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Engineering Pluripotent Stem Cells-Derived Inner Ear Organoids With Enhanced Maturation and Reproducibility by Micro-Topographical Cues.
Inner ear organoids (IEOs) derived from pluripotent stem cells (PSCs) provide a promising platform for modeling neurosensory disorders and hearing loss; however, conventional systems often exhibit substantial structural variability, incomplete maturation, and limited reproducibility due to insufficient control of early organoid morphogenesis. Here, we demonstrate that micro-topographical cues applied during initial IEO formation enhance the development and functional maturation of PSC-derived IEOs. This microengineering strategy introduces temporally defined microscale geometric confinement to regulate early cell-cell and cell-extracellular matrix (ECM) interactions, thereby promoting epithelial organization and developmental fidelity. Microengineered IEOs (M-IEOs) exhibit improved reproducibility and neurosensory maturation, including increased hair cell-like populations, stereocilia-like structures and kinocilium-like features exhibiting a characteristic (9 × 2) + 2 microtubule organization. Functionally, M-IEOs exhibit enhanced electrophysiological responsiveness, supported by complementary transcriptomic and in situ analyses indicating activation of inner ear lineage maturation pathways. Furthermore, we demonstrate the versatility of M-IEOs by integrating them with a microfluidic vascular system to model vascular-epithelial interactions and inflammatory responses, highlighting its potential for disease modeling and pharmacological screening. Together, these findings establish transient micro-topographical guidance as an instructive regulator of inner ear organoid development and provide a robust, vascular-compatible platform for neurosensory research, disease modeling, and translational screening applications.
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FGF8-mediated gene regulation affects regional identity in human cerebral organoids.
The morphogen FGF8 establishes graded positional cues imparting regional cellular responses via modulation of early target genes. The roles of FGF signaling and its effector genes remain poorly characterized in human experimental models mimicking early fetal telencephalic development. We used hiPSC-derived cerebral organoids as an in vitro platform to investigate the effect of FGF8 signaling on neural identity and differentiation. We found that FGF8 treatment increases cellular heterogeneity, leading to distinct telencephalic and mesencephalic-like domains that co-develop in multi-regional organoids. Within telencephalic regions, FGF8 affects the anteroposterior and dorsoventral identity of neural progenitors and the balance between GABAergic and glutamatergic neurons, thus impacting spontaneous neuronal network activity. Moreover, FGF8 efficiently modulates key regulators responsible for several human neurodevelopmental disorders. Overall, our results show that FGF8 signaling is directly involved in both regional patterning and cellular diversity in human cerebral organoids and in modulating genes associated with normal and pathological neural development.
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Establishment of patient-derived organoid models of lower-grade glioma.
BACKGROUND: Historically, creating patient-derived models of lower-grade glioma (LGG) has been challenging, contributing to few experimental platforms that support laboratory-based investigations of this disease. Although organoid modeling approaches have recently been employed to create in vitro models of high-grade glioma (HGG), it is unknown whether this approach can be successfully applied to LGG. METHODS: In this study, we developed an optimized protocol for the establishment of organoids from LGG primary tissue samples by utilizing physiologic (5%) oxygenation conditions and employed it to produce the first known suite of these models. To assess their fidelity, we surveyed key biological features of patient-derived organoids using metabolic, genomic, histologic, and lineage marker gene expression assays. RESULTS: Organoid models were created with a success rate of 91% (n = 20/22) from primary tumor samples across glioma histological subtypes and tumor grades (WHO Grades 1-4), and a success rate of 87% (13/15) for WHO Grade 1-3 tumors. Patient-derived organoids recapitulated stemness, proliferative, and tumor-stromal composition profiles of their respective parental tumor specimens. Cytoarchitectural, mutational, and metabolic traits of parental tumors were also conserved. Importantly, LGG organoids were maintained in vitro for weeks to months and reanimated after biobanking without loss of integrity. CONCLUSIONS: We report an efficient method for producing faithful in vitro models of LGG. New experimental platforms generated through this approach are well positioned to support preclinical studies of this disease, particularly those related to tumor immunology, tumor-stroma interactions, identification of novel drug targets, and personalized assessments of treatment response profiles.
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[Organoids and organ-on-a-chip: novel strategies for TCM against respiratory virus-induced cranial nerve injury].
