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Human iPSC-Derived Microglia Integrate Into Cerebral Organoids and Assume an In Vivo-Like Phenotype.
Microglia, the brain-resident macrophages, are critically involved in numerous physiological and pathological brain processes, including neoplasms, epilepsy, and neurodegeneration. However, investigating microglial function is notoriously difficult because they are extremely sensitive to changes in their environment and drastically alter their transcriptional state and morphology once they are isolated from the brain and cultured in vitro. In vivo experiments in mice are likewise limited because of vast differences between mouse and human microglia, particularly regarding the expression of disease-associated genes. To overcome this issue, we developed a highly controlled in vitro cerebral organoid platform where human microglia adopt an in vivo-like phenotype. This approach allows long-term studies and high-throughput analysis of in vivo-like human microglia suitable for disease modeling and drug testing.
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Mimicking the Biological Sense of Taste In Vitro Using a Taste Organoids-on-a-Chip System.
Thanks to the gustatory system, humans can experience the flavors in foods and drinks while avoiding the intake of some harmful substances. Although great advances in the fields of biotechnology, microfluidics, and nanotechnologies have been made in recent years, this astonishing recognition system can hardly be replaced by any artificial sensors designed so far. Here, taste organoids are coupled with an extracellular potential sensor array to form a novel bioelectronic organoid and developed a taste organoids-on-a-chip system (TOS) for highly mimicking the biological sense of taste ex vivo with high stability and repeatability. The taste organoids maintain key taste receptors expression after the third passage and high cell viability during 7 days of on-chip culture. Most importantly, the TOS not only distinguishs sour, sweet, bitter, and salt stimuli with great specificity, but also recognizes varying concentrations of the stimuli through an analytical method based on the extraction of signal features and principal component analysis. It is hoped that this bioelectronic tongue can facilitate studies in food quality controls, disease modelling, and drug screening.
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Robust, Long-Term Culture of Endoderm-Derived Hepatic Organoids for Disease Modeling.
Organoid technologies have become a powerful emerging tool to model liver diseases, for drug screening, and for personalized treatments. These applications are, however, limited in their capacity to generate functional hepatocytes in a reproducible and efficient manner. Here, we generated and characterized the hepatic organoid (eHEPO) culture system using human induced pluripotent stem cell (iPSC)-derived EpCAM-positive endodermal cells as an intermediate. eHEPOs can be produced within 2 weeks and expanded long term (>16 months) without any loss of differentiation capacity to mature hepatocytes. Starting from patient-specific iPSCs, we modeled citrullinemia type 1, a urea cycle disorder caused by mutations in the argininosuccinate synthetase (ASS1) enzyme. The disease-related ammonia accumulation phenotype in eHEPOs could be reversed by the overexpression of the wild-type ASS1 gene, which also indicated that this model is amenable to genetic manipulation. Thus, eHEPOs are excellent unlimited cell sources to generate functional hepatic organoids in a fast and efficient manner.
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Compressive molding of engineered tissues via thermoresponsive hydrogel devices.
Biofabrication of tissues requires sourcing appropriate combinations of cells, and then arranging those cells into a functionally-useful construct. Recently, organoids with diverse cell populations have shown great promise as building blocks from which to assemble more complex structures. However, organoids typically adopt spherical or uncontrolled morphologies, which intrinsically limit the tissue structures that can be produced using this bioassembly technique. Here, we develop microfabricated smart hydrogel platforms in thermoresponsive poly(N-isopropylacrylamide) to compressively mold microtissues such as spheroids or organoids into customized forms, on demand. These Compressive Hydrogel Molders (CHyMs) compact at cell culture temperatures to force loaded tissues into a new shape, and then expand to release the tissues for downstream applications. As a first demonstration, breast cancer spheroids were biaxially compacted in cylindrical cavities, and uniaxially compacted in rectangular ones. Spheroid shape changes persisted after the tissues were released from the CHyMs. We then demonstrate long-term molding of spherical brain organoids in ring-shaped CHyMs over one week. Fused bridges formed only when brain organoids were encased in Matrigel, and the resulting ring-shaped organoids expressed tissue markers that correspond with expected differentiation profiles. These results demonstrate that tissues differentiate appropriately even during long-term molding in a CHyM. This platform hence provides a new tool to shape pre-made tissues as desired, via temporary compression and release, allowing an exploration of alternative organoid geometries as building blocks for bioassembly applications.
