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Human nasal organoids sustain reproducible airway epithelial differentiation and influenza virus-host innate immune interaction.
BACKGROUND: The cultures of human nasal epithelial cells (NECs) are good surrogate models for studying immune responses. Three-dimensional (3D) organoids have emerged as an alternative for nasal epithelium, serving as robust platforms for modeling respiratory viral infections. We aimed to generate human NEC organoids and to determine their cellular characteristics as an in vitro model for air-liquid interface (ALI) culture and influenza A virus (IAV) infection. METHODS: Primary human nasal mucosal tissues were obtained from healthy donors undergoing nasal surgery, and nasal epithelial cell (NEC) organoids were established using Matrigel-based three-dimensional culture. Differentiated NEC organoids and air-liquid interface (ALI) cultures were characterized by histology, immunostaining, electron microscopy, and quantitative real-time PCR, followed by influenza A virus (A/WS/33, H1N1) infection assays. In addition, single-cell RNA sequencing (scRNA-seq) was performed to analyze cellular heterogeneity and differentiation trajectories of NEC organoids. RESULTS: We successfully developed human NEC organoids with using a Matrigel-based 3D culture system that recapitulates the structural characteristics and cellular composition of the human nasal epithelium. Histological results, real-time PCR, and single-cell RNA sequencing (scRNA-seq) revealed that the organoids consisted of seven different cell types and displayed a difference in the composition between the cells. The organoids displayed a much higher rate of basal progenitor cells whereas ciliated cells were less dominant. Unlike ALI cultures in which passage (P) 2 was the limit, the organoids exhibited successful and subsequent passages up to P5. Our data determined that NEC organoids are an adequate in vitro model for IAV infection and showed a sharp induction of mRNA levels for interferons (IFNs) and IFN-stimulated genes following IAV infection. CONCLUSIONS: This study's results demonstrate that human NEC organoids serve as a robust in vitro model, successfully recapitulating the biological characteristics of nasal epithelium, and could be an adequate tool for exploring distinct IFN-related innate immune responses following influenza infection.
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Modeling Mycobacterium tuberculosis pathogenesis in lung epithelial organoids reveals strain-specific host responses and intercellular crosstalk.
Emerging studies have identified alveolar epithelial cells as a conducive niche for Mycobacterium tuberculosis (Mtb) replication and spread during early infection. However, the host-pathogen interactions and intercellular crosstalk within the lung epithelial microenvironment remain inadequately understood. Here, we developed a lung epithelial organoid coculture model and exposed it to the virulent H37Rv strain or the avirulent Bacillus Calmette-Guérin (BCG) strain to investigate tuberculosis (TB) pathogenesis. Transcriptomic analyses revealed that Mtb infection markedly alters cell death patterns in organoids and modulates signal transduction pathways in peripheral blood mononuclear cells (PBMCs). Western blot indicates the H37Rv strain induced ferroptosis, autophagy, and apoptosis while suppressing necroptosis in organoids. In contrast, BCG predominantly enhanced autophagy. PBMCs also exhibited strain-specific responses, with BCG strongly activating the Hippo and Notch signaling pathways, whereas H37Rv primarily engaged the tumor necrosis factor (TNF) signaling pathway. Furthermore, Mtb significantly reshaped the paracrine and autocrine signaling dynamics between PBMCs and organoids. NicheNet network analysis identified TNFSF15 and brain-derived neurotrophic factor (BDNF), induced by H37Rv, as key mediators. Experimentally, overexpression of TNFSF15 and BDNF suggested that TNFSF15 from organoids promoted BDNF expression in PBMCs via paracrine signaling. In turn, BDNF from PBMCs then inhibited ferroptosis in organoids, contributing to restrict Mtb growth. Overall, our study provides a conceptual framework for understanding the mechanisms of TB pathogenesis within alveolar epithelial cells and offers valuable insights to prevent and control TB transmission in humans.
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Single-Cell Sequencing of Human Neural Organoids in a Model of Intracerebral Hemorrhage Reveals Temporally Dynamic Responses to Blood.
