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Brain exposure to SARS-CoV-2 virions perturbs synaptic homeostasis.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with short- and long-term neurological complications. The variety of symptoms makes it difficult to unravel molecular mechanisms underlying neurological sequalae after coronavirus disease 2019 (COVID-19). Here we show that SARS-CoV-2 triggers the up-regulation of synaptic components and perturbs local electrical field potential. Using cerebral organoids, organotypic culture of human brain explants from individuals without COVID-19 and post-mortem brain samples from individuals with COVID-19, we find that neural cells are permissive to SARS-CoV-2 to a low extent. SARS-CoV-2 induces aberrant presynaptic morphology and increases expression of the synaptic components Bassoon, latrophilin-3 (LPHN3) and fibronectin leucine-rich transmembrane protein-3 (FLRT3). Furthermore, we find that LPHN3-agonist treatment with Stachel partially restored organoid electrical activity and reverted SARS-CoV-2-induced aberrant presynaptic morphology. Finally, we observe accumulation of relatively static virions at LPHN3-FLRT3 synapses, suggesting that local hindrance can contribute to synaptic perturbations. Together, our study provides molecular insights into SARS-CoV-2-brain interactions, which may contribute to COVID-19-related neurological disorders.
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Low-cost and scalable projected light-sheet microscopy for the high-resolution imaging of cleared tissue and living samples.
Light-sheet fluorescence microscopy (LSFM) is a widely used technique for imaging cleared tissue and living samples. However, high-performance LSFM systems are typically expensive and not easily scalable. Here we introduce a low-cost, scalable and versatile LSFM framework, which we named 'projected light-sheet microscopy' (pLSM), with high imaging performance and small device and computational footprints. We characterized the capabilities of pLSM, which repurposes readily available consumer-grade components, optimized optics, over-network control architecture and software-driven light-sheet modulation, by performing high-resolution mapping of cleared mouse brains and of post-mortem pathological human brain samples, and via the molecular phenotyping of brain and blood-vessel organoids derived from human induced pluripotent stem cells. We also report a method that leverages pLSM for the live imaging of the dynamics of sparsely labelled multi-layered bacterial pellicle biofilms at an air-liquid interface. pLSM can make high-resolution LSFM for biomedical applications more accessible, affordable and scalable.
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Temporal uncoupling of radial glia lineage progression in cortical organoids.
Radial glial progenitors (RGPs) produce all excitatory neurons in the developing cerebral cortex. Mosaic analysis with double markers (MADM)-based lineage tracing in vivo has revealed a quantitative framework of RGP lineage progression1. Here we established MADM technology2,3 in mouse embryonic stem cells to probe RGP lineage progression in a self-organizing cortical organoid system. We found that RGPs exhibit a high level of plasticity in proliferative potential in organoids rather than strict temporally stereotyped lineage progression as observed in vivo. RGPs in organoids showed increased lineage restriction, diminishing cell-type diversity in clones of cortical projection neurons, despite uniform single-cell transcriptional signatures of RGPs and a unitary lineage trajectory. Thus, critical non-cell-autonomous cues that are absent in self-organizing systems and/or the genuine stem cell niche are essential for faithful temporal control of RGP lineage progression and the generation of clonal cortical cell-type diversity.
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Generation of Functional GnRH-Responsive Pituitary-like Organoids from Ovine Embryonic Stem Cells.
The pituitary gland is a master regulator of endocrine and reproductive physiology; however, the lack of physiologically relevant in vitro models in livestock species has limited studies of pituitary development and endocrine function. Although pituitary organoids have been generated from human and mouse pluripotent stem cells, comparable models remain unavailable in large animal species. Here, we established functional pituitary-like organoids from ovine embryonic stem cells (oESCs) using a three-dimensional differentiation strategy. The resulting organoids self-organized into Rathke's pouch-like structures and acquired pituitary progenitor characteristics, as demonstrated by the robust expression of LHX3, PITX1, ISLET1 and PROP1. Compared with conventional two-dimensional differentiation, the organoids displayed enhanced structural organization and improved lineage specification. Notably, the organoids exhibited functional gonadotroph maturation, with GnRH treatment significantly upregulating FSHB and LHB expression, as confirmed by immunohistochemistry, qPCR and Western blot. SMART-Seq transcriptome analysis revealed progressive loss of pluripotency-associated signatures and activation of pituitary developmental programs, including sustained expression of lineage-associated regulators such as PAX6. Collectively, this study establishes the first ovine pituitary-like organoid model and demonstrates its capacity to generate functional GnRH-responsive gonadotroph cells. This platform provides a valuable resource for investigating pituitary development, reproductive endocrinology, livestock fertility, and endocrine disease mechanisms in a large-animal context.
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Human cerebral organoids as a therapeutic drug screening model for Creutzfeldt-Jakob disease.
Creutzfeldt-Jakob Disease (CJD) is a fatal, currently incurable, neurodegenerative disease. The search for candidate treatments would be greatly facilitated by the availability of human cell-based models of prion disease. Recently, an induced pluripotent stem cell derived human cerebral organoid model was shown to take up and propagate human CJD prions. This model offers new opportunities to screen drug candidates for the treatment of human prion diseases in an entirely human genetic background. Here we provide the first evidence that human cerebral organoids can be a viable model for CJD drug screening by using an established anti-prion compound, pentosan polysulfate (PPS). PPS delayed prion propagation in a prophylactic-like treatment paradigm and also alleviated propagation when applied following establishment of infection in a therapeutic-like treatment paradigm. This study demonstrates the utility of cerebral organoids as the first human 3D cell culture system for screening therapeutic drug candidates for human prion diseases.
