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Patient-derived models of UBA5-associated encephalopathy identify defects in neurodevelopment and highlight potential therapeutic avenues.
UBA5 encodes for the E1 enzyme of the UFMylation cascade, which plays an essential role in endoplasmic reticulum (ER) homeostasis. The clinical phenotypes of UBA5-associated encephalopathy include developmental delays, epilepsy, and intellectual disability. To date, there is no humanized neuronal model to study the cellular and molecular consequences of UBA5 pathogenic variants. We developed and characterized patient-derived cortical organoid cultures from two patients with compound heterozygous variants in UBA5. Both shared the same missense variant, which encodes a hypomorphic allele (p.A371T), along with a nonsense variant (p.G267* or p.A123fs*4). Single-cell RNA sequencing of 100-day organoids identified defects in GABAergic interneuron development. We demonstrated aberrant neuronal firing and reduction in size of patient-derived organoids. Mechanistically, we showed that ER homeostasis is perturbed along with an exacerbated unfolded protein response pathway in engineered U87-MG cells and patient-derived organoids expressing UBA5 pathogenic variants. We also assessed two potential therapeutic modalities that augmented UBA5 protein abundance to rescue aberrant molecular and cellular phenotypes. We assessed SINEUP, a long noncoding RNA that augments translation efficiency, and CRISPRa, a modified CRISPR-Cas9 approach to augment transcription efficiency to increase UBA5 protein production. Our study provides a humanized model that allows further investigations of UBA5 variants in the brain and highlights promising approaches to alleviate cellular aberrations for this rare, developmental disorder.
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Gene Correction Reverses Ciliopathy and Photoreceptor Loss in iPSC-Derived Retinal Organoids from Retinitis Pigmentosa Patients.
Retinitis pigmentosa (RP) is an irreversible, inherited retinopathy in which early-onset nyctalopia is observed. Despite the genetic heterogeneity of RP, RPGR mutations are the most common causes of this disease. Here, we generated induced pluripotent stem cells (iPSCs) from three RP patients with different frameshift mutations in the RPGR gene, which were then differentiated into retinal pigment epithelium (RPE) cells and well-structured retinal organoids possessing electrophysiological properties. We observed significant defects in photoreceptor in terms of morphology, localization, transcriptional profiling, and electrophysiological activity. Furthermore, shorted cilium was found in patient iPSCs, RPE cells, and three-dimensional retinal organoids. CRISPR-Cas9-mediated correction of RPGR mutation rescued photoreceptor structure and electrophysiological property, reversed the observed ciliopathy, and restored gene expression to a level in accordance with that in the control using transcriptome-based analysis. This study recapitulated the pathogenesis of RPGR using patient-specific organoids and achieved targeted gene therapy of RPGR mutations in a dish as proof-of-concept evidence.
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Neuron culture from adult goldfish.
Neurons and glia from the central nervous system of the adult teleost Carassius auratus have been grown as explant cultures of minced brain tissue and as trypsin dissociated cells. These cultures exhibit extensive neurite growth from two neuronal types, have organotypic ultrastructure, and contain electrically active cells. Autoradiographic data indicate that these neurons do not divide in culture, and histological evidence suggests that some mature neurons survive explantation and regenerate processes. However, explantation of brain fragments not containing undifferentiated cells, localized in the ventricular and subventricular zones in the brains of fish, resulted in mesenchymal and glial cell cultures only. Therefore, a contribution to the population of cells in culture by undifferentiated cells must be considered. The cultured neurons remained viable for at least 19 weeks and ultrastructural and electrophysiological data indicate synaptic interaction between cells in explant cultures.
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Development of a three-dimensional organoid model to explore early retinal phenotypes associated with Alzheimer's disease.
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of Aβ plaques and neurofibrillary tangles, resulting in synaptic loss and neurodegeneration. The retina is an extension of the central nervous system within the eye, sharing many structural similarities with the brain, and previous studies have observed AD-related phenotypes within the retina. Three-dimensional retinal organoids differentiated from human pluripotent stem cells (hPSCs) can effectively model some of the earliest manifestations of disease states, yet early AD-associated phenotypes have not yet been examined. Thus, the current study focused upon the differentiation of hPSCs into retinal organoids for the analysis of early AD-associated alterations. Results demonstrated the robust differentiation of retinal organoids from both familial AD and unaffected control cell lines, with familial AD retinal organoids exhibiting a significant increase in the Aβ42:Aβ40 ratio as well as phosphorylated Tau protein, characteristic of AD pathology. Further, transcriptional analyses demonstrated the differential expression of many genes and cellular pathways, including those associated with synaptic dysfunction. Taken together, the current study demonstrates the ability of retinal organoids to serve as a powerful model for the identification of some of the earliest retinal alterations associated with AD.
