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HiPSC-derived multi-organoids-on-chip system for safety assessment of antidepressant drugs.
The poor predictive power of existing preclinical models has spurred efforts to develop human-relevant models for accurate assessment of drug safety. In this work, we developed a multi-organoids-on-a-chip system derived from human induced pluripotent stem cells (hiPSCs), which allows for the assessment of the cardiac safety of an antidepressant drug, following liver metabolism in vitro. This liver-heart organoids-on-chip device contains compartmentalized chambers separated by a porous membrane, which permits the co-culture of 3D human liver organoids in the upper multi-well chamber and cardiac organoids in the bottom micropillar array simultaneously. The co-cultured liver and heart organoids on chip maintained good viability and human organ-specific functions respectively, including the synthesis of albumin and urea of liver organoids, and the beating function of cardiac organoids. In particular, the liver organoids displayed proper metabolic capabilities with high expression of CYP450 enzyme genes. Clomipramine, a widely used antidepressant drug, can be metabolized into an active metabolite (desmethylclomipramine) through the hepatic CYP450 enzymes of liver organoids on chip identified by mass spectrometry. After exposure to 1 μM clomipramine in the liver chamber for 24 h and 48 h, the co-cultured heart organoids in the bottom layer showed significantly reduced cell viability, impaired functions of cardiac beating and calcium flux, indicating the hepatic metabolism-dependent cardiotoxicity induced by clomipramine. By combining stem cell biology and microengineered technology, this proposed hiPSC-derived multi-organoids-on-a-chip system can reflect human organ-specific functions, as well as the complex process of drug metabolism and responses at the multi-organ level. It may provide a novel platform for the assessment of drug effectiveness and safety in vitro.
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A Drug Screening Pipeline Using 2D and 3D Patient-Derived In Vitro Models for Pre-Clinical Analysis of Therapy Response in Glioblastoma.
Glioblastoma is one of the most common and lethal types of primary brain tumor. Despite aggressive treatment with chemotherapy and radiotherapy, tumor recurrence within 6-9 months is common. To overcome this, more effective therapies targeting cancer cell stemness, invasion, metabolism, cell death resistance and the interactions of tumor cells with their surrounding microenvironment are required. In this study, we performed a systematic review of the molecular mechanisms that drive glioblastoma progression, which led to the identification of 65 drugs/inhibitors that we screened for their efficacy to kill patient-derived glioma stem cells in two dimensional (2D) cultures and patient-derived three dimensional (3D) glioblastoma explant organoids (GBOs). From the screening, we found a group of drugs that presented different selectivity on different patient-derived in vitro models. Moreover, we found that Costunolide, a TERT inhibitor, was effective in reducing the cell viability in vitro of both primary tumor models as well as tumor models pre-treated with chemotherapy and radiotherapy. These results present a novel workflow for screening a relatively large groups of drugs, whose results could lead to the identification of more personalized and effective treatment for recurrent glioblastoma.
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Protocol for modeling injury-induced regeneration in retinal organoids and isolating hNRSCs for subretinal transplantation in rd10 mice.
Human retinal organoids (hROs) provide a platform for modeling injury and assessing regenerative capacity in vitro. Here, we present a protocol for stepwise generation of hROs from human embryonic stem cells (hESCs). We describe steps for microsurgical induction of neural retina (NR) injury, isolation of CPAMD8+ human neural retinal stem-like cells (hNRSCs) using flow cytometry, and their subretinal transplantation into rd10 mice. By isolating and subretinally injecting hESC-derived neural retinal stem-like cells, this protocol enables subsequent functional studies in retinal degeneration models. For complete details on the use and execution of this protocol, please refer to Liu et al.1.
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Disrupting glioblastoma networks with tumor treating fields (TTFields) in in vitro models.
