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Glioblastoma organoids derived from patient tumor tissueas a model for studying extracellular communication and microRNAs in the tumor microenvironment.
Glioblastoma (GBM) is the most aggressive primary brain tumor in adult patients and is characterized by significant cellular heterogeneity and complex interactions between tumor cells and the tumor microenvironment. Extracellular vesicles (EVs) and the microRNAs (miRNAs) they carry are important mediators of intercellular signaling and, at the same time, a potential source of biomarkers useful in the diagnosis of gliomas. Experimental models capable of preserving the biological properties of the original tumor are essential for studying these processes. In this study, we prepared glioblastoma organoids (GBOs) derived from patient tumor tissue and analyzed extracellular miRNAs present in EVs isolated from conditioned media. Concurrently, we analyzed miRNAs contained in EVs isolated from cerebrospinal fluid (CSF) of patients with gliomas of varying degrees of malignancy. A total of 12 GBOs cultures were successfully established, long-term cultured, and cryopreserved. Immunohistochemical analysis confirmed the presence of markers typical of glioblastoma cells (Nestin, GFAP, Sox2). In early-stage organoids, cell populations representing the tumor microenvironment were also detected, including endothelial cells (CD31) and microglia (CD163). In some cases, reactive astrocytes and neurons were also present. Highly abundant miRNAs were identified in EVs isolated from conditioned media of GBO and primary GBM cultures, including hsa-miR-21-5p, members of the let-7 family, hsa-miR-16-5p, hsa-miR-125b-5p, and others. These miRNAs were simultaneously detected in EVs isolated from the CSF of patients with GBM and high-grade gliomas. In contrast, miR-204-5p, which was predominantly present in CSF samples, was minimally represented in EVs from tumor cultures, suggesting a possible origin of this miRNA in non-tumor brain tissue. The presented data support the use of GBOs derived from patient tumor tissue as a suitable model for studying intercellular communication and extracellular signaling in GBM and simultaneously demonstrate the potential of EV-associated miRNAs as biomarkers of the tumor microenvironment.
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Patient-derived organoids in human cancer: a platform for fundamental research and precision medicine.
Cancer is associated with a high degree of heterogeneity, encompassing both inter- and intra-tumor heterogeneity, along with considerable variability in clinical response to common treatments across patients. Conventional models for tumor research, such as in vitro cell cultures and in vivo animal models, demonstrate significant limitations that fall short of satisfying the research requisites. Patient-derived tumor organoids, which recapitulate the structures, specific functions, molecular characteristics, genomics alterations and expression profiles of primary tumors. They have been efficaciously implemented in illness portrayal, mechanism exploration, high-throughput drug screening and assessment, discovery of innovative therapeutic targets and potential compounds, and customized treatment regimen for cancer patients. In contrast to conventional models, tumor organoids offer an intuitive, dependable, and efficient in vitro research model by conserving the phenotypic, genetic diversity, and mutational attributes of the originating tumor. Nevertheless, the organoid technology also confronts the bottlenecks and challenges, such as how to comprehensively reflect intra-tumor heterogeneity, tumor microenvironment, tumor angiogenesis, reduce research costs, and establish standardized construction processes while retaining reliability. This review extensively examines the use of tumor organoid techniques in fundamental research and precision medicine. It emphasizes the importance of patient-derived tumor organoid biobanks for drug development, screening, safety evaluation, and personalized medicine. Additionally, it evaluates the application of organoid technology as an experimental tumor model to better understand the molecular mechanisms of tumor. The intent of this review is to explicate the significance of tumor organoids in cancer research and to present new avenues for the future of tumor research.
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Human meibomian gland organoids to study epithelial homeostasis and dysfunction.
Meibomian glands (MGs) are holocrine glands that secrete lipids to maintain the homeostasis of the ocular surface, and their dysfunction leads to dry eye disease. Herein, we established long-term 3D organoid culture for murine and human MGs, which retained the cell lineages and lipid-producing ability. The organoids mimicked the drug treatment responses and generated functional MGs after orthotopic transplantation. Inspired by organoid cultures, we found FGF10 eye drops could rescue all-trans retinoic acid-induced MG dysfunction in mice. Besides, nicotinamide uniquely hampered the human MG organoid expansion by inhibiting FGF10 signaling. Single-cell atlas and lipidome not only aligned the delineated cell types and featured lipids between human MGs and organoids, but also highlighted MAPK signaling inhibition enhanced acinar cell differentiation and functional maturation of MG organoids. In summary, this study established an organoid platform to explore epithelial homeostasis and dysfunction of MGs, facilitating drug development and regenerative medicine for dry eye disease.
