critical for the socially acceptable advance of brain organoid models of cognition.

First Organoid Intelligence (OI) workshop to form an OI community.

The brain is arguably the most powerful computation system known. It is extremely efficient in processing large amounts of information and can discern signals from noise, adapt, and filter faulty information all while running on only 20 watts of power. The human brain's processing efficiency, progressive learning, and plasticity are unmatched by any computer system. Recent advances in stem cell technology have elevated the field of cell culture to higher levels of complexity, such as the development of three-dimensional (3D) brain organoids that recapitulate human brain functionality better than traditional monolayer cell systems. Organoid Intelligence (OI) aims to harness the innate biological capabilities of brain organoids for biocomputing and synthetic intelligence by interfacing them with computer technology. With the latest strides in stem cell technology, bioengineering, and machine learning, we can explore the ability of brain organoids to compute, and store given information (input), execute a task (output), and study how this affects the structural and functional connections in the organoids themselves. Furthermore, understanding how learning generates and changes patterns of connectivity in organoids can shed light on the early stages of cognition in the human brain. Investigating and understanding these concepts is an enormous, multidisciplinary endeavor that necessitates the engagement of both the scientific community and the public. Thus, on Feb 22-24 of 2022, the Johns Hopkins University held the first Organoid Intelligence Workshop to form an OI Community and to lay out the groundwork for the establishment of OI as a new scientific discipline. The potential of OI to revolutionize computing, neurological research, and drug development was discussed, along with a vision and roadmap for its development over the coming decade.

Brain organoids and organoid intelligence from ethical, legal, and social points of view.

Human brain organoids, aka cerebral organoids or earlier "mini-brains", are 3D cellular models that recapitulate aspects of the developing human brain. They show tremendous promise for advancing our understanding of neurodevelopment and neurological disorders. However, the unprecedented ability to model human brain development and function in vitro also raises complex ethical, legal, and social challenges. Organoid Intelligence (OI) describes the ongoing movement to combine such organoids with Artificial Intelligence to establish basic forms of memory and learning. This article discusses key issues regarding the scientific status and prospects of brain organoids and OI, conceptualizations of consciousness and the mind-brain relationship, ethical and legal dimensions, including moral status, human-animal chimeras, informed consent, and governance matters, such as oversight and regulation. A balanced framework is needed to allow vital research while addressing public perceptions and ethical concerns. Interdisciplinary perspectives and proactive engagement among scientists, ethicists, policymakers, and the public can enable responsible translational pathways for organoid technology. A thoughtful, proactive governance framework might be needed to ensure ethically responsible progress in this promising field.

Neuronal cultures playing Pong: First steps toward advanced screening and biological computing.

In this issue of Neuron, Kagan et al.1 implement learning-in-a-dish as an important step toward organoid intelligence. These systems may complement the study of molecular and cellular mechanisms of cognition and allow innovations in pharmacological and toxicological studies of neurodevelopmental or neurodegenerative disorders as well as advances in biological computing.

Shell microelectrode arrays (MEAs) for brain organoids.

Brain organoids are important models for mimicking some three-dimensional (3D) cytoarchitectural and functional aspects of the brain. Multielectrode arrays (MEAs) that enable recording and stimulation of activity from electrogenic cells offer notable potential for interrogating brain organoids. However, conventional MEAs, initially designed for monolayer cultures, offer limited recording contact area restricted to the bottom of the 3D organoids. Inspired by the shape of electroencephalography caps, we developed miniaturized wafer-integrated MEA caps for organoids. The optically transparent shells are composed of self-folding polymer leaflets with conductive polymer-coated metal electrodes. Tunable folding of the minicaps' polymer leaflets guided by mechanics simulations enables versatile recording from organoids of different sizes, and we validate the feasibility of electrophysiology recording from 400- to 600-µm-sized organoids for up to 4 weeks and in response to glutamate stimulation. Our studies suggest that 3D shell MEAs offer great potential for high signal-to-noise ratio and 3D spatiotemporal brain organoid recording.

Quality criteria for in vitro human pluripotent stem cell-derived models of tissue-based cells.

The advent of the technology to isolate or generate human pluripotent stem cells provided the potential to develop a wide range of human models that could enhance understanding of mechanisms underlying human development and disease. These systems are now beginning to mature and provide the basis for the development of in vitro assays suitable to understand the biological processes involved in the multi-organ systems of the human body, and will improve strategies for diagnosis, prevention, therapies and precision medicine. Induced pluripotent stem cell lines are prone to phenotypic and genotypic changes and donor/clone dependent variability, which means that it is important to identify the most appropriate characterization markers and quality control measures when sourcing new cell lines and assessing differentiated cell and tissue culture preparations for experimental work. This paper considers those core quality control measures for human pluripotent stem cell lines and evaluates the state of play in the development of key functional markers for their differentiated cell derivatives to promote assurance of reproducibility of scientific data derived from pluripotent stem cell-based systems.

Human IPSC 3D brain model as a tool to study chemical-induced dopaminergic neuronal toxicity.

