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Application of an organoid-based model to explore Helicobacter pylori-human gastric epithelium interaction in vitro.
Helicobacter pylori infection causes histopathologic changes in gastric epithelial cells, resulting in conditions such as gastritis, gastric ulcers, and ultimately, gastric cancer. To date, various experimental models, including cell lines and animal studies, have been employed to investigate these pathological processes. However, each model presents its limitations. This study compared the re-cultured three-dimensional organoids from infected single cells, apical-out, and two-dimensional (2D) organoids models to better understand the mechanisms underlying the epithelial changes caused by H. pylori infection in the human stomach. Thus, we analyzed the epithelial cell responses, inflammatory mediator expressions, apical-junctional complex alterations, and H. pylori infection interactions in these organoid models. Moreover, we revealed that the high accessibility and experimental efficiency of the apical-out and 2D models enable easier manipulation and faster analysis of H. pylori infection compared to the single-cell infection model. These results indicate the potential of various organoid models in H. pylori infection studies to provide essential data that help in understanding the mechanisms of epithelial changes and in developing new therapeutic strategies for gastric pathology.
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A 3D Human Neuron-on-Chip Platform to Monitor Neuronal Injury Responses.
Traumatic brain injury (TBI) is a major cause of neurological dysfunction and long-term neurodegeneration, yet the intrinsic neuronal contributions to TBI pathophysiology remain incompletely defined. Here, we present a novel Neuron-on-Chip microfluidic device and weight-drop impactor platform that can be used to mechanically injure mature human prefrontal cortex neurons (hPFCs) embedded in three-dimensional (3D) hydrogels, enabling the study of injury responses in pure neuronal cultures. We assessed real-time calcium dynamics across 13 metrics of single-cell and network activity, revealing a biphasic injury response: an early phase (0.5-24 h) characterized by excitotoxicity, hyper-synchronized bursting, and network collapse; and a late phase (8 d) marked by sustained depolarization and structural remodeling. Secretome profiling revealed progressive elevations in extracellular pT181 and total Tau from days 1 to 5 post-injury. Cytokine analysis identified early (24 h) elevations in IP-10, IL-10, IFNα2, and NCAM, and late increases (8 d) in CXCL9 and MPO, linking neuronal activity changes to stage-specific inflammatory signaling. Immunocytochemistry and immunoblotting confirmed temporally ordered upregulation of calpain-1 and active caspase-3 (days 1-3), phosphorylated Tau (AT8+, days 5-8), and neurofibrillary tangle-like Tau aggregates (NFT+, day 8). These findings establish our platform as a scalable microphysiological model for probing the dynamic cellular and molecular sequelae of neuronal response to injury, offering insights into neurodegeneration and opportunities for therapeutic discovery.
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Silver nanoparticles exposure induces developmental neurotoxicity in hiPSC-derived cerebral organoids.
Silver nanoparticles (AgNPs) are used in various research fields. Although the neurotoxicity of AgNPs has been explored in animal models and 2D cell-culture models, including human stem cells, these models cannot accurately mimic the development of the human brain. Therefore, the potential mechanisms of AgNPs-induced developmental neurotoxicity in humans are still largely unclear. In this study, cerebral organoids derived from induced pluripotent stem cells were treated with 0.1 μg/mL or 0.5 μg/mL AgNPs for 7 days. At the low concentration (0.1 μg/mL), AgNPs increased the cell proliferation and inhibited the neural apoptosis in the organoids, but impaired the cilium assembly and elongation, which may perturb the cell cycle and induce abnormal cerebral-organoid growth. Conversely, at the high concentration (0.5 μg/mL), AgNPs significantly inhibited cell proliferation and induced apoptosis in cerebral organoids. High-concentration AgNPs reduced the expression and co-localization of the cytoskeleton proteins F-actin, myosin, and tubulin, thereby perturbing neurite growth. In conclusion, AgNPs exposure induces developmental neurotoxic effects in cerebral organoids and is thus a potential congenital risk factor.
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Intestinal organoids as advanced modeling platforms to study the role of host-microbiome interaction in homeostasis and disease.
After birth, animals are colonized by a diverse community of microorganisms. The digestive tract is known to contain the largest number of microbiome in the body. With emergence of the gut-brain axis, the importance of gut microbiome and its metabolites in host health has been extensively studied in recent years. The establishment of organoid culture systems has contributed to studying intestinal pathophysiology by replacing current limited models. Owing to their architectural and functional complexity similar to a real organ, co-culture of intestinal organoids with gut microbiome can provide mechanistic insights into the detrimental role of pathobiont and the homeostatic function of commensal symbiont. Here organoid-based bacterial co-culture techniques for modeling host-microbe interactions are reviewed. This review also summarizes representative studies that explore impact of enteric microorganisms on intestinal organoids to provide a better understanding of host-microbe interaction in the context of homeostasis and disease. [BMB Reports 2023; 56(1): 15-23].
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High-throughput single-cell transcriptomics on organoids.