BACKGROUND: Intracerebral hemorrhage leads to significant morbidity and mortality due to primary mechanical and secondary neurotoxic injury to brain parenchyma. Timing of surgical evacuation to ensure optimal outcomes is controversial, with recent evidence suggesting early intervention improves functional outcome. Here, we characterize the impact of blood-induced secondary injury on diverse brain cell types in a scalable organoid model of intracerebral hemorrhage. METHODS: Human neural organoids consisting of excitatory neurons, inhibitory neurons, neural progenitor cells, astrocytes, endothelial cells, and microglia were generated from induced pluripotent stem cell-derived cells and treated with 5% blood for either 6 or 24 hours. Organoids were dissociated and analyzed by single-cell RNA sequencing. RESULTS: Single-cell sequencing of 96 725 cells across 18 organoids resolved intermediate progenitors, neural progenitor cells, microglia, inhibitory neurons, endothelial cells, excitatory neurons, and astrocytes. Twenty-four-hour exposure to blood induced cellular reactivity, whereas 6-hour exposure did not. Intermediate progenitors, endothelial cells, and astrocytes were the most reactive to blood and exhibited gene expression patterns corresponding to astrocyte reactivity, angiogenesis, and progenitor cell ischemic excitotoxicity. Drug repurposing analysis identified neurotransmitter-modulating, vasculature-remodeling, and protein synthesis pathways as potential therapeutic targets, mitigating blood-induced neurotoxicity. CONCLUSIONS: Blood exposure induces transcriptomic changes in a temporal and cell type-specific manner, particularly mediated by astrocyte reactivity, angiogenesis, and impairment of neurogenesis. Early and complete removal of blood after 6 hours of blood exposure mitigates secondary neurotoxicity seen in 24-hour exposure to blood. Transcriptomic signatures of blood-mediated neurotoxicity may potentially be reversed by antiadrenergic, dopamine agonist, and vasodilatory mechanisms.
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Human stem cell models to unravel brain cancer.
Pre-clinical animal models of human brain tumors have been invaluable tools for studying cancer pathogenesis and exploring novel treatment modalities. Such models recapitulate important aspects of the human disease such as the stem-progenitor-differentiated cell hierarchy. Although powerful, we argue that animal models are inherently limited in their ability to phenocopy certain important aspects of human brain tumor biology. We specifically highlight the inability of mouse models to generate certain forms aggressive pediatric medulloblastoma likely owing to cellular, anatomic, and genetic differences between the human and mouse brains. Additionally, we review some limitations of human brain tumor derived cell lines and outline why they are a sub-optimal system for purposes of pre-clinical modeling. Below, we present the case for human stem cell-based models of brain tumors, focusing mainly on glioblastoma and medulloblastoma. Drawing on several recently published studies, we review the exciting progress that has been made towards modeling human brain tumors using two-dimensional adherent stem cell cultures and three-dimensional organoids. We identify the important advances arrived at using these human stem cell-based models and suggest opportunities for future work in this direction. In this review article, we aim to highlight the utility and promises of human stem cell-based models of brain tumors as a complementary system to traditional transgenic animal and cell line systems.
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A human intestinal organoid derived from fetal human colon cells model for studying enteroviral pathogenesis.
Human enteroviruses, including enterovirus 71 (EV71), cause hand, foot, and mouth disease (HFMD) and may lead to severe neurological diseases in infants. Enteroviruses first infect the gastrointestinal tract and then spread to the main organs, such as the liver, lungs, heart, and brain. Human intestinal organoids (HIOs) provide a physiologically relevant model for studying enterovirus infections. Unlike traditional two-dimensional (2D) monolayer cultures, HIOs maintain complex epithelial cell diversity and three-dimensional (3D) architecture, allowing for a more accurate representation of in vivo viral-host interactions. In this study, we developed efficient and stable HIOs based on fetal human primary colon cells using the 3D culture system. We discriminated cultured HIOs containing goblet, enteroendocrine, and Paneth cells, and examined the replication efficiency of enteroviruses in HIOs compared to 2D monolayer cultures. HIOs were infected with enteroviruses (EV71, coxsackievirus B3, echovirus 6), and viral replication was assessed using molecular and imaging techniques, which exhibit a higher level of dynamic viral replication in HIOs than in 2D culture. The replication level of enteroviruses increased about 10-fold in HIOs with the virus titre in HIOs was 5-10 times higher than that in 2D cell cultures (p < 0.05). Also, our findings demonstrate that goblet cells serve as a primary site of viral replication. This observation highlights the importance of cellular microenvironments in enteroviral infections and provides insights into gut-specific viral tropism. Collectively, we established an infection model with human intestinal organoids for enteroviruses, providing new opportunities into evaluating enterovirus-related antiviral drugs and modelling enterovirus-associated diseases.
