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Structural Analysis of Cerebral Organoids Using Confocal Microscopy and Transmission/Scanning Electron Microscopy.
Cerebral organoid cultures from human-induced pluripotent stem cells are widely used to study complex human brain development; however, there is still limited ultrastructural information regarding the development. In this study, we examined the structural details of cerebral organoids using various microscopy techniques. Two protocols were chosen as representative methods for the development of brain organoids: the classic whole-cerebral organoid (Whole-CO) culture technique, and the air-liquid interface-cerebral organoid (ALI-CO) culture technique. Immunostained confocal laser scanning microscopy (CLSM) revealed the formation of the CTIP2- and TBR1-positive cortical deep layer on days 90 and 150, depending on the developmental progress of both methods. Furthermore, the presence of astrocytes and oligodendrocytes was verified through immunostained CLSM utilizing two-dimensional and three-dimensional reconstruction images after a 150-day period. Transmission electron microscopy analysis revealed nanometer-resolution details of the cellular organelles and neuron-specific structures including synapses and myelin. Large-area scanning electron microscopy confirmed the well-developed neuronal connectivity from each culture method on day 150. Using those microscopy techniques, we clearly showed significant details within two representative culture protocols, the Whole-CO and ALI-CO culture methods. These multi-level images provide ultrastructural insight into the features of cerebral organoids depending on the developmental stage.
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Microglia modulate neurodevelopment in human neuroimmune organoids.
Dissecting contributions of microglia to human brain development and disease pathogenesis requires modeling interactions between these microglia and their local environment. In this issue of Cell Stem Cell, Popova et al. (2021) propose a transcriptomic "microglia report card" and create a neuroimmune organoid to model complex interactions involving human microglia.
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Advanced human developmental toxicity and teratogenicity assessment using human organoid models.
Tremendous progress has been made in the field of toxicology leading to the advance of developmental toxicity assessment. Conventional animal models and in vitro two-dimensional models cannot accurately describe toxic effects and predict actual in vivo responses due to obvious inter-species differences between humans and animals, as well as the lack of a physiologically relevant tissue microenvironment. Human embryonic stem cell (hESC)- and induced pluripotent stem cell (iPSC)-derived three-dimensional organoids are ideal complex and multicellular organotypic models, which are indispensable in recapitulating morphogenesis, cellular interactions, and molecular processes of early human organ development. Recently, human organoids have been used for drug discovery, chemical toxicity and safety in vitro assessment. This review discusses the recent advances in the use of human organoid models, (i.e., brain, retinal, cardiac, liver, kidney, lung, and intestinal organoid models) for developmental toxicity and teratogenicity assessment of distinct tissues/organs following exposure to pharmaceutical compounds, heavy metals, persistent organic pollutants, nanomaterials, and ambient air pollutants. Combining next-generation organoid models with innovative engineering technologies generates novel and powerful tools for developmental toxicity and teratogenicity assessment, and the rapid progress in this field is expected to continue.
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Functional recovery through the plastic adaptation of organoid grafts in cortical tissue.
The lack of effective therapeutic options for patients suffering from neurological impairments related to acquired brain damage requires novel translational strategies, among which transplantation of neural tissue is receiving strong attention. One of the most recent developments involves the implantation of brain organoid models, derived from embryonic or induced pluripotent stem cells, into damaged rodent cortices. While this approach is gaining popularity, the extent of graft integration within the host tissue remains poorly understood. This review aims to examine whether xenotransplanting organoids into cortical tissue induces functional recovery and plastic adaptation to the damaged implantation sites. Physiological indications of grafted organoid plasticity and integration into the host included viability, corticogenesis, vascularisation, growth, and the development of area-specific morphological identities. The functional integration into host neural circuitry has been probed by tracing of axonal projection growth according to implantation sites, but also through observations of spontaneous, stimulus evoked, and selectively tuned activity of grafted neurons. Finally, some studies also investigated whether the engraftment procedure facilitated behavioural recovery in tasks requiring motor, memory, or reward-seeking functions. Overall, organoid grafts show signs of progressive anatomical, functional, and behaviourally-relevant integration within the damaged host cortices. Yet, further investigation is necessary before this transplantation approach can be actually translated into a robust method to achieve functional restoration in patients suffering from brain damage.
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A fully automated high-throughput workflow for 3D-based chemical screening in human midbrain organoids.
Three-dimensional (3D) culture systems have fueled hopes to bring about the next generation of more physiologically relevant high-throughput screens (HTS). However, current protocols yield either complex but highly heterogeneous aggregates ('organoids') or 3D structures with less physiological relevance ('spheroids'). Here, we present a scalable, HTS-compatible workflow for the automated generation, maintenance, and optical analysis of human midbrain organoids in standard 96-well-plates. The resulting organoids possess a highly homogeneous morphology, size, global gene expression, cellular composition, and structure. They present significant features of the human midbrain and display spontaneous aggregate-wide synchronized neural activity. By automating the entire workflow from generation to analysis, we enhance the intra- and inter-batch reproducibility as demonstrated via RNA sequencing and quantitative whole mount high-content imaging. This allows assessing drug effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow.
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Transcriptomic Mapping of Neural Diversity, Differentiation and Functional Trajectory in iPSC-Derived 3D Brain Organoid Models.
