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A neuroanatomical analysis of spinal cord injury in the rhesus monkey (Macaca mulatta).
Thirteen rhesus monkeys were subjected to impacts of either 200, 300, 400, oe 500 g-cm, on the dorsal surface of the spinal cord (Allen 1911). Six monkeys served as controls to determine the results of transection of the cord as well as the optimal survival time, and 2 additional subjects underwent the operative procedures only. Most of the animals were sacrificed 1 week postoperatively and the brains and spinal cords were processed by the Fink and Heimer (1967) technique for degenerating axons. Two contrils and 1 experimental subject, sacrificed at 1 week, were prepared for electron-microscopic analysis...
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Neuronal excitatory-to-inhibitory balance is altered in cerebral organoid models of genetic neurological diseases.
The neuro-physiological properties of individuals with genetic pre-disposition to neurological disorders are largely unknown. Here we aimed to explore these properties using cerebral organoids (COs) derived from fibroblasts of individuals with confirmed genetic mutations including PRNPE200K, trisomy 21 (T21), and LRRK2G2019S, which are associated with Creutzfeldt Jakob disease, Down Syndrome, and Parkinson's disease. We utilized no known disease/healthy COs (HC) as normal function controls. At 3-4 and 6-10 months post-differentiation, COs with mutations showed no evidence of disease-related pathology. Electrophysiology assessment showed that all COs exhibited mature neuronal firing at 6-10 months old. At this age, we observed significant changes in the electrophysiology of the COs with disease-associated mutations (dCOs) as compared with the HC, including reduced neuronal network communication, slowing neuronal oscillations, and increased coupling of delta and theta phases to the amplitudes of gamma oscillations. Such changes were linked with the detection of hypersynchronous events like spike-and-wave discharges. These dysfunctions were associated with altered production and release of neurotransmitters, compromised activity of excitatory ionotropic receptors including receptors of kainate, AMPA, and NMDA, and changed levels and function of excitatory glutamatergic synapses and inhibitory GABAergic synapses. Neuronal properties that modulate GABAergic inhibition including the activity of Na-K-Cl cotransport 1 (NKCC1) in Cl- homeostasis and the levels of synaptic and extra-synaptic localization of GABA receptors (GABARs) were altered in the T21 COs only. The neurosteroid allopregnanolone, a positive modulator of GABARs, was downregulated in all the dCOs. Treatment with this neurosteroid significantly improved the neuronal communication in the dCOs, possibly through improving the GABAergic inhibition. Overall, without the manifestation of any disease-related pathology, the genetic mutations PRNPE200K, T21, and LRRK2G2019S significantly altered the neuronal network communication in dCOs by disrupting the excitatory-to-inhibitory balance.
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In vivo development of immune tissue in human intestinal organoids transplanted into humanized mice.
Human intestinal organoids (HIOs) derived from pluripotent stem cells provide a valuable model for investigating human intestinal organogenesis and physiology, but they lack the immune components required to fully recapitulate the complexity of human intestinal biology and diseases. To address this issue and to begin to decipher human intestinal-immune crosstalk during development, we generated HIOs containing immune cells by transplanting HIOs under the kidney capsule of mice with a humanized immune system. We found that human immune cells temporally migrate to the mucosa and form cellular aggregates that resemble human intestinal lymphoid follicles. Moreover, after microbial exposure, epithelial microfold cells are increased in number, leading to immune cell activation determined by the secretion of IgA antibodies in the HIO lumen. This in vivo HIO system with human immune cells provides a framework for future studies on infection- or allergen-driven intestinal diseases.
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Large-scale and innervated functional human gut tissues for transplantation via transient spheroid confinement.
Key limitations of current human gastrointestinal organoids include incomplete physiological maturation and the need for complex, time-consuming assembloid approaches to integrate a functional nervous system for transplantation. Here we present a confined culture system (CCS) method that generates large-scale, elongated and functional human small intestinal, colonic and gastric tissues with a de novo enteric nervous system (ENS). We use a 3D-printed scaffolding tray to restrict spheroid fusion and growth, promoting the spontaneous co-development of a functional ENS. Transcriptomic and electrophysiological data demonstrate selective neuromuscular function and the presence of excitatory and inhibitory neurons within the tissues. When compared to traditional methods, the CCS expedites tissue maturation for transplantation, yielding organoids that are up to ten times larger, reaching widths of 8 cm after 10 weeks, and exhibit enhanced engraftment rate. CCS organoids integrate and adapt to murine luminal environment while maintaining barrier integrity and functional capacity. The CCS methodology simplifies current protocols while accelerating the production of complex, functional and clinically relevant gut tissues.
