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TBX2 is a neuroblastoma core regulatory circuitry component enhancing MYCN/FOXM1 reactivation of DREAM targets.
Chromosome 17q gains are almost invariably present in high-risk neuroblastoma cases. Here, we perform an integrative epigenomics search for dosage-sensitive transcription factors on 17q marked by H3K27ac defined super-enhancers and identify TBX2 as top candidate gene. We show that TBX2 is a constituent of the recently established core regulatory circuitry in neuroblastoma with features of a cell identity transcription factor, driving proliferation through activation of p21-DREAM repressed FOXM1 target genes. Combined MYCN/TBX2 knockdown enforces cell growth arrest suggesting that TBX2 enhances MYCN sustained activation of FOXM1 targets. Targeting transcriptional addiction by combined CDK7 and BET bromodomain inhibition shows synergistic effects on cell viability with strong repressive effects on CRC gene expression and p53 pathway response as well as several genes implicated in transcriptional regulation. In conclusion, we provide insight into the role of the TBX2 CRC gene in transcriptional dependency of neuroblastoma cells warranting clinical trials using BET and CDK7 inhibitors.
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Chick fetal organ spheroids as a model to study development and disease.
BACKGROUND: Organ culture models have been used over the past few decades to study development and disease. The in vitro three-dimensional (3D) culture system of organoids is well known, however, these 3D systems are both costly and difficult to culture and maintain. As such, less expensive, faster and less complex methods to maintain 3D cell culture models would complement the use of organoids. Chick embryos have been used as a model to study human biology for centuries, with many fundamental discoveries as a result. These include cell type induction, cell competence, plasticity and contact inhibition, which indicates the relevance of using chick embryos when studying developmental biology and disease mechanisms. RESULTS: Here, we present an updated protocol that enables time efficient, cost effective and long-term expansion of fetal organ spheroids (FOSs) from chick embryos. Utilizing this protocol, we generated FOSs in an anchorage-independent growth pattern from seven different organs, including brain, lung, heart, liver, stomach, intestine and epidermis. These three-dimensional (3D) structures recapitulate many cellular and structural aspects of their in vivo counterpart organs and serve as a useful developmental model. In addition, we show a functional application of FOSs to analyze cell-cell interaction and cell invasion patterns as observed in cancer. CONCLUSION: The establishment of a broad ranging and highly effective method to generate FOSs from different organs was successful in terms of the formation of healthy, proliferating 3D organ spheroids that exhibited organ-like characteristics. Potential applications of chick FOSs are their use in studies of cell-to-cell contact, cell fusion and tumor invasion under defined conditions. Future studies will reveal whether chick FOSs also can be applicable in scientific areas such as viral infections, drug screening, cancer diagnostics and/or tissue engineering.
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Development of the serotonin release assay with enterochromaffin cell-rich human intestinal organoids for the risk evaluation of drug-induced emesis.
Drug-induced emesis is one of the major symptoms of gastrointestinal toxicity. Preclinical risk assessment of emesis has been challenging owing to the lack of suitable animal models or in vitro assay systems. One of the triggers of emesis is an excessive serotonin (5-HT) release from enterochromaffin (EC) cells in the intestinal tract, which activates the vomiting center in the brain stem and elicits the vomiting reflex. Here, we propose a 5-HT release assay using human jejunal organoids that are preferentially differentiated into EC cells. In this assay, EC cell-rich organoids were stimulated with emetic risk drugs, and 5-HT release into the media was quantified, along with cellular ATP level and LDH leakage. Anaplastic lymphoma kinase (ALK)/ROS1 tyrosine kinase inhibitors (TKIs) have been reported to exhibit different probabilities of emesis in clinical settings. We demonstrated that 5-HT release from EC cell-rich organoids could qualitatively distinguish between drugs with high or low emetic risk. The results indicated that 5-HT release by ALK/ROS1-TKIs tended to occur prior to the onset of LDH leakage from EC cell-rich organoids, suggesting that 5-HT release was not simply a result of cell bursting. Epidermal growth factor receptor TKIs exhibit a lower potency for inducing 5-HT release, which is consistent with their low emetic risk. In summary, the use of EC cell-rich organoids in 5-HT release assays may prove to be a valuable tool in predicting drug-induced emesis in humans during drug development.
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Engineering fibronectin-templated multi-component fibrillar extracellular matrices to modulate tissue-specific cell response.
Cells assemble fibronectin, the major extracellular matrix (ECM) protein, into fibrillar matrices, which serve as 3D architectural scaffolds to provide, together with other ECM proteins tissue-specific environments. Although recent approaches enable to bioengineer 3D fibrillar fibronectin matrices in vitro, it remains elusive how fibronectin can be co-assembled with other ECM proteins into complex 3D fibrillar matrices that recapitulate tissue-specific compositions and cellular responses. Here, we introduce the engineering of fibrillar fibronectin-templated 3D matrices that can be complemented with other ECM proteins, including vitronectin, collagen, and laminin to resemble ECM architectures observed in vivo. For the co-assembly of different ECM proteins, we employed their innate fibrillogenic mechanisms including shear forces, pH-dependent electrostatic interactions, or specific binding domains. Through recapitulating various tissue-specific ECM compositions and morphologies, the large scale multi-composite 3D fibrillar ECM matrices can guide fibroblast adhesion, 3D fibroblast tissue formation, or tissue morphogenesis of epithelial cells. In other examples, we customize multi-composite 3D fibrillar matrices to support the growth of signal propagating neuronal networks and of human brain organoids. We envision that these 3D fibrillar ECM matrices can be tailored in scale and composition to modulate tissue-specific responses across various biological length scales and systems, and thus to advance manyfold studies of cell biological systems.