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Tumor microenvironment-preserving gliosarcoma organoids as an in vitro preclinical platform: a comparative analysis with glioblastoma models.
BACKGROUND: Gliosarcoma (GS) is a rare variant of glioblastoma (GBM), characterized by biphasic glial and sarcomatous histology and poor prognosis. Despite its distinct clinical features, GS remains underrepresented in glioma research due to the lack of biologically faithful and experimentally tractable models. METHODS: We established patient-derived gliosarcoma organoids (GSOs) from freshly resected tumors using a suspension culture system without enzymatic dissociation. Histological, molecular, and functional properties were evaluated using H&E staining, immunohistochemistry, whole-exome sequencing, 3D invasion assays, and single-cell RNA sequencing (scRNA-seq). Drug response assays were performed using temozolomide and ANA-12 (NTRK2 inhibitor). RESULTS: GSOs preserved key histological features, genetic alterations, and tumor microenvironmental cell populations from the original tumors. They stable growth and viability over extended culture periods and maintained integrity following cryopreservation and recovery, supporting their long-term utility. 3D invasion assays further revealed infiltrative behavior, consistent with the aggressive nature of GS. Histological and molecular analyses revealed that GSOs retained glial and mesenchymal differentiation, diverse non-malignant stromal cells, and case-specific somatic alterations. Comparative scRNA-seq revealed distinct transcriptional programs: GSOs were enriched for fibroblast-like and oligodendrocyte progenitor-like states, while glioblastoma organoids (GBOs) displayed astrocyte-like differentiation and high connectivity signatures. Functional assays confirmed consistent sensitivity of GSOs to temozolomide, and selective therapeutic response to NTRK2 inhibition was observed in the GSO harboring an NTRK2 alteration, supporting the utility for genomic-context-guided therapy. CONCLUSION: Together, GSOs constitute a tractable and translationally relevant model that faithfully reflects the cellular complexity, genetic landscape, and tumor-specific heterogeneity. This model addresses a critical gap in GS biology and supports its integration into precision oncology.
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A novel integrated system using patient-derived glioma cerebral organoids and xenografts for disease modeling and drug screening.
A physiologically relevant glioma tumor model is important to the study of disease progression and screening drug candidates. However, current preclinical glioma models lack the brain microenvironment, and the established tumor cell lines do not represent glioma biology and cannot be used to evaluate the therapeutic effect. Here, we reported a real-time integrated system by generating 3D ex vivo cerebral organoids and in vivo xenograft tumors based on glioma patient-derived tissues and cells. Our system faithfully recapitulated the histological features, response to chemotherapy drugs, and clinical progression of their corresponding parental tumors. Additionally, our model successfully identified a case from a grade II astrocytoma patient with typical grade IV GBM features in both organoids and xenograft models, which mimicked the disease progression of this patient. Further genomic and transcriptomic characterization was associated with individual clinical features. We have demonstrated the "GBM-&Normal-like" signature to predict prognosis. In conclusion, we developed an integrated system of parallel models from patient-derived glioma cerebral organoids and xenografts for understanding the glioma biology and prediction of response to chemotherapy drugs, which might lead to a new strategy for personalized treatment for this deadly disease.
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Self-organizing human dual neural organoids model regional disease defects and teratogenicity.
The human nervous system emerges during early embryogenesis through coordinated, self-organizing differentiation of peripheral and central lineages. A key event in this process is the emergence of neural crest from the dorsal neural tube, which gives rise to major peripheral nervous system (PNS) structures. However, current in vitro models face challenges in recapitulating this process, limiting mechanistic insights into human central-peripheral neurogenesis. Here, we report a method for generating human peripheral-central dual neural organoids (hPCOs), a self-organizing organoid system that recapitulates the co-development of peripheral sensory ganglia and dorsal spinal cord. Using hPCOs, we uncover an unexpected bidirectional role of Wnt signaling in orchestrating peripheral and central fate determination. Modeling with hPCOs reveals nociceptor defects in human congenital insensitivity to pain. Furthermore, integrating hPCOs with image analysis enables the visual assessment of teratogenic risks. Together, hPCOs provide a physiologically relevant platform for investigating human self-organized central nervous system (CNS)-PNS co-development, disease mechanisms, and drug discovery.
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Modeling Human Nonalcoholic Fatty Liver Disease (NAFLD) with an Organoids-on-a-Chip System.
