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Modeling heart failure by induced pluripotent stem cell-derived organoids.
Cardiac organoids offer significant advantages for in vitro studies, as their 3D structure and cellular composition more closely replicate tissue complexity compared to 2D models. This is particularly relevant for studying complex diseases like heart failure (HF), which involve multiple cell types and cardiac structures. Thus, the primary aim of this study was to produce self-assembled, scaffold-free cardiac organoids from induced pluripotent stem cells (iPSCs), capable of simulating key aspects of HF in vitro. Gene expression analysis confirmed a transition from stemness markers (OCT4, NANOG) to cardiac markers (TNNT2, DES), validating their cardiac phenotype. To induce hallmark HF features, endothelin-1 (ET-1) treatment was applied. Key findings indicate that this experimental model successfully reproduced HF pathological markers, including the upregulation of genes encoding atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and the cytoskeletal protein α-skeletal actin (ACTA1), along with changes in microRNA (miR) expression profiles. Functionally, ET-1 treatment reduced organoid contractility, indicating a decline in contractile function-a hallmark of HF. Furthermore, histological analyses by Thioflavin T (ThT) staining, ThT fluorescence assay and filter trap assay on protein extracts demonstrated protein aggregation following ET-1 treatment. Co-administration of various nutraceuticals was shown to mitigate these effects. These findings underscore the value of this ET-1-stimulated cardiac organoid model as a powerful platform for studying HF mechanisms and evaluating novel therapeutic approaches.
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Single-cell multimodal transcriptomics to study neuronal diversity in human stem cell-derived brain tissue and organoid models.
Advances in human cell reprogramming and induced pluripotent stem cell technologies generate tremendous potential for neuroscience studies in health and disease, while the neuroscientist toolbox for engineering a range of brain tissues and neuronal cell types is rapidly expanding. Here, we discuss how the emergence of new single-cell genomics methods may help benchmarking and optimizing the tissue engineering process. The inherent heterogeneity and variability of reprogrammed brain tissue may conceal important disease mechanisms if not accounted for by rigorous experimental design. Single-cell genomics methods may address this technical challenge and ultimately improve the development of new therapeutics for neurological and psychiatric disorders.
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CRISPR screens in human neural organoids and assembloids.
Studying the molecular mechanisms underlying the assembly of the human nervous system remains a significant challenge. The ability to generate neural cells from pluripotent stem cells, combined with advanced genome-editing techniques, provides unprecedented opportunities to uncover the biology of human neurodevelopment and disease. Organoids and assembloids enable the in vitro modeling of previously inaccessible developmental processes, such as the specification and migration of human neurons, including the integration of cortical interneurons from the ventral into the dorsal forebrain. Here, we present a detailed protocol that combines pooled CRISPR-Cas9 screening with neural organoid and assembloid models and illustrate how it can be applied to map hundreds of disease genes onto cellular pathways and specific aspects of human neural development. Our protocol outlines key steps, from planning and optimizing genetic perturbations to designing readouts for neuronal generation and migration, conducting the screening and validating candidate genes. The screening experiments take ~3 months to complete and require expertise in stem cell culture and neural differentiation, genetic engineering of human induced pluripotent stem cell lines, fluorescence-activated cell sorting and next-generation sequencing and analyses. The integration of genetic screening and human cellular models constitutes a powerful platform for investigating the mechanisms of human brain development and disease, paving the way for the discovery of novel therapeutics.
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Genetically modified organoids for tissue engineering and regenerative medicine.
To date, genetically modified organoids are emerging as a promising 3D modeling tool aimed at solving genetically relevant clinical and biomedical problems for regenerative medicine and tissue engineering. As an optimal vehicle for gene delivery, genetically modified organoids can enhance or reduce the expression of target genes through virus and non-virus-based gene transfection methods to achieve tissue regeneration. Animal experiments and preclinical studies have demonstrated the beneficial role of genetically modified organoids in various aspects of organ regeneration, including thymus, lacrimal glands, brain, lung, kidney, photoreceptors, etc. Furthermore, the technology offers a potential treatment option for various diseases, such as Fabry disease, non-alcoholic steatohepatitis, and Lynch syndrome. Nevertheless, the uncertain safety of genetic modification, the risk of organoid application, and bionics of current genetically modified organoids are still challenging. This review summarizes the researches on genetically modified organoids in recent years, and describes the transfection methods and functions of genetically modified organoids, then introduced their applications at length. Also, the limitations and future development directions of genetically modified organoids are included.
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A novel human organoid model system reveals requirement of TCF4 for oligodendroglial differentiation.
Heterozygous mutations of TCF4 in humans cause Pitt-Hopkins syndrome, a neurodevelopmental disease associated with intellectual disability and brain malformations. Although most studies focus on the role of TCF4 in neural stem cells and neurons, we here set out to assess the implication of TCF4 for oligodendroglial differentiation. We discovered that both monoallelic and biallelic mutations in TCF4 result in a diminished capacity to differentiate human neural progenitor cells toward myelinating oligodendrocytes through the forced expression of the transcription factors SOX10, OLIG2, and NKX6.2. Using this experimental strategy, we established a novel organoid model, which generates oligodendroglial cells within a human neurogenic tissue-like context. Also, here we found a reduced ability of TCF4 heterozygous cells to differentiate toward oligodendroglial cells. In sum, we establish a role of human TCF4 in oligodendrocyte differentiation and provide a model system, which allows to dissect the disease etiology in a human tissue-like context.
