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Altered lipid metabolism marks glioblastoma stem and non-stem cells in separate tumor niches.
Glioblastoma (GBM) displays marked cellular and metabolic heterogeneity that varies among cellular microenvironments within a tumor. Metabolic targeting has long been advocated as a therapy against many tumors including GBM, but how lipid metabolism is altered to suit different microenvironmental conditions and whether cancer stem cells (CSCs) have altered lipid metabolism are outstanding questions in the field. We interrogated gene expression in separate microenvironments of GBM organoid models that mimic the transition between nutrient-rich and nutrient-poor pseudopalisading/perinecrotic tumor zones using spatial-capture RNA-sequencing. We revealed a striking difference in lipid processing gene expression and total lipid content between diverse cell populations from the same patient, with lipid enrichment in hypoxic organoid cores and also in perinecrotic and pseudopalisading regions of primary patient tumors. This was accompanied by regionally restricted upregulation of hypoxia-inducible lipid droplet-associated (HILPDA) gene expression in organoid cores and pseudopalisading regions of clinical GBM specimens, but not lower-grade brain tumors. CSCs have low lipid droplet accumulation compared to non-CSCs in organoid models and xenograft tumors, and prospectively sorted lipid-low GBM cells are functionally enriched for stem cell activity. Targeted lipidomic analysis of multiple patient-derived models revealed a significant shift in lipid metabolism between GBM CSCs and non-CSCs, suggesting that lipid levels may not be simply a product of the microenvironment but also may be a reflection of cellular state. CSCs had decreased levels of major classes of neutral lipids compared to non-CSCs, but had significantly increased polyunsaturated fatty acid production due to high fatty acid desaturase (FADS1/2) expression which was essential to maintain CSC viability and self-renewal. Our data demonstrate spatially and hierarchically distinct lipid metabolism phenotypes occur clinically in the majority of patients, can be recapitulated in laboratory models, and may represent therapeutic targets for GBM.
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Biotechnical Multiscale Engineering of Scaffolds for Stem Cell and Organoid Research.
The publication describes complex support structures and scaffolds for stem cells and organoids. Consideration of geometric and structural parameters has an influence on stem cell development and organogenesis comparable to that of molecular genetics and biochemical parameters. Two essential representatives are discussed here in more detail: hematopoietic stem cells (HSCs) and brain organoids. Due to their ability to fully regenerate the blood system, HSCs are used for stem cell transplantations. Therefore, efficient approaches to create an artificial but close-to-nature stem cell niche in vitro for amplification of HSCs are highly desirable. Apart from biochemical and biological factors, geometrical and biomechanical parameters are important. Biotechnological multiscale engineering is able to mimic the HSC niche, improving their amplification. Another highly dynamic process underlying hierarchic orders is organogenesis. Entire organs develop from individual cells that are spatially arranged in precise patterns and exposed to chemical, mechanical, and structural stimuli. The significance of the different scales during their development is explained using human brain organoids. Here, geometrically suitable structures improve biochemical differentiation protocols. Such technical hybrid systems can foster research of a rather inaccessible organ and possibly serve as a platform for more energy-efficient computing devices, such as organoid automata, hence, orgamats.
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Pluripotent stem cell derived intestinal organoids with an enteric nervous system.
The use of human pluripotent stem cells (hPSCs) and differentiation techniques offer new ways to generate specific tissue. It is now possible to differentiate hPSC into human intestinal organoids that include an enteric nervous system. Using step-wise differentiation processes, we generate innervated intestinal organoids that form three-dimensional structures bearing an epithelium, neurons and glial cells embedded in a supporting mesenchyme. Innervated organoids further develop to a complex structure with similar organization and cellular differentiation as the developing intestine. These tools open up new fields of application in the study of the development and pathophysiology of enteric neuropathies. Herein, we describe the generation of both human intestinal organoids and vagal neural crest cells from hPSC and their combination into an innervated organoid. We also discuss technical considerations for these experiments, and highlight advantages and limitations of the system.
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Therapeutic Transplantation of Human Central Nervous System Organoids for Neural Reconstruction.
Damage to the central nervous system (CNS) often leads to irreversible neurological deficits, and there are currently few effective treatments available. However, recent advancements in regenerative medicine have identified CNS organoids as promising therapeutic options for addressing CNS injuries. These organoids, composed of various neurons and supporting cells, have shown potential for direct repair at injury sites. CNS organoids resemble the structure and function of actual brain tissue, which allows them to adapt and function well within the physiological environment when transplanted into injury sites. Research findings suggest that CNS organoids can replace damaged neurons, form new neural connections, and promote neural recovery. This review highlights the emerging benefits, evaluates preclinical transplantation outcomes, and explores future strategies for optimizing neuroregeneration using CNS organoids. With continued research and technological advancements, these organoids could provide new hope for patients suffering from neurological deficits.
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Intravital Multimodal Imaging of Human Cortical Organoid Transplantation in a Mouse Model of Chronic Stroke.
