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Computational fluid dynamic analysis of physical forces playing a role in brain organoid cultures in two different multiplex platforms.
BACKGROUND: Organoid cultivation in suspension culture requires agitation at low shear stress to allow for nutrient diffusion, which preserves tissue structure. Multiplex systems for organoid cultivation have been proposed, but whether they meet similar shear stress parameters as the regularly used spinner flask and its correlation with the successful generation of brain organoids has not been determined. RESULTS: Here we used computational fluid dynamics (CFD) to simulate two multiplex culture conditions: steering plates on an orbital shaker and the use of a previously described bioreactor. The bioreactor had low speed and high shear stress regions that may affect cell aggregate growth, depending on volume, whereas the computed variables of the steering plates were closer to those of the spinning flask. CONCLUSION: Our protocol improves the initial steps of the standard brain organoid formation, and the produced organoids displayed regionalized brain structures, including retinal pigmented cells. Overall, we conclude that suspension culture on orbital steering plates is a cost-effective practical alternative to previously described platforms for the cultivation of brain organoids for research and multiplex testing.
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Neural Responses to Hypoxic Injury in a Vascularized Cerebral Organoid Model.
Hypoxic injury (HI) in the prenatal period often causes neonatal neurological disabilities. Due to the difficulty in obtaining clinical samples, the molecular and cellular mechanisms remain unclear. Here we use vascularized cerebral organoids to investigate the hypoxic injury phenotype and explore the intercellular interactions between vascular and neural tissues under hypoxic conditions. Our results indicate that fused vascularized cerebral organoids exhibit broader hypoxic responses and larger decreases in panels of neural development-related genes when exposed to low oxygen levels compared to single cerebral organoids. Interestingly, vessels also exhibit neural protective effects on T-box brain protein 2+ intermediate progenitors (IPs), which are markedly lost in HI cerebral organoids. Furthermore, we identify the role of bone morphogenic protein signaling in protecting IPs. Thus, this study has established an in vitro organoid system that can be used to study the contribution of vessels to brain injury under hypoxic conditions and provides a strategy for the identification of intervention targets.
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The epidermal growth factor receptor variant type III mutation frequently found in gliomas induces astrogenesis in human cerebral organoids.
OBJECTIVES: The epidermal growth factor receptor variant type III (EGFRvIII) is the most common mutation of EGFR in glioblastoma multiforme (GBM) and is found in approximately 25% of all GBMs. Intriguingly, EGFRvIII is mostly found in GFAP+ astrocytic tumour cells in the brain, suggesting connection of EGFRvIII to astrogenesis. In this study, we explored whether EGFRvIII mutation facilitates astrogenesis in human development setting. MATERIALS AND METHODS: Using CRISPR-Cas9, we generated EGFRvIII mutations in H9-hESCs. Wild type (wt) H9-hESCs were used as an isogenic control. Next, we generated cerebral organoids using the wt and EGFRvIII-hESCs and examined the astrogenic differentiation of the brain organoids. RESULTS: EGFRvIII-organoids showed abundant astrocytes (GFAP+ , S100β+ ), while no astrocytes were detected in wt hESC-derived organoids at day 49. On the contrary, TUJ1+ neurons were more abundant in the wt-organoids than the EGFRvIII-organoids. This result suggested that constitutively active EGFRvIII promoted astrogenesis at the expense of neurogenesis. In addition, the EGFRvIII-organoids were larger in size and retained more Ki67+ cells than wt-organoids, indicating enhanced cell proliferation by the mutation. The EGFRvIII-organoids displayed massive apoptotic cell death after treatment with temozolomide and hence, could be used for evaluation of anti-GBM drugs. CONCLUSIONS: EGFRvIII mutation-induced astrogenesis and massive cell proliferation in a human brain development model. These results provide us new insights into the mechanisms relating EGFRvIII mutation-mediated gliogenesis and gliomagenesis.
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The Application of the Tissue Microarray (TMA) Technology to Analyze Cerebral Organoids.