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Pharmacological reversal of synaptic and network pathology in human MECP2-KO neurons and cortical organoids.
Duplication or deficiency of the X-linked MECP2 gene reliably produces profound neurodevelopmental impairment. MECP2 mutations are almost universally responsible for Rett syndrome (RTT), and particular mutations and cellular mosaicism of MECP2 may underlie the spectrum of RTT symptomatic severity. No clinically approved treatments for RTT are currently available, but human pluripotent stem cell technology offers a platform to identify neuropathology and test candidate therapeutics. Using a strategic series of increasingly complex human stem cell-derived technologies, including human neurons, MECP2-mosaic neurospheres to model RTT female brain mosaicism, and cortical organoids, we identified synaptic dysregulation downstream from knockout of MECP2 and screened select pharmacological compounds for their ability to treat this dysfunction. Two lead compounds, Nefiracetam and PHA 543613, specifically reversed MECP2-knockout cytologic neuropathology. The capacity of these compounds to reverse neuropathologic phenotypes and networks in human models supports clinical studies for neurodevelopmental disorders in which MeCP2 deficiency is the predominant etiology.
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Comparative Single-Cell Transcriptomics of Human Neuroblastoma and Preclinical Models Reveals Conservation of an Adrenergic Cell State.
Transgenic mice and organoid models, such as three-dimensional tumoroid cultures, have emerged as powerful tools for investigating cancer development and targeted therapies. Yet, the extent to which these preclinical models recapitulate the cellular identity of heterogeneous malignancies, like neuroblastoma, remains to be validated. In this study, we characterized the transcriptional landscape of TH-MYCN tumors by single-cell RNA sequencing and developed ex vivo tumoroids. Integrated analysis with murine fetal adrenal samples confirmed that both TH-MYCN tumors and tumoroids closely mirror the cellular profiles of normal embryonic sympathoblasts and chromaffin cells. Comprehensive comparison between tumors from patients with neuroblastoma and TH-MYCN mice demonstrated similarities in adrenergic tumor cell composition. Ex vivo tumoroid cultures displayed histologic resemblance and shared transcriptional profiles with the originating TH-MYCN tumors and human neuroblastoma tumors. Importantly, subpopulations within tumoroids exhibited gene expression associated with poor survival of patients with neuroblastoma. Notably, recurrent observations of a low-proliferative chromaffin phenotype connected to the highly proliferative sympathetic phenotype suggested that pushing sympathoblasts into a chromaffin-like state may offer an interesting therapeutic strategy for neuroblastoma. Together, this study not only deepens our understanding of a widely used transgenic mouse neuroblastoma model but also introduces an ex vivo model that maintains critical adrenergic cell state identity, thereby enhancing its translational potential for neuroblastoma research. Significance: Transgenic mouse models and ex vivo tumoroids, characterized through single-cell RNA sequencing, faithfully recapitulate neuroblastoma cellular identity, offering a useful platform for investigating potential therapeutic strategies.
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Modelling viral encephalitis caused by herpes simplex virus 1 infection in cerebral organoids.
Herpes simplex encephalitis is a life-threatening disease of the central nervous system caused by herpes simplex viruses (HSVs). Following standard of care with antiviral acyclovir treatment, most patients still experience various neurological sequelae. Here we characterize HSV-1 infection of human brain organoids by combining single-cell RNA sequencing, electrophysiology and immunostaining. We observed strong perturbations of tissue integrity, neuronal function and cellular transcriptomes. Under acyclovir treatment viral replication was stopped, but did not prevent HSV-1-driven defects such as damage of neuronal processes and neuroepithelium. Unbiased analysis of pathways deregulated upon infection revealed tumour necrosis factor activation as a potential causal factor. Combination of anti-inflammatory drugs such as necrostatin-1 or bardoxolone methyl with antiviral treatment prevented the damages caused by infection, indicating that tuning the inflammatory response in acute infection may improve current therapeutic strategies.
