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1
Logan, Sarah, Thiago Arzua, Yasheng Yan, Congshan Jiang, Xiaojie Liu, Lai-Kang Yu, Qing-Song Liu, and Xiaowen Bai. "Dynamic Characterization of Structural, Molecular, and Electrophysiological Phenotypes of Human-Induced Pluripotent Stem Cell-Derived Cerebral Organoids, and Comparison with Fetal and Adult Gene Profiles." Cells 9, no. 5 (May 23, 2020): 1301. http://dx.doi.org/10.3390/cells9051301.
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Background: The development of 3D cerebral organoid technology using human-induced pluripotent stem cells (iPSCs) provides a promising platform to study how brain diseases are appropriately modeled and treated. So far, understanding of the characteristics of organoids is still in its infancy. The current study profiled, for the first time, the electrophysiological properties of organoids at molecular and cellular levels and dissected the potential age equivalency of 2-month-old organoids to human ones by a comparison of gene expression profiles among cerebral organoids, human fetal and adult brains. Results: Cerebral organoids exhibit heterogeneous gene and protein markers of various brain cells, such as neurons, astrocytes, and vascular cells (endothelial cells and smooth muscle cells) at 2 months, and increases in neural, glial, vascular, and channel-related gene expression over a 2-month differentiation course. Two-month organoids exhibited action potentials, multiple channel activities, and functional electrophysiological responses to the anesthetic agent propofol. A bioinformatics analysis of 20,723 gene expression profiles showed the similar distance of gene profiles in cerebral organoids to fetal and adult brain tissues. The subsequent Ingenuity Pathway Analysis (IPA) of select canonical pathways related to neural development, network formation, and electrophysiological signaling, revealed that only calcium signaling, cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) signaling in neurons, glutamate receptor signaling, and synaptogenesis signaling were predicted to be downregulated in cerebral organoids relative to fetal samples. Nearly all cerebral organoid and fetal pathway phenotypes were predicted to be downregulated compared with adult tissue. Conclusions: This novel study highlights dynamic development, cellular heterogeneity and electrophysiological activity. In particular, for the first time, electrophysiological drug response recapitulates what occurs in vivo, and neural characteristics are predicted to be highly similar to the human brain, further supporting the promising application of the cerebral organoid system for the modeling of the human brain in health and disease. Additionally, the studies from these characterizations of cerebral organoids in multiple levels and the findings from gene comparisons between cerebral organoids and humans (fetuses and adults) help us better understand this cerebral organoid-based cutting-edge platform and its wide uses in modeling human brain in terms of health and disease, development, and testing drug efficacy and toxicity.
2
Estridge, R. Chris, Jennifer E. O’Neill, and Albert J. Keung. "Matrigel Tunes H9 Stem Cell-Derived Human Cerebral Organoid Development." Organoids 2, no. 4 (October 5, 2023): 165–76. http://dx.doi.org/10.3390/organoids2040013.
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Human cerebral organoids are readily generated from human embryonic stem cells and human induced pluripotent stem cells and are useful in studying human neurodevelopment. Recent work with human cerebral organoids have explored the creation of different brain regions and the impacts of soluble and mechanical cues. Matrigel is a gelatinous, heterogenous mixture of extracellular matrix proteins, morphogens, and growth factors secreted by Engelbreth-Holm-Swarm mouse sarcoma cells. It is a core component of almost all cerebral organoid protocols, generally supporting neuroepithelial budding and tissue polarization; yet, its roles and effects beyond its general requirement in organoid protocols are not well understood, and its mode of delivery is variable, including the embedding of organoids within it or its delivery in soluble form. Given its widespread usage, we asked how H9 stem cell-derived hCO development and composition are affected by Matrigel dosage and delivery method. We found Matrigel exposure influences organoid size, morphology, and cell type composition. We also showed that greater amounts of Matrigel promote an increase in the number of choroid plexus (ChP) cells, and this increase is regulated by the BMP4 pathway. These results illuminate the effects of Matrigel on human cerebral organoid development and the importance of delivery mode and amount on organoid phenotype and composition.
3
He, Zhisong, Ashley Maynard, Akanksha Jain, Tobias Gerber, Rebecca Petri, Hsiu-Chuan Lin, Malgorzata Santel, et al. "Lineage recording in human cerebral organoids." Nature Methods 19, no. 1 (December 30, 2021): 90–99. http://dx.doi.org/10.1038/s41592-021-01344-8.