Oxidative stress is caused by an imbalance between the generation and detoxification of reactive oxygen and nitrogen species (ROS/RNS). This imbalance plays an important role in brain aging and age-related neurodegenerative diseases. In the context of Parkinson's disease (PD), the sensitivity of dopaminergic neurons in the substantia nigra pars compacta to oxidative stress is considered a key factor of PD pathogenesis. Here we study the effect of different oxidative stress-inducing compounds (6-OHDA, MPTP or MPP+) on the population of dopaminergic neurons in an iPSC-derived human brain 3D model (aka BrainSpheres). Treatment with 6-OHDA, MPTP or MPP+ at 4 weeks of differentiation disrupted the dopaminergic neuronal phenotype in BrainSpheres at (50, 5000, 1000 µM respectively). 6-OHDA increased ROS production and decreased mitochondrial function most efficiently. It further induced the greatest changes in gene expression and metabolites related to oxidative stress and mitochondrial dysfunction. Co-culturing BrainSpheres with an endothelial barrier using a transwell system allowed the assessment of differential penetration capacities of the tested compounds and the damage they caused in the dopaminergic neurons within the BrainSpheres In conclusion, treatment with compounds known to induce PD-like phenotypes in vivo caused molecular deficits and loss of dopaminergic neurons in the BrainSphere model. This approach therefore recapitulates common animal models of neurodegenerative processes in PD at similarly high doses. The relevance as tool for drug discovery is discussed.

Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Second International Conference on 3Rs Research and Progress, Hyderabad, 2021.

The fact that animal models fail to replicate human disease faithfully is now being widely accepted by researchers across the globe. As a result, they are exploring the use of alternatives to animal models. The time has come to refine our experimental practices, reduce the numbers and eventually replace the animals used in research with human-derived and human-relevant 3-D disease models. Oncoseek Bio-Acasta Health, which is an innovative biotechnology start-up company based in Hyderabad and Vishakhapatnam, India, organises an annual International Conference on 3Rs Research and Progress. In 2021, this conference was on 'Advances in Research Animal Models and Cutting-Edge Research in Alternatives'. This annual conference is a platform that brings together eminent scientists and researchers from various parts of the world, to share recent advances from their research in the field of alternatives to animals including new approach methodologies, and to promote practices to help refine animal experiments where alternatives are not available. This report presents the proceedings of the conference, which was held in hybrid mode (i.e. virtual and in-person) in November 2021.

The Future of 3D Brain Cultures in Developmental Neurotoxicity Testing.

Human brain is undoubtedly the most complex organ in the body. Thus, it is difficult to develop adequate and at the same time human relevant test systems and models to cover the aspects of brain homeostasis and even more challenging to address brain development. Animal tests for Developmental Neurotoxicity (DNT) have been devised, but because of complex underlying mechanisms of neural development, and interspecies differences, there are many limitations of animal-based approaches. The high costs, high number of animals used per test and technical difficulties of these tests are prohibitive for routine DNT chemical screening. Therefore, many potential DNT chemicals remain unidentified. New approach methodologies (NAMs) are needed to change this. Experts in the field have recommended the use of a battery of human in vitro tests to be used for the initial prioritization of high-risk environmental chemicals for DNT testing. Microphysiological systems (MPS) of the brain mimic the in vivo counterpart in terms of cellular composition, recapitulation of regional architecture and functionality. These systems amendable to use in a DNT test battery with promising features such as (i) complexity, (ii) closer recapitulation of in vivo response and (iii) possibility to multiplex many assays in one test system, which can increase throughput and predictivity for human health. The resent progress in 3D brain MPS research, advantages, limitations and future perspectives are discussed in this review.

Oligodendrogenesis and myelination tracing in a CRISPR/Cas9-engineered brain microphysiological system.

Introduction: Oligodendrocytes (OLs) are the myelin-forming cells of the central nervous system (CNS). Although OLs can be differentiated from human-induced pluripotent stem cells (hiPSCs), the in vitro modeling of axon myelination in human cells remains challenging. Brain microphysiological systems (bMPS, e.g. organoids) are complex three-dimensional (3D) cultures that offer an ideal system to study this process as OLs differentiate in a more in vivo-like environment; surrounded by neurons and astrocytes, which support the myelination of axons. Methods: Here, we take advantage of CRISPR/Cas9 technology to generate a hiPSC line in which proteolipid protein 1 (PLP1), an OLs marker, is tagged with super-fold GFP (sfGFP). While generating the PLP1-sfGFP reporter, we used reverse transfection and obtained higher Knock-In (KI) efficiency compared to forward transfection (61-72 vs. 46%). Results: After validation of the KI and quality control of the PLP1-sfGFP line, selected clones were differentiated into bMPS, and the fidelity, specificity, and function of the tagged PLP protein were verified in this model. We tracked different stages of oligodendrogenesis in the verified lines based on PLP1-sfGFP+ cells' morphology, and the presence of PLP1-sfGFP surrounding axons during bMPS' differentiation. Finally, we challenged the bMPS with cuprizone and quantified changes in both the percentage of PLP1-sfGFP expressing cells and the intensity of GFP expression. Discussion: This work demonstrates an efficient method for generating hiPSC KI lines and the description of a new 3D model to study OL differentiation, migration, and maturation both during in vitro neurodevelopment as well as in response to environmental chemicals or disease-associated stressors.