PURPOSE: This study investigates the biological effect of Tumor Treating Fields (TTFields) on key drivers of glioblastoma's malignancy-tumor microtube (TM) formation-and on the function and overall integrity of the tumor cell network. METHOD: Using a two-dimensional monoculture GB cell network model (2DTM) of primary glioblastoma cell (GBC) cultures (S24, BG5 or T269), we evaluated the effects of TTFields on cell density, interconnectivity and structural integrity of the tumor network. We also analyzed calcium (Ca2+) transient dynamics and network morphology, validating findings in patient-derived tumoroids and brain tumor organoids. RESULTS: In the 2DTM assay, TTFields reduced cell density by 85-88% and disrupted network interconnectivity, particularly in cells with multiple TMs. A "crooked TM" phenotype emerged in 5-6% of treated cells, rarely seen in controls. Ca2+ transients were significantly compromised, with global Ca2+ activity reduced by 51-83%, active and periodic cells by over 50%, and intercellular co-activity by 52% in S24, and almost completely in BG5 GBCs. The effects were more pronounced at 200 kHz compared to a 50 kHz TTFields. Similar reductions in Ca2+ activity were observed in patient-derived tumoroids. In brain tumor organoids, TTFields significantly reduced tumor cell proliferation and infiltration. CONCLUSION: Our comprehensive study provides new insights into the multiple effects of Inovitro-modeled TTFields on glioma progression, morphology and network dynamics in vitro. Future in vivo studies to verify our in vitro findings may provide the basis for a deeper understanding and optimization of TTFields as a therapeutic modality in the treatment of GB.
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Cardiac organoids bridge translational gaps in 3R-compliant safety testing.
Cardiotoxicity remains a leading cause of late-stage drug attrition and post-marketing withdrawal, underscoring persistent limitations in current preclinical safety assessment paradigms. Traditional animal models are constrained by interspecies differences in electrophysiology, ion channel composition, myocardial remodeling, and pharmacokinetics, while two-dimensional human induced pluripotent stem cel-derived cardiomyocytes (hiPSC-CMs) lack tissue-level, functional maturity, organization and multicellular complexity. These limitations create a translational gap between cellular assays and whole-organ physiology. Human cardiac organoids represent an emerging mesoscopic platform that bridges this gap by integrating three-dimensional architecture, electromechanical coupling, and multicellular interactions within a human genetic context. Unlike isolated cardiomyocytes, cardiac organoids support coordinated conduction, arrhythmic susceptibility, force generation, extracellular matrix remodeling, and emerging neurocardiac crosstalk. These properties position them as promising tools for implementing the 3Rs (Replacement, Reduction, Refinement) in early-stage cardiovascular drug development. This review critically evaluates the biological fidelity, functional capabilities, and disease modeling relevance of human cardiac organoids, and examines their potential integration into safety pharmacology frameworks. We propose that cardiac organoids constitute a critical intermediate layer between reductionist cellular systems and whole-organ models. Finally, we delineate the scientific and regulatory steps, including standardization and validation against established frameworks, required to transition them from exploratory research tools to robust platforms for predictive and ethically aligned cardiovascular safety assessments.
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Establishment and molecular characterisation of patient-derived organoids for primary central nervous system lymphoma.
Primary central nervous system lymphoma (PCNSL) exhibits substantial intratumoural and intertumoural heterogeneity, complicating the development of effective treatment methods. Existing in vitro models fail to simulate the cellular and mutational diversity of native tumours and require prolonged generation times. Therefore, we developed a culture method for patient-derived PCNSL organoids (CLOs) and evaluated the organoids through extensive molecular characterisation, histopathological analysis, single-nucleus RNA sequencing, bulk RNA sequencing and whole-exome sequencing. These CLOs accurately mimicked the histological attributes, gene expression landscapes and mutational profiles of their original tumours. Single-nucleus RNA sequencing also revealed that CLOs maintained cell-type heterogeneity and the molecular signatures of their original tumours. CLOs were generated within 2 weeks, demonstrating rapid development and reliability. Therapeutic profiling was performed on three selected CLOs treated with four standard drugs. The CLOs exhibited specific sensitivity to methotrexate, and resistance to dexamethasone, ibrutinib and rituximab, suggesting that CLOs may be valuable tools for reflecting drug sensitivities. Taken together, these results emphasise that CLOs effectively emulate the key characteristics of PCNSL, increasing the understanding of the genetic landscape of this complex disease. CLOs provide a rapid and reliable platform for exploring individualised treatment strategies, potentially accelerating the transition of research findings to clinical practice.
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Blood vessel organoids generated by base editing and harboring single nucleotide variation in Notch3 effectively recapitulate CADASIL-related pathogenesis.