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AbstractInduced pluripotent stem cell (iPSC)-derived organoids provide models to study human organ development. Single-cell transcriptomics enable highly resolved descriptions of cell states within these systems; however, approaches are needed to directly measure lineage relationships. Here we establish iTracer, a lineage recorder that combines reporter barcodes with inducible CRISPR–Cas9 scarring and is compatible with single-cell and spatial transcriptomics. We apply iTracer to explore clonality and lineage dynamics during cerebral organoid development and identify a time window of fate restriction as well as variation in neurogenic dynamics between progenitor neuron families. We also establish long-term four-dimensional light-sheet microscopy for spatial lineage recording in cerebral organoids and confirm regional clonality in the developing neuroepithelium. We incorporate gene perturbation (iTracer-perturb) and assess the effect of mosaic TSC2 mutations on cerebral organoid development. Our data shed light on how lineages and fates are established during cerebral organoid formation. More broadly, our techniques can be adapted in any iPSC-derived culture system to dissect lineage alterations during normal or perturbed development.
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Gomez-Jones, Tashaé, and Robert M. Kao. "Ethical Dimensions of Human Organoids Research." American Biology Teacher 83, no. 9 (November 2021): 575–78. http://dx.doi.org/10.1525/abt.2021.83.9.575.
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Over the past decade, the development of three-dimensional mammalian cell organization—called human organoids—from stem cells has provided a framework for future clinical therapies. As human organoid research progresses, we also need to keep in mind the cross-cultural and ethical dimensions of human organoids research. Our review article aims to examine the ethical dimensions of cerebral human organoids and provide an ethical framework guide within human organoids research.
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Bao, Zhongyuan, Kaiheng Fang, Zong Miao, Chong Li, Chaojuan Yang, Qiang Yu, Chen Zhang, Zengli Miao, Yan Liu, and Jing Ji. "Human Cerebral Organoid Implantation Alleviated the Neurological Deficits of Traumatic Brain Injury in Mice." Oxidative Medicine and Cellular Longevity 2021 (November 22, 2021): 1–16. http://dx.doi.org/10.1155/2021/6338722.
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Traumatic brain injury (TBI) causes a high rate of mortality and disability, and its treatment is still limited. Loss of neurons in damaged area is hardly rescued by relative molecular therapies. Based on its disease characteristics, we transplanted human embryonic stem cell- (hESC-) derived cerebral organoids in the brain lesions of controlled cortical impact- (CCI-) modeled severe combined immunodeficient (SCID) mice. Grafted organoids survived and differentiated in CCI-induced lesion pools in mouse cortical tissue. Implanted cerebral organoids differentiated into various types of neuronal cells, extended long projections, and showed spontaneous action, as indicated by electromyographic activity in the grafts. Induced vascularization and reduced glial scar were also found after organoid implantation, suggesting grafting could improve local situation and promote neural repair. More importantly, the CCI mice’s spatial learning and memory improved after organoid grafting. These findings suggest that cerebral organoid implanted in lesion sites differentiates into cortical neurons, forms long projections, and reverses deficits in spatial learning and memory, a potential therapeutic avenue for TBI.
6
Camp, J. Gray, Farhath Badsha, Marta Florio, Sabina Kanton, Tobias Gerber, Michaela Wilsch-Bräuninger, Eric Lewitus, et al. "Human cerebral organoids recapitulate gene expression programs of fetal neocortex development." Proceedings of the National Academy of Sciences 112, no. 51 (December 7, 2015): 15672–77. http://dx.doi.org/10.1073/pnas.1520760112.
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Cerebral organoids—3D cultures of human cerebral tissue derived from pluripotent stem cells—have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and previously unidentified interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single-cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.
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Yakoub, Abraam M., and Mark Sadek. "Development and Characterization of Human Cerebral Organoids." Cell Transplantation 27, no. 3 (March 2018): 393–406. http://dx.doi.org/10.1177/0963689717752946.