Investigating TCM for preventing and treating respiratory virus-induced nerve injury urgently requires highly biomimetic models that can replicate the complex structure and physiological microenvironment of the body. Conventional two-dimensional cell models and animal experiments present significant limitations, hindering a comprehensive understanding of the complicated interactions between drug components and their targets. As cutting-edge technologies, organoids and organ-on-a-chip systems address these shortcomings by respectively modeling three-dimensional cellular architecture, inter-organ interactions, and dynamic microenvironments, thereby establishing novel technological platforms for pharmaceutical research. This review systematically summarized common respiratory viruses that induce cranial nerve injury. It analyzed the mechanism by which respiratory virus infections damage the lungs and, through direct injury and indirect invasion of the vascular system, lead to cranial nerve injury. By synthesizing literature from the past five years, the paper focused on the application and specific cases utilizing cutting-edge technologies like respiratory tract organoids, brain organoids, and related organ-on-a-chip in studying virus-induced nerve injury. The aim is to provide new perspectives and methodologies for future drug development against respiratory viruses and to formulate innovative research strategies for TCM-based interventions.
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Patient-derived glioblastoma organoids as real-time avatars for assessing responses to clinical CAR-T cell therapy.
Patient-derived tumor organoids have been leveraged for disease modeling and preclinical studies but rarely applied in real time to aid with interpretation of patient treatment responses in clinics. We recently demonstrated early efficacy signals in a first-in-human, phase 1 study of dual-targeting chimeric antigen receptor (CAR)-T cells (EGFR-IL13Rα2 CAR-T cells) in patients with recurrent glioblastoma. Here, we analyzed six sets of patient-derived glioblastoma organoids (GBOs) treated concurrently with the same autologous CAR-T cell products as patients in our phase 1 study. We found that CAR-T cell treatment led to target antigen reduction and cytolysis of tumor cells in GBOs, the degree of which correlated with CAR-T cell engraftment detected in patients' cerebrospinal fluid (CSF). Furthermore, cytokine release patterns in GBOs mirrored those in patient CSF samples over time. Our findings highlight a unique trial design and GBOs as a valuable platform for real-time assessment of CAR-T cell bioactivity and insights into immunotherapy efficacy.
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Establishment of patient-derived three-dimensional organoid culture in renal cell carcinoma.
PURPOSE: Renal cell carcinoma is a heterogeneous kidney cancer, and over 403,000 cases were reported worldwide in 2018. Current methods for studying renal cell carcinoma are limited to two-dimensional (2D) culture of primary cell lines and patient-derived xenograft models. Numerous studies have suggested that 2D culture poorly represents the diversity, heterogeneity, and drug-resistance of primary tumors. The time and cost associated with patient-derived xenograft models poses a realistic barrier to their clinical utility. As a biomimetic model, patient-derived three-dimensional (3D) organoid culture can overcome these disadvantages and bridge the gap between in vitro cell culture and in vivo patient-derived xenograft models. Here, we establish a patient-derived 3D organoid culture system for clear cell renal cell carcinoma and demonstrate the biomimetic characteristics of our model with respect to both primary kidney cancer and conventional 2D culture. MATERIALS AND METHODS: Normal renal tissues and tumor tissues were collected from patients with clear cell renal cell carcinoma. The dissociated cells were cultured as conventional 2D culture and 3D organoid culture. The biomimetic characteristic of the two cultures were compared. RESULTS: Compared with 2D culture, the 3D organoid cultures retained the characteristic lipid-rich, clear cell morphology of clear cell renal cell carcinoma. Carbonic anhydrase 9 and vimentin were validated as biomarkers of renal cell carcinoma. Expression of the two validated biomarkers was more enhanced in 3D organoid culture. CONCLUSIONS: Patient-derived 3D organoid culture retains the characteristics of renal cell carcinoma with respect to morphology and biomarker expression.
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Engineered thoracic spinal cord organoids for transplantation after spinal cord injury.