"Multi-Omics" technologies have contributed greatly to the understanding of various diseases by enabling researchers to accurately and rapidly investigate the molecular circuitry that connects cellular systems. The tissue-engineered, three-dimensional (3D), in vitro disease model "organoid" integrates the "omics" results in a model system, elucidating the complex links between genotype and phenotype. These 3D structures have been used to model cancer, infectious disease, toxicity, and neurological disorders. Here, we describe the advantage of using the tissue microarray (TMA) technology to analyze human-induced pluripotent stem cell-derived cerebral organoids. Compared with the conventional processing of individual samples, sectioning and staining of TMA slides are faster and can be automated, decreasing labor and reagent costs. The TMA technology faithfully captures cell morphology variations and detects specific biomarkers. The use of this technology can scale up organoid research results in at least two ways: (1) in the number of specimens that can be analyzed simultaneously and (2) in the number of consecutive sections that can be produced for analysis with different probes and antibodies.
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Complementing Western Bioethics: Nishida Kitarō's Relational Ontology and the Future of Brain Organoid Research.
This paper explores the complementarity of Nishida Kitarō's relational ontology with Western bioethics in addressing novel moral dilemmas arising from advanced neural technologies, including brain organoids, human-animal chimeras, and biocomputational systems. Nishida's philosophy transcends the subject-object dichotomy by emphasizing the interconnectedness and co-creative nature of all beings, thereby reframing traditional questions of moral status, personhood, and consent. We examine recent scientific advances and policy debates to demonstrate that Nishida's approach not only enriches ethical understanding but also provides more nuanced guidance that can account for both the technological dynamism and cultural diversity of contemporary research. We argue that this relational, context-dependent ethics, grounded in humility and continuous reflection, fosters an inclusive attitude toward emerging forms of life. Nishida's framework coexists harmoniously with Western philosophy and deepens our grasp of the relational networks that interconnect humans, animals, and even machine- or organoid-based intelligence. We propose an agile, forward-looking model of ethics to respond to the transformative developments of neural innovation.
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Putting Two Heads Together to Build a Better Brain.
3D organoids enable in vitro human brain development models, but they have not yet recapitulated some essential features of brain circuit formation. Recently, several studies appearing in Nature, Nature Methods, and Cell Stem Cell generated fused organoid models of inhibitory and excitatory neuron development, which can now achieve functional circuit integration.
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A Label-Free Ultraviolet Photoacoustic Microscopy Enables Nuclear Imaging and Toxicity Assessment in an Intact Brain Organoid.
Fetal alcohol spectrum disorders (FASDs) are caused by prenatal exposure to ethanol (EtOH), leading to developmental brain abnormalities. Cortical organoids derived from human-induced pluripotent stem cells provide physiologically relevant models to study such neurotoxic effects. However, accurate imaging of nuclear morphology, a key indicator of cytotoxicity and developmental impairment, remains difficult. Traditional fluorescence-based techniques rely on staining and sectioning, limiting throughput and potentially altering native structures. Herein, we present an ultraviolet photoacoustic microscopy system that targets endogenous nucleic acids at 266 nm excitation, resulting in a lateral resolution of 278 nm, which is sufficient to resolve individual nuclei in situ. The system integrates precise z-axis scanning for focal-plane alignment, enabling high-resolution depth-resolved imaging of subvolumes within 3D intact live organoids without physical sectioning. Using EtOH-treated cortical organoids as a model of FASD-associated neurotoxicity, we observed significant reductions in nuclear area, diameter, and circularity by 46.1%, 20.8%, and 6.0%, respectively, indicating structural damage consistent with apoptosis and impaired neurodevelopment. This method is the first to demonstrate label-free nuclear imaging in intact live brain organoids, providing a robust and preparation-free platform for probing disease-relevant phenotypes, accelerating drug screening, and enabling early toxicity assessments in neurodevelopmental disorder models.
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Shaping early neural development by timed elevated tissue oxygen tension: Insights from multiomic analysis on human cerebral organoids.