Chronic stroke poses enduring neurological deficits, while experimental strategies based on human induced pluripotent stem cell-derived cortical organoids (COs) remain limited by the lack of tools for longitudinal in vivo graft monitoring. Here, we introduce the Multimodal Imaging Platform for Organoid Tracking (MIPOT), integrating MRI, bioluminescence imaging, surgical microscopy, and intravital two-photon fluorescence microscopy to monitor CO transplantation in chronic stroke lesions in mice. MIPOT enabled confirmation of initial graft placement within cleaned infarct cavities, longitudinal assessment of viability-associated bioluminescence signals, and high-resolution visualization of graft-derived cells and short-term cellular dynamics. Bioluminescence imaging revealed a progressive decline in graft-associated signals that stabilized at approximately 25% by two weeks, while endpoint histology confirmed limited persistence of human graft-derived cells at later time points. Together, MIPOT provides a multimodal framework for monitoring organoid transplantation in the injured brain and supports future optimization of graft survival, maturation, and host-graft integration.
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The proteomic architecture of schizophrenia iPSC-derived cerebral organoids reveals alterations in GWAS and neuronal development factors.
Schizophrenia (Scz) is a brain disorder that has a typical onset in early adulthood but otherwise maintains unknown disease origins. Unfortunately, little progress has been made in understanding the molecular mechanisms underlying neurodevelopment of Scz due to ethical and technical limitations in accessing developing human brain tissue. To overcome this challenge, we have previously utilized patient-derived Induced Pluripotent Stem Cells (iPSCs) to generate self-developing, self-maturating, and self-organizing 3D brain-like tissue known as cerebral organoids. As a continuation of this prior work, here we provide an architectural map of the developing Scz organoid proteome. Utilizing iPSCs from n = 25 human donors (n = 8 healthy Ctrl donors, and n = 17 Scz patients), we generated 3D cerebral organoids, employed 16-plex isobaric sample-barcoding chemistry, and simultaneously subjected samples to comprehensive high-throughput liquid-chromatography/mass-spectrometry (LC/MS) quantitative proteomics. Of 3,705 proteins identified by high-throughput proteomic profiling, we identified that just ~2.62% of the organoid global proteomic landscape was differentially regulated in Scz organoids. In sum, just 43 proteins were up-regulated and 54 were down-regulated in Scz patient-derived organoids. Notably, a range of neuronal factors were depleted in Scz organoids (e.g., MAP2, TUBB3, SV2A, GAP43, CRABP1, NCAM1 etc.). Based on global enrichment analysis, alterations in key pathways that regulate nervous system development (e.g., axonogenesis, axon development, axon guidance, morphogenesis pathways regulating neuronal differentiation, as well as substantia nigra development) were perturbed in Scz patient-derived organoids. We also identified prominent alterations in two novel GWAS factors, Pleiotrophin (PTN) and Podocalyxin (PODXL), in Scz organoids. In sum, this work serves as both a report and a resource that researchers can leverage to compare, contrast, or orthogonally validate Scz factors and pathways identified in observational clinical studies and other model systems.
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Single-cell sequencing and organoids: applications in organ development and disease.
The integration of single-cell sequencing and organoid technologies has been transformative for biomedical research, enabling investigations of organ development, disease mechanisms, and therapeutic innovation at even finer resolutions. Organoids serve as 3D in vitro models that replicate the structural and functional complexity of human tissues, while single-cell sequencing can resolve cellular heterogeneity, transcriptional dynamics, and lineage trajectories at high resolution. This review systematically explores the synergistic potential of these two technologies across multiple domains. First, it describes their application in studying the developmental mechanisms of organs including the brain, lungs, heart, liver, intestines, and kidneys, revealing key signaling pathways and cellular interaction networks. Then, it details their application in studying in vitro models of various diseases, including neurodegenerative disorders, genetic diseases, infectious diseases, metabolic syndrome, and tumors, advancing the in-depth analysis of pathological mechanisms. By leveraging patient-derived organoid biobanks, combining these two technologies can accelerate drug screening and precision, while utilizing transplantable tissue constructs to pioneer regenerative medicine strategies. This review also highlights the strengths of combining these two technologies in dynamically decoding cellular behavior and communication networks. By constructing physiologically relevant multifunctional research platforms, the integration of single-cell sequencing with organoid models will accelerate the elucidation of disease mechanisms and drive innovative breakthroughs in precision medicine and regenerative medicine. Looking ahead, the deep integration of single-cell sequencing with organoids, combined with cutting-edge technologies such as spatial transcriptomics and gene editing, will continue to propel life sciences toward a transformative leap from descriptive research to mechanism-driven, precision-oriented, and personalized approaches.
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Comparative Analysis of Ultra-Low Versus Hydrogel Growing Conditions for Patient-Derived Glioma Organoids.