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Central nervous system organoids for modeling neurodegenerative diseases.
Recent advances in induced pluripotent stem cell (iPSC) technology have allowed researchers to generate neurodegenerative disease-specific iPSCs and use the cells to derive a variety of relevant cell populations for laboratory modeling and drug testing. Nevertheless, these efforts have faced challenges related to immaturity and lack of complex developmental niches in the derived cell populations, limiting the utility of these in vitro models of neurodegenerative disease. Such limitations may be overcome by using human iPSC technology to generate three-dimensional (3D) brain organoids, which better recapitulate in vivo tissue architecture than traditional neuronal cultures to provide more complex and representative disease models and drug testing systems. In this review, we focus on the application of pluripotent stem cell-derived central nervous system (CNS) organoids to model neurodegenerative diseases. We first summarize recent progress in generating and characterizing various CNS organoids from pluripotent stem cells. We then review the application of CNS organoids for modeling several different human neurodegenerative diseases. We also describe several novel pathological mechanisms and drugs that were studied using patient iPSC-derived CNS organoids. Finally, we discuss remaining challenges and emerging opportunities for the use of 3D brain organoids for in vitro modeling of CNS development and neurodegeneration.
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Human Spinal Organoid-on-a-Chip to Model Nociceptive Circuitry for Pain Therapeutics Discovery.
The discovery of new pain therapeutics targeting human nociceptive circuitry is an emerging, exciting, and rewarding field. However, current models for evaluating prospective new therapeutics [e.g., animals and two-dimensional (2D) in vitro cultures] fail to fully recapitulate the complexity of human nociceptive neuron and dorsal horn neuron biology, significantly limiting the development of novel pain therapeutics. Here, we report human spinal organoid-on-a-chip devices for modeling the biology and electrophysiology of human nociceptive neurons and dorsal horn interneurons in nociceptive circuitry. Our device can be simply made through the integration of a membrane with a three-dimensional (3D)-printed organoid holder. By combining air-liquid interface culture and spinal organoid protocols, our devices can differentiate human stem cells into human sensori-spinal-cord organoids with dorsal spinal cord interneurons and sensory neurons. By easily transferring from culture well plates to the multiple-electrode array (MEA) system, our device also allows the plug-and-play measurement of organoid activity for testing nociceptive modulators (e.g., mustard oil, capsaicin, velvet ant venom, etc.). Our organoid-on-a-chip devices are cost-efficient, scalable, easy to use, and compatible with conventional well plates, allowing the plug-and-play measurement of spinal organoid electrophysiology. By the integration of human sensory-spinal-cord organoids with our organoid-on-a-chip devices, our method may hold the promising potential to screen and validate novel therapeutics for human pain medicine discovery.
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Exposure to the Amino Acids Histidine, Lysine, and Threonine Reduces mTOR Activity and Affects Neurodevelopment in a Human Cerebral Organoid Model.
Evidence of the impact of nutrition on human brain development is compelling. Previous in vitro and in vivo results show that three specific amino acids, histidine, lysine, and threonine, synergistically inhibit mTOR activity and behavior. Therefore, the prenatal availability of these amino acids could be important for human neurodevelopment. However, methods to study the underlying mechanisms in a human model of neurodevelopment are limited. Here, we pioneer the use of human cerebral organoids to investigate the impact of amino acid supplementation on neurodevelopment. In this study, cerebral organoids were exposed to 10 mM and 50 mM of the amino acids threonine, histidine, and lysine. The impact was determined by measuring mTOR activity using Western blots, general cerebral organoid size, and gene expression by RNA sequencing. Exposure to threonine, histidine, and lysine led to decreased mTOR activity and markedly reduced organoid size, supporting findings in rodent studies. RNA sequencing identified comprehensive changes in gene expression, with enrichment in genes related to specific biological processes (among which are mTOR signaling and immune function) and to specific cell types, including proliferative precursor cells, microglia, and astrocytes. Altogether, cerebral organoids are responsive to nutritional exposure by increasing specific amino acid concentrations and reflect findings from previous rodent studies. Threonine, histidine, and lysine exposure impacts the early development of human cerebral organoids, illustrated by the inhibition of mTOR activity, reduced size, and altered gene expression.