Experimental models of the central nervous system (CNS) are imperative for developmental and pathophysiological studies of neurological diseases. Among these models, three-dimensional (3D) induced pluripotent stem cell (iPSC)-derived brain organoid models have been successful in mitigating some of the drawbacks of 2D models; however, they are plagued by high organoid-to-organoid variability, making it difficult to compare specific gene regulatory pathways across 3D organoids with those of the native brain. Single-cell RNA sequencing (scRNA-seq) transcriptome datasets have recently emerged as powerful tools to perform integrative analyses and compare variability across organoids. However, transcriptome studies focusing on late-stage neural functionality development have been underexplored. Here, we combine and analyze 8 brain organoid transcriptome databases to study the correlation between differentiation protocols and their resulting cellular functionality across various 3D organoid and exogenous brain models. We utilize dimensionality reduction methods including principal component analysis (PCA) and uniform manifold approximation projection (UMAP) to identify and visualize cellular diversity among 3D models and subsequently use gene set enrichment analysis (GSEA) and developmental trajectory inference to quantify neuronal behaviors such as axon guidance, synapse transmission and action potential. We showed high similarity in cellular composition, cellular differentiation pathways and expression of functional genes in human brain organoids during induction and differentiation phases, i.e., up to 3 months in culture. However, during the maturation phase, i.e., 6-month timepoint, we observed significant developmental deficits and depletion of neuronal and astrocytes functional genes as indicated by our GSEA results. Our results caution against use of organoids to model pathophysiology and drug response at this advanced time point and provide insights to tune in vitro iPSC differentiation protocols to achieve desired neuronal functionality and improve current protocols.
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Trans-omic profiling uncovers molecular controls of early human cerebral organoid formation.
Defining the molecular networks orchestrating human brain formation is crucial for understanding neurodevelopment and neurological disorders. Challenges in acquiring early brain tissue have incentivized the use of three-dimensional human pluripotent stem cell (hPSC)-derived neural organoids to recapitulate neurodevelopment. To elucidate the molecular programs that drive this highly dynamic process, here, we generate a comprehensive trans-omic map of the phosphoproteome, proteome, and transcriptome of the exit of pluripotency and neural differentiation toward human cerebral organoids (hCOs). These data reveal key phospho-signaling events and their convergence on transcriptional factors to regulate hCO formation. Comparative analysis with developing human and mouse embryos demonstrates the fidelity of our hCOs in modeling embryonic brain development. Finally, we demonstrate that biochemical modulation of AKT signaling can control hCO differentiation. Together, our data provide a comprehensive resource to study molecular controls in human embryonic brain development and provide a guide for the future development of hCO differentiation protocols.
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[Current progress in brain organoid research for human biology].
Understanding human brain development, function, disease, and drug responses remains a central challenge in neuroscience and medicine. However, detailed analyses of living human brain tissue are technically and ethically difficult, and conventional studies have therefore relied largely on animal models and two-dimensional culture systems. Nevertheless, substantial differences exist between humans and animals in developmental processes, cellular composition, neural circuit, and pharmacological responses. Brain organoids are three-dimensional neural tissues generated from human embryonic stem cells or induced pluripotent stem cells, and they can recapitulate aspects of neural progenitor differentiation, cellular diversity, tissue architecture, and neuronal activity. Owing to these properties, brain organoids provide a valuable platform for human biology research. In this article, we review the development of brain organoid research and discuss recent advances including neuronal activity measurements, neural circuit models using assembloids and connectoids, reconstruction of human-enriched cell types and gene programs, analyses of inter-individual variability, toxicological studies, disease modeling, and drug evaluation. Furthermore, we outline the current status and future perspectives of human biology research using brain organoids, with particular attention to challenges such as heterogeneity, limited maturation, the absence of vascular and immune systems, standardization, quality control, and ethical considerations.
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Investigating receptor-mediated antibody transcytosis using blood-brain barrier organoid arrays.
BACKGROUND: The pathways that control protein transport across the blood-brain barrier (BBB) remain poorly characterized. Despite great advances in recapitulating the human BBB in vitro, current models are not suitable for systematic analysis of the molecular mechanisms of antibody transport. The gaps in our mechanistic understanding of antibody transcytosis hinder new therapeutic delivery strategy development. METHODS: We applied a novel bioengineering approach to generate human BBB organoids by the self-assembly of astrocytes, pericytes and brain endothelial cells with unprecedented throughput and reproducibility using micro patterned hydrogels. We designed a semi-automated and scalable imaging assay to measure receptor-mediated transcytosis of antibodies. Finally, we developed a workflow to use CRISPR/Cas9 gene editing in BBB organoid arrays to knock out regulators of endocytosis specifically in brain endothelial cells in order to dissect the molecular mechanisms of receptor-mediated transcytosis. RESULTS: BBB organoid arrays allowed the simultaneous growth of more than 3000 homogenous organoids per individual experiment in a highly reproducible manner. BBB organoid arrays showed low permeability to macromolecules and prevented transport of human non-targeting antibodies. In contrast, a monovalent antibody targeting the human transferrin receptor underwent dose- and time-dependent transcytosis in organoids. Using CRISPR/Cas9 gene editing in BBB organoid arrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis. CONCLUSIONS: Human BBB organoid arrays are a robust high-throughput platform that can be used to discover new mechanisms of receptor-mediated antibody transcytosis. The implementation of this platform during early stages of drug discovery can accelerate the development of new brain delivery technologies.
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Patients' perspectives on the derivation and use of organoids.
Organoid research is enhancing understanding of human development and diseases as well as aiding in medication development and selection, raising hopes for even more future therapeutic options. Nevertheless, this work raises important ethical issues and there is a paucity of data regarding patients' perspectives on them. We report on 60 interviews with adult patients or parents of pediatric patients from diverse disease populations who receive medical care at a major academic research institution in the United States. Interviewees expressed broad support for organoid development and use. However, patients viewed brain organoids, and sometimes gonadal organoids, as morally distinct; and some organoid research poses moral concerns. Nonetheless, patients generally understood the potential value of such research and approved of it, provided it was aimed at good intent and conducted with ethical oversight and a robust consent process. These data should help inform conceptual and policy deliberations about appropriate organoid use.