Oxygen plays a critical role in early neural development in brains, particularly before establishment of complete vasculature; however, it has seldom been investigated due to technical limitations. This study uses an in vitro human cerebral organoid model with multiomic analysis, integrating advanced microscopies and single-cell RNA sequencing, to monitor tissue oxygen tension during neural development. Results reveal a key period between weeks 4 and 6 with elevated intra-organoid oxygen tension, altered energy homeostasis, and rapid neurogenesis within the organoids. The timed oxygen tension elevation can be suppressed by hypoxia treatment or silencing of neuroglobin gene. This study provides insights into the role of oxygen in early neurogenesis from functional, genotypic, phenotypic, and proteomic aspects. These findings highlight the significance of the timed tissue oxygen tension elevation in neurogenesis and provide insights into the role of neuroglobin in neural development, with potential implications for understanding neurodegenerative diseases and therapeutic strategies.
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FACS-Mediated Isolation of Neuronal Cell Populations From Virus-Infected Human Embryonic Stem Cell-Derived Cerebral Organoid Cultures.
Organoids-or pluripotent stem cell-derived in vitro-grown simplified mini organs-have become a tremendously important model to study human organ development and disease. To restrict the noise inherent to the heterogeneous cell mixtures derived from organoid cultures, we developed a new technique of fluorescence-assisted cell sorting (FACS) of virus-infected cerebral organoid cultures. This method still includes the advantage of growing cells in a more natural environment than traditional cell culture, but now renders samples suitable for downstream cell type-specific multi-omics analyses. The protocol starts from stem cell-derived mature brain organoids and includes steps for: preparing the culture for viral infection, production of the viral stocks, FACS sample preparation, and gating and sorting implementation. The protocol has been developed for Zika virus infection, but can be extrapolated to other viruses or fluorescent marker expression as illustrated in an alternate protocol using a single-cycle lentivirus expressing a fluorescent reporter protein. © 2018 by John Wiley & Sons, Inc.
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A Hybrid 2D-to-3D in vitro Differentiation Platform Improves Outcomes of Cerebral Cortical Organoid Generation in hiPSCs.
Three-dimensional (3D) cerebral cortical organoids are popular in vitro cellular model systems widely used to study human brain development and disease, compared to traditional stem cell-derived methods that use two-dimensional (2D) monolayer cultures. Despite the advancements made in protocol development for cerebral cortical organoid derivation over the past decade, limitations due to biological, mechanistic, and technical variables remain in generating these complex 3D cellular systems. Building from our previously established differentiation system, we have made modifications to our existing 3D cerebral cortical organoid protocol that resolve several of these technical and biological challenges when working with diverse groups of human induced pluripotent stem cell (hiPSC) lines. This improved protocol blends a 2D monolayer culture format for the specification of neural stem cells and expansion of neuroepithelial progenitor cells with a 3D system for improved self-aggregation and subsequent organoid development. Furthermore, this "hybrid" approach is amenable to both an accelerated cerebral cortical organoid protocol as well as an alternative long-term differentiation protocol. In addition to establishing a hybrid technical format, this protocol also offers phenotypic and morphological characterization of stage-specific cellular profiles using antibodies and fluorescent-based dyes for live cell imaging. © 2024 Wiley Periodicals LLC. Basic Protocol 1: hiPSC-based 2D monolayer specification into neural stem cells (NSCs) Basic Protocol 2: Serial passaging and 2D monolayer expansion of neuroepithelial progenitor cells (NPCs) Support Protocol 1: Direct cryopreservation and rapid thawing of NSCs and NPCs Basic Protocol 3: Bulk aggregation of 3D neurospheres and accelerated cerebral cortical organoid differentiation Alternate Protocol 1: Bulk aggregation of 3D neurospheres and long-term cerebral cortical organoid differentiation Support Protocol 2: High-throughput 3D neurosphere formation and 2D neurosphere migration assay Support Protocol 3: LIVE/DEAD stain cell imaging assay of 3D neurospheres Support Protocol 4: NeuroFluor NeuO live cell dye for 3D cerebral cortical organoids.