BACKGROUND: Most data related to high-grade glioma and glioblastoma (GBM) 3D models refer to ultra-low/suspension cultivation systems, for several reasons: easy to initiate, high penetrability of treatments, ease of harvesting for downstream analysis. However, the major limitation of these models is the absence of the extracellular matrix, which enables the specific, local invasiveness of these aggressive brain tumors. AIM: This study provides a comparison between ultra-low and several extracellular matrix (ECM) and hydrogel culture conditions for various grades of patient-derived glioma organoids, in terms of cell growth, invasiveness, and release of cytokines/chemokines, as a model for downstream analysis of soluble factors. MATERIALS AND METHODS: Patient-derived glioma organoids were initiated in three different conditions: ultra-low attachment(UL), 3D Matrigel, and dextran-based hydrogels. Human endothelial cells were used for tube assay co-cultures and human normal monocyte cell line SC to assess monocyte invasion and interaction with tumor organoids. RESULTS: UL conditions supported organoid formation with better growth rate and easy passaging, and reliable detection of secreted cytokines and chemokines, closely mirroring qualitative secretory profiles observed in tumor explants. In contrast, ECM-based systems enabled invasive growth patterns and co-culture assays, but with a restricted immune cell infiltration and with a reduced detectable level of soluble factors. While qualitative cytokine signatures were preserved across culture platforms, substantial quantitative differences were observed, highlighting the influence of matrix composition on measurable outputs. Preliminary data showed that organoid-forming capacity indicated no clear association with Isocitrate Dehydrogenase 1 (IDH1) mutational status in the tested samples. CONCLUSION: Our findings indicate that UL and ECM-based models provide complementary information and recommend a dual-platform cell culture initiation strategy to maximize robustness and translational relevance in patient-derived glioma research.
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Microglia determine an immune-challenged environment and facilitate ibuprofen action in human retinal organoids.
Prenatal immune challenges pose significant risks to human embryonic brain and eye development. However, our knowledge about the safe usage of anti-inflammatory drugs during pregnancy is still limited. While human induced pluripotent stem cells (hIPSC)-derived brain organoid models have started to explore functional consequences upon viral stimulation, these models commonly lack microglia, which are susceptible to and promote inflammation. Furthermore, microglia are actively involved in neuronal development. Here, we generate hIPSC-derived microglia precursor cells and assemble them into retinal organoids. Once the outer plexiform layer forms, these hIPSC-derived microglia (iMG) fully integrate into the retinal organoids. Since the ganglion cell survival declines by this time in 3D-retinal organoids, we adapted the model into 2D and identify that the improved ganglion cell number significantly decreases only with iMG presence. In parallel, we applied the immunostimulant POLY(I:C) to mimic a fetal viral infection. While POLY(I:C) exposure alters the iMG phenotype, it does not hinder their interaction with ganglion cells. Furthermore, iMG significantly enhance the supernatant's inflammatory secretome and increase retinal cell proliferation. Simultaneous exposure with the non-steroidal anti-inflammatory drug (NSAID) ibuprofen dampens POLY(I:C)-mediated changes of the iMG phenotype and ameliorates cell proliferation. Remarkably, while POLY(I:C) disrupts neuronal calcium dynamics independent of iMG, ibuprofen rescues this effect only if iMG are present. Mechanistically, ibuprofen targets the enzymes cyclooxygenase 1 and 2 (COX1/PTGS1 and COX2/PTGS2) simultaneously, from which iMG mainly express COX1. Selective COX1 blockage fails to restore the calcium peak amplitude upon POLY(I:C) stimulation, suggesting ibuprofen's beneficial effect depends on the presence and interplay of COX1 and COX2. These findings underscore the importance of microglia in the context of prenatal immune challenges and provide insight into the mechanisms by which ibuprofen exerts its protective effects during embryonic development.
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Heart Organoids and Engineered Heart Tissues: Novel Tools for Modeling Human Cardiac Biology and Disease.
Organoids are three-dimensional in vitro cell constructs that recapitulate organ properties and structure to a significant extent. They constitute particularly useful models to study unapproachable states in humans, such as embryonic and fetal development, or early disease progression in adults. In recent years organoids have been implemented to model a wide range of different organs and disease conditions. However, the technology for their fabrication and application to cardiovascular studies has been lagging significantly when compared to other organoid types (e.g., brain, pancreas, kidney, intestine). This is a surprising fact since cardiovascular disease (CVD) and congenital heart disease (CHD) constitute the leading cause of mortality and morbidity in the developed world, and the most common birth defect in humans, respectively, and collectively constitute one of the largest unmet medical needs in the modern world. There is a critical need to establish in vitro models of the human heart that faithfully recapitulate its biology and function, thus enabling basic and translational studies to develop new therapeutics. Generating heart organoids that truly resemble the heart has proven difficult due to its complexity, but significant progress has been made recently to overcome this obstacle. In this review, we will discuss progress in novel heart organoid generation methods, the advantages and disadvantages of each approach, and their translational applications for advancing cardiovascular studies and the treatment of heart disorders.