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Human organoids in basic research and clinical applications.
Organoids are three-dimensional (3D) miniature structures cultured in vitro produced from either human pluripotent stem cells (hPSCs) or adult stem cells (AdSCs) derived from healthy individuals or patients that recapitulate the cellular heterogeneity, structure, and functions of human organs. The advent of human 3D organoid systems is now possible to allow remarkably detailed observation of stem cell morphogens, maintenance and differentiation resemble primary tissues, enhancing the potential to study both human physiology and developmental stage. As they are similar to their original organs and carry human genetic information, organoids derived from patient hold great promise for biomedical research and preclinical drug testing and is currently used for personalized, regenerative medicine, gene repair and transplantation therapy. In recent decades, researchers have succeeded in generating various types of organoids mimicking in vivo organs. Herein, we provide an update on current in vitro differentiation technologies of brain, retinal, kidney, liver, lung, gastrointestinal, cardiac, vascularized and multi-lineage organoids, discuss the differences between PSC- and AdSC-derived organoids, summarize the potential applications of stem cell-derived organoids systems in the laboratory and clinic, and outline the current challenges for the application of organoids, which would deepen the understanding of mechanisms of human development and enhance further utility of organoids in basic research and clinical studies.
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Matched three-dimensional organoids and two-dimensional cell lines of melanoma brain metastases mirror response to targeted molecular therapy.
Despite advances in the treatment paradigm for patients with metastatic melanoma, melanoma brain metastasis (MBM) continues to represent a significant treatment challenge. The study of MBM is limited, in part, by shortcomings in existing preclinical models. Surgically eXplanted Organoids (SXOs) are ex vivo, three-dimensional cultures prepared from primary tissue samples with minimal processing that recapitulate genotypic and phenotypic features of parent tumors without an artificial extracellular scaffold. MBM SXOs were created by a novel protocol incorporating techniques for establishing glioma and cutaneous melanoma organoids. A BRAFV600K-mutant and BRAF-wildtype MBM sample were collected directly from the operating room. Organoids were cultured in an optimized culture medium without an artificial extracellular scaffold. Concurrently, matched patient-derived cell lines were created. Organoid growth was observed within 3-4 weeks, and MBM SXOs retained histological features of the parent tissue, including pleomorphic epithelioid cells with abundant cytoplasm, large nuclei, focal melanin accumulation, and strong SOX10 positivity. After sufficient growth, organoids could be manually parcellated to increase the number of replicates. Matched SXOs and cell lines demonstrated sensitivity to BRAF and MEK inhibitors. Further study using SXOs may improve the translational relevance of preclinical studies and enable the study of the metastatic melanoma tumor microenvironment.
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Patient-derived pediatric brain tumor orthotopic xenografts and tumor organoids faithfully recapitulate primary tumors.
Extensive molecular analyses by many groups have revealed a heterogenous landscape of embryonal brain tumors, but patient-derived tumor organoid (TO) model development has remained limited. Here, we describe the establishment of TO and TO xenografts (TOX) from patient-derived orthotopic xenografts (PDOXs) of medulloblastoma, embryonal tumor with multilayer rosettes, and atypical teratoid rhabdoid tumors. DNA methylation, bulk- and single-cell RNA sequencing, and whole-genome sequencing demonstrated that TOs and TOXs faithfully recapitulate the epigenetic, transcriptomic, and genetic landscape of PDOXs, as well as replicate the intratumor cellular heterogeneity of PDOXs that are often lost in established cell lines. We show that TOs and PDOXs have similar drug responses. The development of embryonal brain TOs will facilitate in vitro functional assays, including high-throughput drug or CRISPR screens, without the need for fresh tumors from tumor-bearing mice. This will accelerate the identification and validation of vulnerabilities and therapeutic strategies for preclinical testing toward clinical trials.
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Tackling the Many Facets of Glioblastoma Heterogeneity.
Glioblastoma is an incurable brain tumor notorious for its heterogeneity. Recent studies in Cell (Jacob et al., 2020) and Cell Stem Cell (Bhaduri et al., 2020) leverage novel glioblastoma organoid models and single-cell RNA-sequencing technologies to tackle glioblastoma's heterogeneous nature, providing new tools and insights into tumor biology.
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Cortical organoid-derived models of the melanoma brain metastatic niche enable prioritization of cancer-targeting drugs.
Effective systemic therapies against brain metastases are severely limited. To understand and target vulnerabilities of human metastases in a brain niche context, we developed reproducible melanoma brain metastasis (MBM) models for metastasis-integrating drug screening. We co-cultured A375 melanoma cells or tumor regional lymph node-derived disseminated cancer cells (DCCs) in close proximity with human induced pluripotent stem cell-derived cortical organoids (hCOs). In these, RNA sequencing revealed an upregulation of metastasis-associated features. First, A375 cells and DCCs were screened against an anti-cancer library containing 315 compounds. Hits were ranked by neurotoxicity, central nervous system permeation, and anti-DCC efficacy. Only a minority of hits effectively targeted A375-MBMs, with the first-in-class XPO1 inhibitor selinexor emerging as top hit. Selinexor also demonstrated efficacy in DCC-MBM models and low toxicity on hCOs, suggesting a promising therapeutic window in clinically applied doses. Collectively, the MBM model provides a tool for identifying candidate therapies counteracting metastatic progression.