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Apolipoprotein E regulates lipid metabolism and α-synuclein pathology in human iPSC-derived cerebral organoids.
APOE4 is a strong genetic risk factor for Alzheimer's disease and Dementia with Lewy bodies; however, how its expression impacts pathogenic pathways in a human-relevant system is not clear. Here using human iPSC-derived cerebral organoid models, we find that APOE deletion increases α-synuclein (αSyn) accumulation accompanied with synaptic loss, reduction of GBA levels, lipid droplet accumulation and dysregulation of intracellular organelles. These phenotypes are partially rescued by exogenous apoE2 and apoE3, but not apoE4. Lipidomics analysis detects the increased fatty acid utilization and cholesterol ester accumulation in apoE-deficient cerebral organoids. Furthermore, APOE4 cerebral organoids have increased αSyn accumulation compared to those with APOE3. Carrying APOE4 also increases apoE association with Lewy bodies in postmortem brains from patients with Lewy body disease. Our findings reveal the predominant role of apoE in lipid metabolism and αSyn pathology in iPSC-derived cerebral organoids, providing mechanistic insights into how APOE4 drives the risk for synucleinopathies.
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A Deep Learning-Driven Framework Integrating Organoid-Based Functional Validation Identifies Universal Neoantigens from Recurrent Glioma Mutations.
UNLABELLED: Glioblastoma (GBM) is the most common malignant intracranial tumor in adults, with a median survival of only 16 to 20 months. Neoantigen therapy has shown advantages in the treatment of GBM, as it improves the immunosuppressive microenvironment within the tumor. However, the identification of truly immunogenic neoantigens remains a major challenge. Current computational prediction tools primarily focus on antigen presentation, whereas algorithms that incorporate T-cell immunogenicity features remain limited. Furthermore, standard validation methods, such as enzyme-linked immunospot (ELISpot) assays, lack physiologic relevance and do not fully recapitulate the tumor microenvironment. In this study, we developed a neoantigen prediction algorithm, TCRscore, based on publicly available datasets by integrating human leukocyte antigen binding and T-cell receptor (TCR) recognition features. Twenty-one patient-derived GBM organoid models were established from isocitrate dehydrogenase wild-type tumors to validate the performance of the algorithm. Predicted neoantigens were evaluated using ELISpot assays, flow cytometry, and in vitro killing assays based on organoid-T cell coculture systems. TCRscore outperformed six existing tools in predicting immunogenic neoepitopes. The organoid models retained the key histologic and transcriptomic features of parental tumors and provided an effective platform for functional validation. Coculture assays confirmed that neoantigen-specific T cells could induce targeted killing in GBM organoids. In particular, the analysis identified that the recurrent PIK3R1G376R mutation contributed to a potential shared neoantigen in GBM. Overall, by integrating TCRscore with organoid-based validation, this study provides a high-fidelity, high-quality GBM neoantigen database with significantly enhanced prediction accuracy. SIGNIFICANCE: A clinically impactful framework that integrates a TCR-aware AI algorithm with glioblastoma organoids enables accurate neoantigen prediction and validation, advancing both personalized and population-level immunotherapy strategies.
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Network Analysis Pipeline for Neuronal 2D and 3D Organoid Multielectrode Recordings.
Multielectrode recordings from 2D or 3D cultures are commonly used to assess neuronal activity including action potential firing and burst rates. Yet, multielectrode recordings, which carry parallel time series, can also be used to infer functional connectivity and to compare network features from neuronal networks within neuronal 2D cultures or 3D organoids. This chapter provides a protocol for using the microelectrode array network analysis pipeline (MEA-NAP). With MEA-NAP, users with limited computational experience can easily evaluate network activity in their microelectrode array (MEA) recordings. Users with more computational experience can customize this open-source tool. Analysis of the functional connectivity and network features in MEA-NAP can provide insight into the development of neural circuits and how disruption of neuronal function, for example, in neurologic and psychiatric disorders, alters network function.