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Studies of human neurodevelopmental disorders and stem cell–based regenerative transplants have been hampered by the lack of a model of the developing human brain. Stem cell–derived neurons suffer major limitations, including the ability to recapitulate the 3-dimensional architecture of a brain tissue and the representation of multiple layers and cell types that contribute to the overall brain functions in vivo. Recently, cerebral organoid technology was introduced; however, such technology is still in its infancy, and its low reproducibility and limitations significantly reduce the reliability of such a model as it currently exists, especially considering the complexity of cerebral-organoid protocols. Here we have tested and compared multiple protocols and conditions for growth of organoids, and we describe an optimized methodology, and define the necessary and sufficient factors that support the development of optimal organoids. Our optimization criteria included organoids’ overall growth and size, stratification and representation of the various cell types, inter-batch variability, analysis of neuronal maturation, and even the cost of the procedure. Importantly, this protocol encompasses a plethora of technical tips that allow researchers to easily reproduce it and obtain reliable organoids with the least variability, and showcases a robust array of approaches to characterize successful organoids. This optimized protocol provides a reliable system for genetic or pharmacological (drug development) screens and may enhance understanding and therapy of human neurodevelopmental disorders, including harnessing the therapeutic potential of stem cell–derived transplants.
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Berdenis van Berlekom, Amber, Raphael Kübler, Jeske W. Hoogeboom, Daniëlle Vonk, Jacqueline A. Sluijs, R. Jeroen Pasterkamp, Jinte Middeldorp, et al. "Exposure to the Amino Acids Histidine, Lysine, and Threonine Reduces mTOR Activity and Affects Neurodevelopment in a Human Cerebral Organoid Model." Nutrients 14, no. 10 (May 23, 2022): 2175. http://dx.doi.org/10.3390/nu14102175.
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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.
9
Shnaider, T. A. "Cerebral organoids: a promising model in cellular technologies." Vavilov Journal of Genetics and Breeding 22, no. 2 (April 8, 2018): 168–78. http://dx.doi.org/10.18699/vj18.344.
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The development of the human brain is a complex multi-stage process including the formation of various types of neural cells and their interactions. Many fundamental mechanisms of neurogenesis have been established due to the studying of model animals. However, significant differences in the brain structure compared to other animals do not allow considering all aspects of the human brain formation, which could play the main role in the development of unique cognitive abilities for human. Four years ago, Lancaster’s group elaborated human pluripotent stem cell-derived three-dimensional cerebral organoid technology, which opened a unique opportunity for researchers to model early stages of human neurogenesis in vitro. Cerebral organoids closely remodel many endogenous brain regions with specific cell composition like ventricular zone with radial glia, choroid plexus, and cortical plate with upper and deeper-layer neurons. Moreover, human brain development includes interactions between different brain regions. Generation of hybrid three-dimensional cerebral organoids with different brain region identity allows remodeling some of them, including long-distance neuronal migration or formation of major axonal tracts. In this review, we consider the technology of obtaining human pluripotent stem cell-derived three-dimensional cerebral organoids with different modifications and with different brain region identity. In addition, we discuss successful implementation of this technology in fundamental and applied research like modeling of different neurodevelopmental disorders and drug screening. Finally, we regard existing problems and prospects for development of human pluripotent stem cell-derived threedimensional cerebral organoid technology.
10
Peng, Xiyao, Lei Wu, Qiushi Li, Yuqing Ge, Tiegang Xu, and Jianlong Zhao. "An Easy-to-Use Arrayed Brain–Heart Chip." Biosensors 14, no. 11 (October 22, 2024): 517. http://dx.doi.org/10.3390/bios14110517.
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Multi-organ chips are effective at emulating human tissue and organ functions and at replicating the interactions among tissues and organs. An arrayed brain–heart chip was introduced whose configuration comprises open culture chambers and closed biomimetic vascular channels distributed in a horizontal pattern, separated from each other by an endothelial barrier based on fibrin matrix. A 300 μm-high and 13.2 mm-long endothelial barrier surrounded each organoid culture chamber, thereby satisfying the material transport requirements. Numerical simulations were used to analyze the construction process of fibrin barriers in order to optimize the structural design and experimental manipulation, which exhibited a high degree of correlation with experiment results. In each interconnective unit, a cerebral organoid, a cardiac organoid, and endothelial cells were co-cultured stably for a minimum of one week. The permeability of the endothelial barrier and recirculating perfusion enabled cross talk between cerebral organoids and cardiac organoids, as well as between organoids and endothelial cells. This was corroborated by the presence of cardiac troponin I (cTnI) in the cerebral organoid culture chamber and the observation of cerebral organoid and endothelial cells invading the fibrin matrix after one week of co-culture. The arrayed chip was simple to manipulate, clearly visible under a microscope, and compatible with automated pipetting devices, and therefore had significant potential for application.