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Stress-free cell aggregation by using the CEPT cocktail enhances embryoid body and organoid fitness.
Embryoid bodies (EBs) and self-organizing organoids derived from human pluripotent stem cells (hPSCs) recapitulate tissue development in a dish and hold great promise for disease modeling and drug development. However, current protocols are hampered by cellular stress and apoptosis during cell aggregation, resulting in variability and impaired cell differentiation. Here, we demonstrate that EBs and various organoid models (e.g., brain, gut, kidney) can be optimized by using the small molecule cocktail named CEPT (chroman 1, emricasan, polyamines, trans-ISRIB), a polypharmacological approach that ensures cytoprotection and cell survival. Application of CEPT for just 24 h during cell aggregation has long-lasting consequences affecting morphogenesis, gene expression, cellular differentiation, and organoid function. Various qualification methods confirmed that CEPT treatment enhanced experimental reproducibility and consistently improved EB and organoid fitness as compared to the widely used ROCK inhibitor Y-27632. Collectively, we discovered that stress-free cell aggregation and superior cell survival in the presence of CEPT are critical quality control determinants that establish a robust foundation for bioengineering complex tissue and organ models.
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A human corticospinal organoid-slice connectoid model informs enhancer strategies for post-injury axon regrowth.
Axon elongation in the mammalian central nervous system (CNS) declines during development, limiting regenerative capacity after birth. Intrinsic regulators of this process are promising repair targets, as immature axons can regrow in tissues otherwise not conducive to regeneration. Yet the precise timing and mechanisms underlying the cessation of axon growth in the human CNS remain unresolved. Here, we developed a three-dimensional human corticospinal motor organoid-slice connectoid platform mimicking the developmental axon elongation program and its subsequent restriction through maturation. Cortical and spinal slices establish functional connections while remaining spatially segregated, enabling cortical cell-type-specific observations without direct confounding effects by spinal cells. Using single-cell transcriptomics, computational analyses, axon regrowth assays, and live imaging, we identified transcriptional alterations contributing to decreased axon growth in maturing human cortical projection neurons. We further demonstrate that this decline can be reversed using compounds and repurposable drugs targeting a maturation-associated transcriptional shift, promoting post-injury axon repair.
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Protocol for derivation of organoids and patient-derived orthotopic xenografts from glioma patient tumors.
Tumor organoids and patient-derived orthotopic xenografts (PDOXs) are some of the most valuable pre-clinical tools in cancer research. In this protocol, we describe efficient derivation of organoids and PDOX models from glioma patient tumors. We provide detailed steps for organoid culture, intracranial implantation, and detection of tumors in the brain. We further present technical adjustments for standardized functional assays and drug testing. For complete details on the use and execution of this protocol, please refer to Golebiewska et al. (2020).
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Generation of Intestinal and Colonic Organoids Derived From Human Pluripotent Stem Cells.
Over the past decade, significant advancements have been made in understanding the developmental mechanisms involved in human gastrointestinal formation, with organoids emerging as key experimental models. These three-dimensional in vitro cellular structures mimic the organization and functions of various gut regions, providing a powerful tool for research. By replicating critical stages of gut development, we can now direct the differentiation of cells into specific gastrointestinal tissues. In this protocol, we outline how to generate two types of organoids derived from human pluripotent stem cells (hPSCs): human intestinal organoids (HIOs) and human colonic organoids (HCOs). First, we induce definitive endoderm formation to produce these organoids and specify midgut/hindgut tissues. Three-dimensional spheroids form spontaneously, can be collected, embedded in an extracellular matrix, and cultured over time. During this phase, the organoid epithelium develops, supported by a mesenchymal layer that promotes maturation and differentiation. After a month of culture, HIOs and HCOs reach a developmental and maturation stage comparable to that of the human fetal intestine. These organoids can be used to study human gastrointestinal development, model diseases, and test therapeutic agents.