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Rectal organoid morphology analysis (ROMA) as a novel physiological assay for diagnostic classification in cystic fibrosis.
BACKGROUND: Diagnosing cystic fibrosis (CF) is not always straightforward, in particular when sweat chloride concentration (SCC) is intermediate and <2 CF-causing CFTR variants are identified. The physiological CFTR assays proposed in the guidelines, nasal potential difference and intestinal current measurement, are not readily available nor feasible at all ages. Rectal organoid morphology analysis (ROMA) was previously shown to discriminate between organoids from subjects with and without CF based on a distinct phenotypical difference: compared with non-CF organoids, CF organoids have an irregular shape and lack a visible lumen. The current study serves to further explore the role of ROMA when a CF diagnosis is inconclusive. METHODS: Organoid morphology was analysed using the previously established ROMA protocol. Two indices were calculated: the circularity index to quantify the roundness of organoids and the intensity ratio as a measure of the presence of a central lumen. RESULTS: Rectal organoids from 116 subjects were cultured and analysed together with the 189 subjects from the previous study. ROMA almost completely discriminated between CF and non-CF. ROMA indices correlated with SCC, pancreatic status and genetics, demonstrating convergent validity. For cases with an inconclusive diagnosis according to current guidelines, ROMA provided additional diagnostic information, with a diagnostic ROMA classification for 18 of 24 (75%). DISCUSSION: ROMA provides additional information to support a CF diagnosis when SCC and genetics are insufficient for diagnostic classification. ROMA is standardised and can be centralised, allowing future inclusion in the diagnostic work-up as first-choice physiological assay in case of an unclear diagnosis.
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Electrophysiological Analysis of Retinal Organoid Development Using 3D Microelectrodes of Liquid Metals.
Despite of the substantial potential of human-derived retinal organoids, the degeneration of retinal ganglion cells (RGCs) during maturation limits their utility in assessing the functionality of later-born retinal cell subtypes. Additionally, conventional analyses primarily rely on fluorescent emissions, which limits the detection of actual cell functionality while risking damage to the 3D cytoarchitecture of organoids. Here, an electrophysiological analysis is presented to monitor RGC development in early to mid-stage retinal organoids, and compare distinct features with fully-mature mouse retina. This approach utilizes high-resolution 3D printing of liquid-metal microelectrodes, enabling precise targeting of specific inner retinal layers within organoids. The adaptable distribution and softness of these microelectrodes facilitate the spatiotemporal recording of inner retinal signals. This study not only demonstrates the functional properties of RGCs in retinal organoid development but also provides insights into their synaptic connectivity, reminiscent of fetal native retinas. Further comparison with fully-mature mouse retina in vivo verifies the organoid features, highlighting the potential of early-stage retinal organoids in biomedical research.
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Establishment of 3D Neuro-Organoids Derived from Pig Embryonic Stem-Like Cells.
Although the human brain would be an ideal model for studying human neuropathology, it is difficult to perform in vitro culture of human brain cells from genetically engineered healthy or diseased brain tissue. Therefore, a suitable model for studying the molecular mechanisms responsible for neurological diseases that can appropriately mimic the human brain is needed. Somatic cell nuclear transfer (SCNT) was performed using an established porcine Yucatan EGFP cell line and whole seeding was performed using SCNT blastocysts. Two Yucatan EGFP porcine embryonic stem-like cell (pESLC) lines were established. These pESLC lines were then used to establish an in vitro neuro-organoids. Aggregates were cultured in vitro until 61 or 102 days after neural induction, neural patterning, and neural expansion. The neuro-organoids were sampled at each step and the expression of the dopaminergic neuronal marker (TH) and mature neuronal marker (MAP2) was confirmed by reverse transcription-PCR. Expression of the neural stem cell marker (PAX6), neural precursor markers (S100 and SOX2), and early neural markers (MAP2 and Nestin) were confirmed by immunofluorescence staining. In conclusion, we successfully established neuro-organoids derived from pESLCs in vitro. This protocol can be used as a tool to develop in vitro models for drug development, patient-specific chemotherapy, and human central nervous system disease studies.