11
Santos, Alexandra C., George Nader, Dana El Soufi El Sabbagh, Karolina Urban, Liliana Attisano, and Peter L. Carlen. "Treating Hyperexcitability in Human Cerebral Organoids Resulting from Oxygen-Glucose Deprivation." Cells 12, no. 15 (July 27, 2023): 1949. http://dx.doi.org/10.3390/cells12151949.
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Human cerebral organoids resemble the 3D complexity of the human brain and have the potential to augment current drug development pipelines for neurological disease. Epilepsy is a complex neurological condition characterized by recurrent seizures. A third of people with epilepsy do not respond to currently available pharmaceutical drugs, and there is not one drug that treats all subtypes; thus, better models of epilepsy are needed for drug development. Cerebral organoids may be used to address this unmet need. In the present work, human cerebral organoids are used along with electrophysiological methods to explore oxygen-glucose deprivation as a hyperexcitability agent. This activity is investigated in its response to current antiseizure drugs. Furthermore, the mechanism of action of the drug candidates is probed with qPCR and immunofluorescence. The findings demonstrate OGD-induced hyperexcitable changes in the cerebral organoid tissue, which is treated with cannabidiol and bumetanide. There is evidence for NKCC1 and KCC2 gene expression, as well as other genes and proteins involved in the complex development of GABAergic signaling. This study supports the use of organoids as a platform for modelling cerebral cortical hyperexcitability that could be extended to modelling epilepsy and used for drug discovery.
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Harary, Paul M., Rachel Blue, Mackenzie Castellanos, Mehek Dedhia, Sarah Hamimi, Dennis Jgamadze, Benjamin Rees, et al. "Human brain organoid transplantation: ethical implications of enhancing specific cerebral functions in small-animal models." Molecular Psychology: Brain, Behavior, and Society 2 (June 6, 2023): 14. http://dx.doi.org/10.12688/molpsychol.17544.1.
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Brain organoids are self-organizing, three-dimensional tissues derived from pluripotent stem cells that recapitulate many aspects of the cellular diversity and architectural features of the developing brain. Recently, there has been growing interest in using human brain organoid transplantation in animal models as a means of addressing the limitations of in vitro culture, such as the lack of vascularization, and to explore the potential of organoids for neural repair. While there has been substantial debate on the ethical implications of brain organoid research, particularly the potential for organoids to exhibit higher-order brain functions such as consciousness, the impact of human organoid grafts on animal hosts has been less extensively discussed. Enhancement of host animal brain function may not be technically feasible at this time, but it is imperative to carefully consider the moral significance of these potential outcomes. Here, we discuss the ethical implications of enhancing somatosensation, motor processes, memory, and basic socialization in small-animal models. We consider the moral implications of such outcomes and if safeguards are needed to accommodate any increased moral status of animals transplanted with human brain organoids.
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Tanaka, Yoshiaki, and In-Hyun Park. "Regional specification and complementation with non-neuroectodermal cells in human brain organoids." Journal of Molecular Medicine 99, no. 4 (March 2, 2021): 489–500. http://dx.doi.org/10.1007/s00109-021-02051-9.
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AbstractAlong with emergence of the organoids, their application in biomedical research has been currently one of the most fascinating themes. For the past few years, scientists have made significant contributions to deriving organoids representing the whole brain and specific brain regions. Coupled with somatic cell reprogramming and CRISPR/Cas9 editing, the organoid technologies were applied for disease modeling and drug screening. The methods to develop organoids further improved for rapid and efficient generation of cerebral organoids. Additionally, refining the methods to develop the regionally specified brain organoids enabled the investigation of development and interaction of the specific brain regions. Recent studies started resolving the issue in the lack of non-neuroectodermal cells in brain organoids, including vascular endothelial cells and microglia, which play fundamental roles in neurodevelopment and are involved in the pathophysiology of acute and chronic neural disorders. In this review, we highlight recent advances of neuronal organoid technologies, focusing on the region-specific brain organoids and complementation with endothelial cells and microglia, and discuss their potential applications to neuronal diseases.