Three-dimensional (3D) tissues grown in culture from human stem cells offer the incredible opportunity to analyze and manipulate human development, and to generate patient-specific models of disease. Methods to sequence DNA and RNA in single cells are being used to analyze these so-called 'organoid' systems in high-resolution. Single-cell transcriptomics has been used to quantitate the similarity of organoid cells to primary tissue counterparts in the brain, intestine, liver, and kidney, as well as identify cell-specific responses to environmental variables and disease conditions. The merging of these two technologies, single-cell genomics and organoids, will have profound impact on personalized medicine in the near future.
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SARS-CoV2 entry factors are expressed in primary human glioblastoma and recapitulated in cerebral organoid models.
PURPOSE: Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults with a median overall survival of only 14.6 months despite aggressive treatment. While immunotherapy has been successful in other cancers, its benefit has been proven elusive in GBM, mainly due to a markedly immunosuppressive tumor microenvironment. SARS-CoV-2 has been associated with the development of a pronounced central nervous system (CNS) inflammatory response when infecting different cells including astrocytes, endothelial cells, and microglia. While SARS-CoV2 entry factors have been described in different tissues, their presence and implication on GBM aggressiveness or microenvironment has not been studied on appropriate preclinical models. METHODS: We evaluated the presence of crucial SARS-CoV-2 entry factors: ACE2, TMPRSS2, and NRP1 in matched surgically-derived GBM tissue, cells lines, and organoids; as well as in human brain derived specimens using immunohistochemistry, confocal pixel line intensity quantification, and transcriptome analysis. RESULTS: We show that patient derived-GBM tissue and cell cultures express SARS-CoV2 entry factors, being NRP1 the most crucial facilitator of SARS-CoV-2 infection in GBM. Moreover, we demonstrate that, receptor expression remains present in our GBM organoids, making them an adequate model to study the effect of this virus in GBM for the potential development of viral therapies in the future. CONCLUSION: Our findings suggest that the SARS-CoV-2 virus entry factors are expressed in primary tissues and organoid models and could be potentially utilized to study the susceptibility of GBM to this virus to target or modulate the tumor microenviroment.
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Fate and state transitions during human blood vessel organoid development.
Human blood vessel organoids (hBVOs) have emerged as a system to model human vascular development and disease. Here, we use single-cell multi-omics together with genetic and signaling pathway perturbations to reconstruct hBVO development. Mesodermal progenitors bifurcate into endothelial and mural fates in vitro, and xenografted BVOs acquire definitive arteriovenous endothelial cell specification. We infer a gene regulatory network and use single-cell genetic perturbations to identify transcription factors (TFs) and receptors involved in cell fate specification, including a role for MECOM in endothelial and mural specification. We assess the potential of BVOs to generate organotypic states, identify TFs lacking expression in hBVOs, and find that induced LEF1 overexpression increases brain vasculature specificity. Finally, we map vascular disease-associated genes to hBVO cell states and analyze an hBVO model of diabetes. Altogether, we provide a comprehensive cell state atlas of hBVO development and illuminate the power and limitation of hBVOs for translational research.
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How Organ-on-a-Chip Technology Can Assist in Studying the Role of the Glymphatic System in Neurodegenerative Diseases.
The lack of a conventional lymphatic system that permeates throughout the entire human brain has encouraged the identification and study of alternative clearance routes within the cerebrum. In 2012, the concept of the glymphatic system, a perivascular network that fluidically connects the cerebrospinal fluid to the lymphatic vessels within the meninges via the interstitium, emerged. Although its exact mode of action has not yet been fully characterized, the key underlying processes that govern solute transport and waste clearance have been identified. This review briefly describes the perivascular glial-dependent clearance system and elucidates its fundamental role in neurodegenerative diseases. The current knowledge of the glymphatic system is based almost exclusively on animal-based measurements, but these face certain limitations inherent to in vivo experiments. Recent advances in organ-on-a-chip technology are discussed to demonstrate the technology's ability to provide alternative human-based in vitro research models. Herein, the specific focus is on how current microfluidic-based in vitro models of the neurovascular system and neurodegenerative diseases might be employed to (i) gain a deeper understanding of the role and function of the glymphatic system and (ii) to identify new opportunities for pharmacological intervention.
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Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells.
An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.
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Ready-to-Use Nanofiber Membrane-Integrated Organ-on-a-Chip via Cryopreservation of Cell-Loaded Microfluidic Chip.
Nanofiber (NF) membrane-integrated organ-on-a-chip platforms enhance in vitro tissue barrier models by mimicking in vivo microenvironments. However, their widespread adoption has been hindered by technical complexities such as manual cell seeding. In this study, we developed ReadyChip, a cryopreserved, preseeded NF membrane-integrated organ-on-a-chip that is immediately usable upon thawing. ReadyChip showed robust post-thaw performance by preserving both the structural characteristics and compositional properties of the ECM-coated NF membrane. Its versatility was demonstrated through vascular, retinal, and blood-brain barrier models, each showing post-thaw cellular viability above 92% and sustained expression of functional markers that remained expressed after 7 days of perfusion culture. By eliminating the need for manual seeding and complex culture processes, ReadyChip offers a ready-to-use platform that enhances efficiency and practicality in both biomedical research and industrial applications.