Nonalcoholic fatty liver disease (NAFLD) is a common metabolic and progressive disease, which has emerged as a major cause of chronic liver disease worldwide. It is characterized by the process ranging from simple steatosis to nonalcoholic steatohepatitis. However, a deep understanding of NAFLD progression remains challenging due to the lack of proper in vitro human disease models. In this work, we proposed a new strategy to establish a human NAFLD model based on a human-induced pluripotent stem cell (hiPSC)-derived liver organoids-on-a-chip system. This system allows us to characterize the pathological features of NAFLD in liver organoids by exposure to free fatty acids (FFAs) in perfused three-dimensional (3D) cultures during a prolonged period. Upon FFA induction, liver organoids exhibited lipid droplet formation and triglyceride accumulation. Moreover, they showed upregulated expressions of lipid metabolism-associated genes, indicating the abnormal lipid metabolic process in NAFLD. The FFA-exposed organoids also showed reactive oxygen species (ROS) production and elevated expression of various inflammatory cytokine genes and fibrogenic markers. These alterations represented the typical biochemical characteristics of NAFLD progression, which may provide insight into the potential mechanisms underlying steatosis. The proposed human NAFLD-on-a-chip model combines stem cell organoids with organs-on-chips, which may provide a promising platform for extending their applications for disease studies and effective therapies.
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Proteome architecture of human-induced pluripotent stem cell-derived three-dimensional organoids as a tool for early diagnosis of neuronal disorders.
BACKGROUND AND OBJECTIVES: Induced pluripotent stem cells (iPSCs) derived three-dimensional (3D) model for rare neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) is emerging as a novel alternative to human diseased tissue to explore the disease etiology and potential drug discovery. In the interest of the same, we have generated a TDP-43-mutated human iPSCs (hiPSCs) derived 3D organoid model of ALS disease. The high-resolution mass spectrometry (MS)-based proteomic approach is used to explore the differential mechanism under disease conditions and the suitability of a 3D model to study the disease. MATERIALS AND METHODS: The hiPSCs cell line was procured from a commercial source, grown, and characterized following standard protocols. The mutation in hiPSCs was accomplished using CRISPR/Cas-9 technology and predesigned gRNA. The two groups of organoids were produced by normal and mutated hiPSCs and subjected to the whole proteomic profiling by high-resolution MS in two biological replicates with three technical replicas of each. RESULTS: The proteomic analysis of normal and mutated organoids revealed the proteins associated with pathways of neurodegenerative disorders, proteasomes, autophagy, and hypoxia-inducible factor-1 signaling. Differential proteomic analysis revealed that the mutation in TDP-43 gene caused proteomic deregulation, which impaired protein quality mechanisms. Furthermore, this impairment may contribute to the generation of stress conditions that may ultimately lead to the development of ALS pathology. CONCLUSION: The developed 3D model represents the majority of candidate proteins and associated biological mechanisms altered in ALS disease. The study also offers novel protein targets that may uncloud the precise disease pathological mechanism and be considered for future diagnostic and therapeutic purposes for various neurodegenerative disorders.
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A Functional Precision Medicine Pipeline Combines Comparative Transcriptomics and Tumor Organoid Modeling to Identify Bespoke Treatment Strategies for Glioblastoma.
Li Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome caused by germline mutations in TP53. TP53 is the most common mutated gene in human cancer, occurring in 30-50% of glioblastomas (GBM). Here, we highlight a precision medicine platform to identify potential targets for a GBM patient with LFS. We used a comparative transcriptomics approach to identify genes that are uniquely overexpressed in the LFS GBM patient relative to a cancer compendium of 12,747 tumor RNA sequencing data sets, including 200 GBMs. STAT1 and STAT2 were identified as being significantly overexpressed in the LFS patient, indicating ruxolitinib, a Janus kinase 1 and 2 inhibitors, as a potential therapy. The LFS patient had the highest level of STAT1 and STAT2 expression in an institutional high-grade glioma cohort of 45 patients, further supporting the cancer compendium results. To empirically validate the comparative transcriptomics pipeline, we used a combination of adherent and organoid cell culture techniques, including ex vivo patient-derived organoids (PDOs) from four patient-derived cell lines, including the LFS patient. STAT1 and STAT2 expression levels in the four patient-derived cells correlated with levels identified in the respective parent tumors. In both adherent and organoid cultures, cells from the LFS patient were among the most sensitive to ruxolitinib compared to patient-derived cells with lower STAT1 and STAT2 expression levels. A spheroid-based drug screening assay (3D-PREDICT) was performed and used to identify further therapeutic targets. Two targeted therapies were selected for the patient of interest and resulted in radiographic disease stability. This manuscript supports the use of comparative transcriptomics to identify personalized therapeutic targets in a functional precision medicine platform for malignant brain tumors.