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Schultz, Emily M., TyAnthony J. Jones, Sibei Xu, Dana D. Dean, Bernd Zechmann, and Kelli L. Barr. "Cerebral Organoids Derived from a Parkinson’s Patient Exhibit Unique Pathogenesis from Chikungunya Virus Infection When Compared to a Non-Parkinson’s Patient." Pathogens 10, no. 7 (July 20, 2021): 913. http://dx.doi.org/10.3390/pathogens10070913.
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(1) Background: Arboviruses of medical and veterinary significance have been identified on all seven continents, with every human and animal population at risk for exposure. Like arboviruses, chronic neurodegenerative diseases, like Alzheimer’s and Parkinson’s disease, are found wherever there are humans. Significant differences in baseline gene and protein expression have been determined between human-induced pluripotent stem cell lines derived from non-Parkinson’s disease individuals and from individuals with Parkinson’s disease. It was hypothesized that these inherent differences could impact cerebral organoid responses to viral infection. (2) Methods: In this study, cerebral organoids from a non-Parkinson’s and Parkinson’s patient were infected with Chikungunya virus and observed for two weeks. (3) Results: Parkinson’s organoids lost mass and exhibited a differential antiviral response different from non-Parkinson’s organoids. Neurotransmission data from both infected non-Parkinson’s and Parkinson’s organoids had dysregulation of IL-1, IL-10, and IL-6. These cytokines are associated with mood and could be contributing to persistent depression seen in patients following CHIKV infection. Both organoid types had increased expression of CXCL10, which is linked to demyelination. (4) Conclusions: The differential antiviral response of Parkinson’s organoids compared with non-Parkinson’s organoids highlights the need for more research in neurotropic infections in a neurologically compromised host.
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Simsa, Robin, Theresa Rothenbücher, Hakan Gürbüz, Nidal Ghosheh, Jenny Emneus, Lachmi Jenndahl, David L. Kaplan, Niklas Bergh, Alberto Martinez Serrano, and Per Fogelstrand. "Brain organoid formation on decellularized porcine brain ECM hydrogels." PLOS ONE 16, no. 1 (January 28, 2021): e0245685. http://dx.doi.org/10.1371/journal.pone.0245685.
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Human brain tissue models such as cerebral organoids are essential tools for developmental and biomedical research. Current methods to generate cerebral organoids often utilize Matrigel as an external scaffold to provide structure and biologically relevant signals. Matrigel however is a nonspecific hydrogel of mouse tumor origin and does not represent the complexity of the brain protein environment. In this study, we investigated the application of a decellularized adult porcine brain extracellular matrix (B-ECM) which could be processed into a hydrogel (B-ECM hydrogel) to be used as a scaffold for human embryonic stem cell (hESC)-derived brain organoids. We decellularized pig brains with a novel detergent- and enzyme-based method and analyzed the biomaterial properties, including protein composition and content, DNA content, mechanical characteristics, surface structure, and antigen presence. Then, we compared the growth of human brain organoid models with the B-ECM hydrogel or Matrigel controls in vitro. We found that the native brain source material was successfully decellularized with little remaining DNA content, while Mass Spectrometry (MS) showed the loss of several brain-specific proteins, while mainly different collagen types remained in the B-ECM. Rheological results revealed stable hydrogel formation, starting from B-ECM hydrogel concentrations of 5 mg/mL. hESCs cultured in B-ECM hydrogels showed gene expression and differentiation outcomes similar to those grown in Matrigel. These results indicate that B-ECM hydrogels can be used as an alternative scaffold for human cerebral organoid formation, and may be further optimized for improved organoid growth by further improving protein retention other than collagen after decellularization.
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Delepine, Chloe, Vincent A. Pham, Hayley W. S. Tsang, and Mriganka Sur. "GSK3ß inhibitor CHIR 99021 modulates cerebral organoid development through dose-dependent regulation of apoptosis, proliferation, differentiation and migration." PLOS ONE 16, no. 5 (May 5, 2021): e0251173. http://dx.doi.org/10.1371/journal.pone.0251173.