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Distinctive genomic signature of neural and intestinal organoids from familial Parkinson's disease patient-derived induced pluripotent stem cells.
AIMS: The leucine-rich repeat kinase 2 (LRRK2) G2019S mutation is the most common genetic cause of Parkinson's disease (PD). There is compelling evidence that PD is not only a brain disease but also a gastrointestinal disorder; nonetheless, its pathogenesis remains unclear. We aimed to develop human neural and intestinal tissue models of PD patients harbouring an LRRK2 mutation to understand the link between LRRK2 and PD pathology by investigating the gene expression signature. METHODS: We generated PD patient-specific induced pluripotent stem cells (iPSCs) carrying an LRRK2 G2019S mutation (LK2GS) and then differentiated into three-dimensional (3D) human neuroectodermal spheres (hNESs) and human intestinal organoids (hIOs). To unravel the gene and signalling networks associated with LK2GS, we analysed differentially expressed genes in the microarray data by functional clustering, gene ontology (GO) and pathway analyses. RESULTS: The expression profiles of LK2GS were distinct from those of wild-type controls in hNESs and hIOs. The most represented GO biological process in hNESs and hIOs was synaptic transmission, specifically synaptic vesicle trafficking, some defects of which are known to be related to PD. The results were further validated in four independent PD-specific hNESs and hIOs by microarray and qRT-PCR analysis. CONCLUSION: We provide the first evidence that LK2GS also causes significant changes in gene expression in the intestinal cells. These hNES and hIO models from the same genetic background of PD patients could be invaluable resources for understanding PD pathophysiology and for advancing the complexity of in vitro models with 3D expandable organoids.
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Ti3 C2 Tx MXene Composite 3D Hydrogel Potentiates mTOR Signaling to Promote the Generation of Functional Hair Cells in Cochlea Organoids.
Organoids have certain cellular composition and physiological features in common with real organs, making them promising models of organ formation, function, and diseases. However, Matrigel, the commonly used animal-derived matrices in which they are developed, has limitations in mechanical adjustability and providing complex physicochemical signals. Here, the incorporation of Ti3 C2 Tx MXene nanomaterial into Matrigel regulates the properties of Matrigel and exhibits satisfactory biocompatibility. The Ti3 C2 Tx MXene Matrigel composites (MXene-Matrigel) regulate the development of Cochlear Organoids (Cochlea-Orgs), particularly in promoting the formation and maturation of organoid hair cells. Additionally, regenerated hair cells in MXene-Matrigel are functional and exhibit better electrophysiological properties compared to hair cells in Matrigel. MXene-Matrigel potentiates the amycin (mTOR) signaling pathway to promote hair cell differentiation, and mTOR signaling inhibition restrains hair cell differentiation. Moreover, MXene-Matrigel facilitates innervation establishment between regenerated hair cells and spiral ganglion neurons (SGNs) growing from the Cochlea modiolus in a co-culture system, as well as promotes synapse formation efficiency. The approach overcomes some limitations of the Matrigel-dependent culture system and greatly accelerates the application of nanomaterials in organoid development and research on therapies for hearing loss.
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A hexamer tandem repeat RNA embedded within an SVA retrotransposon drives R-loop formation and neurodegeneration.
Retrotransposon activation is emerging as a significant factor in neurodegenerative disease pathogenesis. SINE-VNTR-Alu (SVAs) are hominid-specific retrotransposons that create genetic variation through insertion polymorphisms and variable short tandem repeat (STR) lengths. We investigate how the SVA (CCCTCT)n STR contributes to the striatal neurodegenerative disorder X-linked dystonia parkinsonism (XDP), where the repeat expansion length within the pathogenic SVA is inversely correlated with age at disease onset. Phenotypic and transcriptomic analysis of XDP and isogenic SVA-deleted striatal organoids reveal that the SVA insertion drives hallmarks of neurodegeneration, including transcriptional dysregulation, decreased neuronal activity, and apoptosis, which are ameliorated by SVA deletion. We identify an (AGAGGG)n hexamer-containing RNA in the XDP-causing SVA that increases expression during organoid maturation and drives R-loop formation in organoids and brain tissue. Knockdown of the hexamer-containing RNA by antisense oligonucleotides rescues apoptosis in XDP organoids. We demonstrate that a retrotransposon-derived tandem repeat RNA may cause neurodegeneration.