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Gnao1 is a molecular switch that regulates the Rho signaling pathway in differentiating neurons.
GNAO1 encodes G protein subunit alpha O1 (Gαo). Pathogenic variations in GNAO1 cause developmental delay, intractable seizures, and progressive involuntary movements from early infancy. Because the functional role of GNAO1 in the developing brain remains unclear, therapeutic strategies are still unestablished for patients presenting with GNAO1-associated encephalopathy. We herein report that siRNA-mediated depletion of Gnao1 perturbs the expression of transcripts associated with Rho GTPase signaling in Neuro2a cells. Consistently, siRNA treatment hampered neurite outgrowth and extension. Growth cone formation was markedly disrupted in monolayer neurons differentiated from iPSCs from a patient with a pathogenic variant of Gαo (p.G203R). This variant disabled neuro-spherical assembly, acquisition of the organized structure, and polarized signals of phospho-MLC2 in cortical organoids from the patient's iPSCs. We confirmed that the Rho kinase inhibitor Y27632 restored these morphological phenotypes. Thus, Gαo determines the self-organizing process of the developing brain by regulating the Rho-associated pathway. These data suggest that Rho GTPase pathway might be an alternative target of therapy for patients with GNAO1-associated encephalopathy.
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Assembloid models of cell-cell interaction to study tissue and disease biology.
Neurodevelopment involves the migration, projection, and integration of various cell types across different regions of the nervous system. Assembloids are self-organizing systems formed by the integration of multiple organoids or cell types. Here, we outline the generation and application of assembloids. We illustrate how assembloids recapitulate critical neurodevelopmental steps, like migration, axon projection, and circuit formation, and how they are starting to provide biological insights into neuropsychiatric disorders. Additionally, we review how assembloids can be used to study properties emerging from cell-cell interactions within non-neural tissues. Overall, assembloid platforms represent a powerful tool for discovering human biology and developing therapeutics.
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Nematosomes in external horizontal cells of white perch (Roccus americana) retina: changes during dark and light adaptation.
Cytoplasmic inclusions resembling nucleoli and termed nematosomes have been described in several areas of the nervous system but not in the retina. Here we report the presence of these structures in the external horizontal cells of white perch (Roccus americana). Nematosomes were larger and more numerous in dark-adapted retinas than in light-adapted ones. An inverse relation of horizontal cell spinules was found in these cells, suggesting that electron-dense material observed in spinules may originate from nematosomes.
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Analogous comparison unravels heightened antiviral defense and boosted viral infection upon immunosuppression in bat organoids.
Horseshoe bats host numerous SARS-related coronaviruses without overt disease signs. Bat intestinal organoids, a unique model of bat intestinal epithelium, allow direct comparison with human intestinal organoids. We sought to unravel the cellular mechanism(s) underlying bat tolerance of coronaviruses by comparing the innate immunity in bat and human organoids. We optimized the culture medium, which enabled a consecutive passage of bat intestinal organoids for over one year. Basal expression levels of IFNs and IFN-stimulated genes were higher in bat organoids than in their human counterparts. Notably, bat organoids mounted a more rapid, robust and prolonged antiviral defense than human organoids upon Poly(I:C) stimulation. TLR3 and RLR might be the conserved pathways mediating antiviral response in bat and human intestinal organoids. The susceptibility of bat organoids to a bat coronavirus CoV-HKU4, but resistance to EV-71, an enterovirus of exclusive human origin, indicated that bat organoids adequately recapitulated the authentic susceptibility of bats to certain viruses. Importantly, TLR3/RLR inhibition in bat organoids significantly boosted viral growth in the early phase after SARS-CoV-2 or CoV-HKU4 infection. Collectively, the higher basal expression of antiviral genes, especially more rapid and robust induction of innate immune response, empowered bat cells to curtail virus propagation in the early phase of infection.
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Construction of Multicellular Neural Tissue Using Three-Dimensional Printing Technology: Cell Interaction.
The study of the human nervous system remains challenging due to its inherent complexity and difficulty in obtaining original samples. Three-dimensional (3D) bioprinting is a rapidly evolving technology in the field of tissue engineering that has made significant contributions to several disciplines, including neuroscience. In order to more accurately reflect the intricate multicellular milieu of the in vivo environment, an increasing number of studies have commenced experimentation with the coprinting of diverse cell types. This article provides an overview of technical details and the application of 3D bioprinting with multiple cell types in the field of neuroscience, focusing on the challenges of coprinting and the research conducted based on multicellular printing. This review discusses cell interactions in coprinting systems, stem cell applications, the construction of brain-like organoids, the establishment of disease models, and the potential for integrating 3D bioprinting with other 3D culture techniques.
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Influence of the centrosome on the structure of nucleated microtubules.
The capacity of the centrosome to influence the lattice structure of nucleated microtubules was studied in vitro. Brain microtubules self-assembled to give predominantly (98%) 14-protofilament microtubules. However, under exactly the same conditions of assembly they grew off of purified centrosomes from neuroblastoma cells to give mostly (82%) 13-protofilament microtubules. Thus, the nucleation sites on the centrosome constrained the microtubule lattice to yield the number of protofilaments usually found in vivo.