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Cerebral organoids generated from human pluripotent stem cells (hiPSCs) are unique in their ability to recapitulate human-specific neurodevelopmental events. They are capable of modeling the human brain and its cell composition, including human-specific progenitor cell types; ordered laminar compartments; and both cell-specific transcriptional signatures and the broader telencephalic transcriptional landscape. The serine/threonine kinase, GSK3β, plays a critical role in neurodevelopment, controlling processes as varied as neurogenesis, morphological changes, polarization, and migration. In the generation of cerebral organoids, inhibition of GSK3β at low doses has been used to increase organoid size and decrease necrotic core. However, little is known of the effects of GSK3β inhibition on organoid development. Here, we demonstrate that while low dose of GSK3β inhibitor CHIR 99021 increases organoid size, higher dose actually reduces organoid size; with the highest dose arresting organoid growth. To examine the mechanisms that may contribute to the phenotypic size differences observed in these treatment groups, we show that low dose of CHIR 99021 increases cell survival, neural progenitor cell proliferation and neuronal migration. A higher dose, however, decreases not only apoptosis but also proliferation, and arrests neural differentiation, enriching the pool of neuroepithelial cells, and decreasing the pools of early neuronal progenitors and neurons. These results reveal new mechanisms of the pleiotropic effects of GSK3β during organoid development, providing essential information for the improvement of organoid production and ultimately shedding light on the mechanisms of embryonic brain development.
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Wong, HakKei. "The importance of cerebral organoid technology in medicine." Highlights in Science, Engineering and Technology 2 (June 22, 2022): 179–85. http://dx.doi.org/10.54097/hset.v2i.572.
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Because of the intricate nature of the nervous systems, neurological diseases have always been one of the least studied areas of pathology and medicine. Currently, there is no cure for these kinds of diseases but only medications or therapies that relieve symptoms and minimise suffering. Thus, cerebral organoids derived from human pluripotent stem cells are produced in order to study the development and pathology of the human brain, especially the embryonic stage, and to model neurological diseases. In this dissertation, I will make a judgement on the appropriate usage of cerebral organoid in investigating neurological disease through exploring and assessing the effectiveness of the cerebral organoids modeling Zika Virus and Alzheimer’s disease and examining the ethical issues arising from this practice.
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Chen, Juan, Haihua Ma, Zhiyu Deng, Qingming Luo, Hui Gong, Ben Long, and Xiangning Li. "Cerebral Organoid Arrays for Batch Phenotypic Analysis in Sections and Three Dimensions." International Journal of Molecular Sciences 24, no. 18 (September 9, 2023): 13903. http://dx.doi.org/10.3390/ijms241813903.
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Organoids can recapitulate human-specific phenotypes and functions in vivo and have great potential for research in development, disease modeling, and drug screening. Due to the inherent variability among organoids, experiments often require a large sample size. Embedding, staining, and imaging each organoid individually require a lot of reagents and time. Hence, there is an urgent need for fast and efficient methods for analyzing the phenotypic changes in organoids in batches. Here, we provide a comprehensive strategy for array embedding, staining, and imaging of cerebral organoids in both agarose sections and in 3D to analyze the spatial distribution of biomarkers in organoids in situ. We constructed several disease models, particularly an aging model, as examples to demonstrate our strategy for the investigation of the phenotypic analysis of organoids. We fabricated an array mold to produce agarose support with microwells, which hold organoids in place for live/dead imaging. We performed staining and imaging of sectioned organoids embedded in agarose and 3D imaging to examine phenotypic changes in organoids using fluorescence micro-optical sectioning tomography (fMOST) and whole-mount immunostaining. Parallel studies of organoids in arrays using the same staining and imaging parameters enabled easy and reliable comparison among different groups. We were able to track all the data points obtained from every organoid in an embedded array. This strategy could help us study the phenotypic changes in organoids in disease models and drug screening.
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Sivitilli, Adam A., Jessica T. Gosio, Bibaswan Ghoshal, Alesya Evstratova, Daniel Trcka, Parisa Ghiasi, J. Javier Hernandez, Jean Martin Beaulieu, Jeffrey L. Wrana, and Liliana Attisano. "Robust production of uniform human cerebral organoids from pluripotent stem cells." Life Science Alliance 3, no. 5 (April 17, 2020): e202000707. http://dx.doi.org/10.26508/lsa.202000707.