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Coordinated differentiation of human intestinal organoids with functional enteric neurons and vasculature.
Human intestinal organoids (HIOs) derived from human pluripotent stem cells co-differentiate both epithelial and mesenchymal lineages in vitro but lack important cell types such as neurons, endothelial cells, and smooth muscle, which limits translational potential. Here, we demonstrate that the intestinal stem cell niche factor, EPIREGULIN (EREG), enhances HIO differentiation with epithelium, mesenchyme, enteric neuroglial populations, endothelial cells, and organized smooth muscle in a single differentiation, without the need for co-culture. When transplanted into a murine host, HIOs mature and demonstrate enteric nervous system function, undergoing peristaltic-like contractions indicative of a functional neuromuscular unit. HIOs also form functional vasculature, demonstrated in vitro using microfluidic devices and in vivo following transplantation, where HIO endothelial cells anastomose with host vasculature. These complex HIOs represent a transformative tool for translational research in the human gut and can be used to interrogate complex diseases as well as for testing therapeutic interventions with high fidelity to human pathophysiology.
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Modeling alcohol-induced neurotoxicity using human induced pluripotent stem cell-derived three-dimensional cerebral organoids.
Maternal alcohol exposure during pregnancy can substantially impact the development of the fetus, causing a range of symptoms, known as fetal alcohol spectrum disorders (FASDs), such as cognitive dysfunction and psychiatric disorders, with the pathophysiology and mechanisms largely unknown. Recently developed human cerebral organoids from induced pluripotent stem cells are similar to fetal brains in the aspects of development and structure. These models allow more relevant in vitro systems to be developed for studying FASDs than animal models. Modeling binge drinking using human cerebral organoids, we sought to quantify the downstream toxic effects of alcohol (ethanol) on neural pathology phenotypes and signaling pathways within the organoids. The results revealed that alcohol exposure resulted in unhealthy organoids at cellular, subcellular, bioenergetic metabolism, and gene expression levels. Alcohol induced apoptosis on organoids. The apoptotic effects of alcohol on the organoids depended on the alcohol concentration and varied between cell types. Specifically, neurons were more vulnerable to alcohol-induced apoptosis than astrocytes. The alcohol-treated organoids exhibit ultrastructural changes such as disruption of mitochondria cristae, decreased intensity of mitochondrial matrix, and disorganized cytoskeleton. Alcohol exposure also resulted in mitochondrial dysfunction and metabolic stress in the organoids as evidenced by (1) decreased mitochondrial oxygen consumption rates being linked to basal respiration, ATP production, proton leak, maximal respiration and spare respiratory capacity, and (2) increase of non-mitochondrial respiration in alcohol-treated organoids compared with control groups. Furthermore, we found that alcohol treatment affected the expression of 199 genes out of 17,195 genes analyzed. Bioinformatic analyses showed the association of these dysregulated genes with 37 pathways related to clinically relevant pathologies such as psychiatric disorders, behavior, nervous system development and function, organismal injury and abnormalities, and cellular development. Notably, 187 of these genes are critically involved in neurodevelopment, and/or implicated in nervous system physiology and neurodegeneration. Furthermore, the identified genes are key regulators of multiple pathways linked in networks. This study extends for the first time animal models of binge drinking-related FASDs to a human model, allowing in-depth analyses of neurotoxicity at tissue, cellular, subcellular, metabolism, and gene levels. Hereby, we provide novel insights into alcohol-induced pathologic phenotypes, cell type-specific vulnerability, and affected signaling pathways and molecular networks, that can contribute to a better understanding of the developmental neurotoxic effects of binge drinking during pregnancy.
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Protocol for validating drug efficacy and safety in scalable 96-well platforms of human cortical organoids and melanoma brain metastases.
Metastatic colonization at secondary organs, such as the brain, represents a critical therapeutic window in cancer progression. Here, we present a protocol for a scalable in vitro screening platform for melanoma brain metastases. We describe steps for generating human cortical organoids in 96-well plates, followed by co-culture with melanoma cells to mimic the colonization process. This platform enables quantitative assessment of drug-induced metastasis regression and safety testing using fluorescence and luminescence readouts, targeting colonized melanoma cells in the brain microenvironment. For complete details on the use and execution of this protocol, please refer to Krieg et al.1.