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The elongation of Mest transcript into MestXL sustains, but does not initiate, the maternal allele bias of its convergent gene Copg2 during neurogenesis.
Precise gene dosage control is critical for cellular identity and development, particularly for imprinted genes, where dosage imbalances are linked to neurodevelopmental disorders. The Mest/Copg2 imprinted locus exemplifies this fine-tuned regulation: while Mest is constitutively paternally expressed, Copg2 shifts from biallelic to maternally-biased expression specifically during neural differentiation, a transition proposed to involve transcriptional interference by the long Mest isoform, MestXL, which extends into Copg2. However, the mechanisms underlying this switch and whether factors beyond MestXL contribute remain unresolved. Using a stem cell-based brain organoid model integrating multi-omic analyses, 3D chromatin mapping, and functional approaches, we challenge the prevailing model by showing that the maternal bias of Copg2 is not solely driven by MestXL-mediated interference. Instead, our data reveal a temporal, stage-specific two-step mechanism: putative enhancer-driven activation of the maternal allele in neural progenitors, followed by MestXL-dependent repression of the paternal allele in neuron-enriched stages. This uncovers an unexpected complexity in imprinted gene dosage regulation during brain development.
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Dynamic behaviour of human neuroepithelial cells in the developing forebrain.
To understand how diverse progenitor cells contribute to human neocortex development, we examined forebrain progenitor behaviour using timelapse imaging. Here we find that cell cycle dynamics of human neuroepithelial (NE) cells differ from radial glial (RG) cells in both primary tissue and in stem cell-derived organoids. NE cells undergoing proliferative, symmetric divisions retract their basal processes, and both daughter cells regrow a new process following cytokinesis. The mitotic retraction of the basal process is recapitulated by NE cells in cerebral organoids generated from human-induced pluripotent stem cells. In contrast, RG cells undergoing vertical cleavage retain their basal fibres throughout mitosis, both in primary tissue and in older organoids. Our findings highlight developmentally regulated changes in mitotic behaviour that may relate to the role of RG cells to provide a stable scaffold for neuronal migration, and suggest that the transition in mitotic dynamics can be studied in organoid models.
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Hypothalamic Contribution to Pituitary Functions Is Recapitulated In Vitro Using 3D-Cultured Human iPS Cells.
The pituitary is a major hormone center that secretes systemic hormones responding to hypothalamus-derived-releasing hormones. Previously, we reported the independent pituitary induction and hypothalamic differentiation of human embryonic stem cells (ESCs). Here, a functional hypothalamic-pituitary unit is generated using human induced pluripotent stem (iPS) cells in vitro. The adrenocorticotropic hormone (ACTH) secretion capacity of the induced pituitary reached a comparable level to that of adult mouse pituitary because of the simultaneous maturation with hypothalamic neurons within the same aggregates. Corticotropin-releasing hormone (CRH) from the hypothalamic area regulates ACTH cells similarly to our hypothalamic-pituitary axis. Our induced hypothalamic-pituitary units respond to environmental hypoglycemic condition in vitro, which mimics a life-threatening situation in vivo, through the CRH-ACTH pathway, and succeed in increasing ACTH secretion. Thus, we generated powerful hybrid organoids by recapitulating hypothalamic-pituitary development, showing autonomous maturation on the basis of interactions between developing tissues.
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Cortical organoids reveal human-specific role of METTL5 in neurodevelopment via regulation of CHCHD2.
Methyltransferase-like 5 (METTL5) catalyzes N6-methyladenosine (m6A) modification on 18S rRNA. In humans, loss-of-function mutations in METTL5 cause severe microcephaly and intellectual disability, whereas Mettl5 knockout (KO) animal models display inconsistent and milder phenotypes. To better model human disease, we generated METTL5-KO human-induced pluripotent stem cell (hiPSC)-derived cortical organoids, which exhibit impaired neural progenitor cell (NPC) proliferation and differentiation, leading to reduced ventricle-like structures and significant reductions in cortical organoid diameter. Mechanistically, Ribo-seq analysis revealed broad translational changes in METTL5-KO NPCs consistent with cellular stress responses rather than transcript-specific translational changes. Single-cell RNA-seq identified downregulation of coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2), a mitochondrial regulator of oxidative metabolism. Overexpression of CHCHD2 in METTL5-KO NPCs rescued proliferation and partially rescued oxidative metabolism in NPCs and ventricle formation in organoids. This highlights a previously uncharacterized connection between CHCHD2, oxidative metabolism, and METTL5-mediated regulation of human neurogenesis.
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CytoCensus, mapping cell identity and division in tissues and organs using machine learning.
A major challenge in cell and developmental biology is the automated identification and quantitation of cells in complex multilayered tissues. We developed CytoCensus: an easily deployed implementation of supervised machine learning that extends convenient 2D 'point-and-click' user training to 3D detection of cells in challenging datasets with ill-defined cell boundaries. In tests on such datasets, CytoCensus outperforms other freely available image analysis software in accuracy and speed of cell detection. We used CytoCensus to count stem cells and their progeny, and to quantify individual cell divisions from time-lapse movies of explanted Drosophila larval brains, comparing wild-type and mutant phenotypes. We further illustrate the general utility and future potential of CytoCensus by analysing the 3D organisation of multiple cell classes in Zebrafish retinal organoids and cell distributions in mouse embryos. CytoCensus opens the possibility of straightforward and robust automated analysis of developmental phenotypes in complex tissues.