Human blood vessel organoids (hBVOs) offer a promising platform for investigating vascular diseases and identifying therapeutic targets. In this study, we focused on in vitro modeling and therapeutic target finding of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common form of hereditary stroke disorder caused by mutations in the NOTCH3 gene. Despite the identification of these mutations, the underlying pathological mechanism is elusive, and effective therapeutic approaches are lacking. CADASIL primarily affects the blood vessels in the brain, leading to ischemic strokes, migraines, and dementia. By employing CRISPR/Cas9 base-editing technology, we generated human induced pluripotent stem cells (hiPSCs) carrying Notch3 mutations. These mutant hiPSCs were differentiated into hBVOs. The NOTCH3 mutated hBVOs exhibited CADASIL-like pathology, characterized by a reduced vessel diameter and degeneration of mural cells. Furthermore, we observed an accumulation of Notch3 extracellular domain (Notch3ECD), increased apoptosis, and cytoskeletal alterations in the NOTCH3 mutant hBVOs. Notably, treatment with ROCK inhibitors partially restored the disconnection between endothelial cells and mural cells in the mutant hBVOs. These findings shed light on the pathogenesis of CADASIL and highlight the potential of hBVOs for studying and developing therapeutic interventions for this debilitating human vascular disorder.
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Nucleation of microtubules in vitro by isolated spindle pole bodies of the yeast Saccharomyces cerevisiae.
Spindle pole bodies (SPBs) were isolated from the yeast Saccharomyces cerevisiae by an adaptation of the Kleinschmidt monolayer technique. Spheroplasts prepared from the cells were lysed on an air-water interface. Spread preparations were picked up on grids, transferred to experimental test solutions, and prepared for whole-mount electron microscopy. Using purified exogenous tubulin from porcine brain tissue, the isolated SPBs were shown to nucleate the assembly of microtubules in vitro. Microtubule growth was directional and primarily onto the intranuclear face of the SPB. Neither the morphology nor the microtubule-initiating capacity of the SPB was affected by treatment with the enzymes DNase, RNase, or phospholipase although both properties were sensitive to trypsin. Analysis of SPBs at various stages of the cell cycle showed that newly replicated SPBs had the capacity to nucleate microtubules. SPBs isolated from exponentially growing cells initiated a subset of the yeast spindle microtubules equivalent to the number of pole-to-pole microtubules seen in vivo. However, SPBs isolated from cells in stationary phase and therefore arrested in G1 nucleated a number of microtubules equal to the total chromosomal and pole-to-pole tubules in the yeast spindle. This may mean that in G1-arrested cells, the SPB is associated with microtubule attachment sites of the yeast chromatin.
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Mini-Review: Induced pluripotent stem cells and the search for new cell-specific ALS therapeutic targets.
Amongst the most important discoveries in ALS pathobiology are the works demonstrating that multiple cell types contribute to disease onset and progression. However, a significant limitation in ALS research is the inability to obtain tissues from ALS patient brain and spinal cord during the course of the disease. In vivo modeling has provided insights into the role of these cell subtypes in disease onset and progression. However, in vivo models also have shortcomings, including the reliance on a limited number of models based upon hereditary forms of the disease. Therefore, using human induced pluripotent stem cells (iPSC) reprogrammed from somatic cells of ALS patients, with both hereditary and sporadic forms of the disease, and differentiated into cell subtypes of both the central nervous system (CNS) and peripheral nervous system (PNS), have become powerful complementary tools for investigating basic mechanisms of disease as well as a platform for drug discovery. Motor neuron and other neuron subtypes, as well as non-neuronal cells have been differentiated from human iPSC and studied for their potential contributions to ALS pathobiology. As iPSC technologies have advanced, 3D modeling with multicellular systems organised in microfluidic chambers or organoids are the next step in validating the pathways and therapeutic targets already identified. Precision medicine approaches with iPSC using either traditional strategies of screening drugs that target a known pathogenic mechanism as well as "blind-to-target" drug screenings that allow for patient stratification based on drug response rather than clinical characteristics are now being employed.
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Distinct discrepancy in breast cancer organoids recapitulation among molecular subtypes revealed by single-cell transcriptomes analysis.