Stem-cell-based neural tissue engineering and spinal cord organoids show promises for spinal cord injury repair. However, the native spinal cord presents cell heterogeneity and a stereotypical spatial structure that makes difficult their recapitulation within an organoid architecture, which requires an assembly encompassing cellular composition, segmental organization and dorsoventral features. Here we engineer a thoracic vertebral segment-specific spinal cord organoid (enTsOrg) model that can precisely match the transplantation site, establish synaptic connections and enhance in vivo neuroelectric conduction. The organoids are generated from fibroblasts-derived induced pluripotent stem cells and a layered double-hydroxide matrix in a basement membrane hydrogel (Matrigel). Grafted in a spinal cord injury mouse model, enTsOrg presents advanced maturation, functionalization and organized distribution of critical neuronal subtypes with thoracic segmental heterogeneity, including various motor neuron and interneuron subtypes, that serve essentially to restore motor functions. Transplantation of enTsOrg can restructure neural circuits in paralysed animals and restore hind-limb motor function. The robust neurological function and therapeutic efficacy of enTsOrg highlight a potential avenue for organoid designing for specific anatomical regions in neurological injury treatments.
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Three-Dimensional Spatiotemporal Modeling of Colon Cancer Organoids Reveals that Multimodal Control of Stem Cell Self-Renewal is a Critical Determinant of Size and Shape in Early Stages of Tumor Growth.
We develop a three-dimensional multispecies mathematical model to simulate the growth of colon cancer organoids containing stem, progenitor and terminally differentiated cells, as a model of early (prevascular) tumor growth. Stem cells (SCs) secrete short-range self-renewal promoters (e.g., Wnt) and their long-range inhibitors (e.g., Dkk) and proliferate slowly. Committed progenitor (CP) cells proliferate more rapidly and differentiate to produce post-mitotic terminally differentiated cells that release differentiation promoters, forming negative feedback loops on SC and CP self-renewal. We demonstrate that SCs play a central role in normal and cancer colon organoids. Spatial patterning of the SC self-renewal promoter gives rise to SC clusters, which mimic stem cell niches, around the organoid surface, and drive the development of invasive fingers. We also study the effects of externally applied signaling factors. Applying bone morphogenic proteins, which inhibit SC and CP self-renewal, reduces invasiveness and organoid size. Applying hepatocyte growth factor, which enhances SC self-renewal, produces larger sizes and enhances finger development at low concentrations but suppresses fingers at high concentrations. These results are consistent with recent experiments on colon organoids. Because many cancers are hierarchically organized and are subject to feedback regulation similar to that in normal tissues, our results suggest that in cancer, control of cancer stem cell self-renewal should influence the size and shape in similar ways, thereby opening the door to novel therapies.
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A standardized patient-derived glioblastoma organoid platform for evaluating radiotherapy efficacy and overcoming resistance.
Patient-derived glioblastoma (GBM) organoids (GBOs) maintain characteristics of their original tumors; however, they have not been widely applied to systematically assess therapy responses at scale or elucidate mechanisms underlying treatment resistance. Here, we develop a standardized reaggregated GBO (rGBO) platform for scalable, quantitative evaluation of radiation responses across 48 patients with GBM. Radiation responses in rGBOs correlate with the survival duration of corresponding patients treated with standard-of-care radiotherapy. Transcriptomic analyses identify gene signatures associated with radiosensitivity and radioresistance. Connectivity mapping with perturbational datasets identifies a panel of compounds with potential for reducing radioresistant and enhancing radiosensitive signatures. Subsequent testing demonstrates that inhibitors of bromodomain and extraterminal (BET) family proteins enhance radiotherapy efficacy in GBO and xenograft models by downregulating the radioresistant signature. Together, our study highlights the potential of rGBOs for standardizing therapeutic screening, evaluating patient-specific responses, identifying associated molecular features, and developing strategies to overcome treatment resistance.
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Extension of retinofugal projections in an assembled model of human pluripotent stem cell-derived organoids.
The development of the visual system involves the coordination of spatial and temporal events to specify the organization of varied cell types, including the elongation of axons from retinal ganglion cells (RGCs) to post-synaptic targets in the brain. Retinal organoids recapitulate many features of retinal development, yet have lacked downstream targets into which RGC axons extend, limiting the ability to model projections of the human visual system. To address these issues, retinal organoids were generated and organized into an in vitro assembloid model of the visual system with cortical and thalamic organoids. RGCs responded to environmental cues and extended axons deep into assembloids, modeling the projections of the visual system. In addition, RGC survival was enhanced in long-term assembloids, overcoming prior limitations of retinal organoids in which RGCs are lost. Overall, these approaches will facilitate studies of human visual system development, as well as diseases or injuries to this critical pathway.