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Long-term exposure to cadmium disrupts neurodevelopment in mature cerebral organoids.
Cadmium (Cd) is a pervasive environmental pollutant. Increasing evidence suggests that Cd exposure during pregnancy can induce adverse neurodevelopmental outcomes. However, due to the limitations of neural cell and animal models, it is challenging to study the developmental neurotoxicity and underlying toxicity mechanism of long-term exposure to environmental pollutants during human brain development. In this study, chronic Cd exposure was performed in human mature cerebral organoids for 49 or 77 days. Our study found that prolonged exposure to Cd resulted in the inhibition of cerebral organoid growth and the disruption of neural differentiation and cortical layer organization. These potential consequences of chronic Cd exposure may include impaired GFAP expression, a reduction in SOX2+ neuronal progenitor cells, an increase in TUJ1+ immature neurons, as well as an initial increase and a subsequent decrease in both TBR2+ intermediate progenitors and CTIP2+ deep layer cortical neurons. Transcriptomic analyses revealed that long-term exposure to Cd disrupted zinc and copper ion homeostasis through excessive synthesis of metallothionein and disturbed synaptogenesis, as evidenced by inhibited postsynaptic protein. Our study employed mature cerebral organoids to evaluate the developmental neurotoxicity induced by long-term Cd exposure.
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Ca2+ signal dynamics in maturing ureteric bud- and collecting duct-derived organoid tubules.
Mechanical signals sensed by human stem cells are transduced via discrete signaling pathways to modulate developmental phenotype and function. Proximal tubules isolated from nephron lineage-derived kidney organoids undergo a developmental increase in abundance and/or activity of the basolateral mechanosensor PIEZO1 and Ca2+ signal transduction pathways (Carrisoza-Gaytan R, Kroll KT, Hiratsuka K, Gupta NR, Morizane R, Lewis JA, Satlin LM. Am J Physiol Cell Physiol 324: C757-C768, 2023). Here, we investigate whether human induced pluripotent stem cell (iPSC)-derived ureteric bud (UB) and collecting duct (CD) organoid cells exhibit a similar developmental increase in PIEZO1 function. Comparison of cells in tubules microdissected from UB and CD organoids cultured for 34-35 days or 62-65 days showed 1) increased intracellular Ca2+ concentration ([Ca2+]i) response to basolateral application of the selective PIEZO1 agonist Yoda1 and 2) decreased time to peak [Ca2+]i with advancing days in culture. Single-cell analyses of the Yoda1-induced [Ca2+]i response revealed 7- to 15-mHz [Ca2+]i oscillations that were more prevalent with advancing days in culture and differentiation (CD vs. UB). Concurrent exposure to inhibitors of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) or the plasma membrane Ca2+-ATPase (PMCA) dampened the amplitude of the Yoda1-induced [Ca2+]i oscillations. Bulk RNA analysis and pathway enrichment analysis revealed broad changes in genes associated with Ca2+ signaling, but not PIEZO1, with advancing days in culture and differentiation. These findings are consistent with a developmental increase in activity of PIEZO1 channels and/or maturation of associated pathways shaping Ca2+ signaling dynamics in maturing UB and CD organoids. Decoding of [Ca2+]i oscillations may identify molecular mechanisms important in morphological and functional differentiation of organoid tubules.NEW & NOTEWORTHY This investigation, focused on analyzing the role of PIEZO1 mechanotransduction in maturing human induced pluripotent stem cell (iPSC)-derived ureteric bud (UB) and collecting duct (CD) kidney organoids, unexpectedly reveals developmentally regulated Ca2+ oscillations and begins to dissect their mechanistic underpinnings. Specifically, transcriptomic analysis reveals that with time in culture and differentiation from UB to CD, organoids progressively acquire the molecular machinery necessary for complex Ca2+ signaling dynamics. These results lead us to speculate that information encoded in oscillatory signals drives renal epithelial differentiation.