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Human cerebral organoid (hCO) models offer the opportunity to understand fundamental processes underlying human-specific cortical development and pathophysiology in an experimentally tractable system. Although diverse methods to generate brain organoids have been developed, a major challenge has been the production of organoids with reproducible cell type heterogeneity and macroscopic morphology. Here, we have directly addressed this problem by establishing a robust production pipeline to generate morphologically consistent hCOs and achieve a success rate of >80%. These hCOs include both a radial glial stem cell compartment and electrophysiologically competent mature neurons. Moreover, we show using immunofluorescence microscopy and single-cell profiling that individual organoids display reproducible cell type compositions that are conserved upon extended culture. We expect that application of this method will provide new insights into brain development and disease processes.
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Li, Xiaodong, Abdullah Shopit, and Jingmin Wang. "A Comprehensive Update of Cerebral Organoids between Applications and Challenges." Oxidative Medicine and Cellular Longevity 2022 (December 5, 2022): 1–10. http://dx.doi.org/10.1155/2022/7264649.
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The basic technology of stem cells has been developed and created organoids, which have established a strong interest in regenerative medicine. Different cell types have been used to generate cerebral organoids, which include interneurons and oligodendrocytes (OLs). OLs are fundamental for brain development. Abundant studies have displayed that brain organoids can recapitulate fundamental and vital features of the human brain, such as cellular regulation and distribution, neuronal networks, electrical activities, and physiological structure. The organoids contain essential ventral brain domains and functional cortical interneurons, which are similar to the developing cortex and medial ganglionic eminence (MGE). So, brain organoids have provided a singular model to study and investigate neurological disorder mechanisms and therapeutics. Furthermore, the blood brain barrier (BBB) organoids modeling contributes to accelerate therapeutic discovery for the treatment of several neuropathologies. In this review, we summarized the advances of the brain organoids applications to investigate neurological disorder mechanisms such as neurodevelopmental and neurodegenerative disorders, mental disorders, brain cancer, and cerebral viral infections. We discussed brain organoids’ therapeutic application as a potential therapeutic unique method and highlighted in detail the challenges and hurdles of organoid models.
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Qiao, Haowen, Wen Zhao, Moujian Guo, Lili Zhu, Tao Chen, Jibo Wang, Xiaodong Xu, Zhentao Zhang, Ying Wu, and Pu Chen. "Cerebral Organoids for Modeling of HSV-1-Induced-Amyloid β Associated Neuropathology and Phenotypic Rescue." International Journal of Molecular Sciences 23, no. 11 (May 26, 2022): 5981. http://dx.doi.org/10.3390/ijms23115981.
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Herpes simplex virus type I (HSV-1) infection is a potential risk factor involved in the Amyloid β (Aβ) associated neuropathology. However, further understanding of the neuropathological effects of the HSV-1 infection is hampered by the limitations of existing infection models due to the distinct differences between human brains and other mammalians’ brains. Here we generated cerebral organoid models derived from pluripotent stem cells to investigate the HSV-induced Aβ associated neuropathology and the role of antiviral drugs in the phenotypic rescue. Our results identified that the HSV-1-infected cerebral organoids recapitulated Aβ associated neuropathology including the multicellular Aβ deposition, dysregulated endogenous AD mediators, reactive gliosis, neuroinflammation, and neural loss, indicating that cerebral organoids offer an opportunity for modeling the interaction of HSV-1 with the complex phenotypes across the genetic, cellular, and tissue levels of the human Alzheimer’s disease (AD). Furthermore, we identified that two antiviral drugs, namely Ribavirin (RBV) and Valacyclovir (VCV), inhibited HSV-1 replication and rescued the neuropathological phenotypes associated with AD in the HSV-1-infected cerebral organoids, implying their therapeutic potential to slow down the progression of AD. Our study provides a high-fidelity human-relevant in-vitro HSV-1 infection model to reconstitute the multiscale neuropathological features associated with AD and discover therapeutic drug candidates relevant to the AD viral hypothesis.
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Roosen, Mieke, Chris Meulenbroeks, Phylicia Stathi, Joris Maas, Julie Morscio, Jens Bunt, and Marcel Kool. "BIOL-11. PRECLINICAL MODELLING OF PEDIATRIC BRAIN TUMORS USING ORGANOID TECHNOLOGY." Neuro-Oncology 25, Supplement_1 (June 1, 2023): i8. http://dx.doi.org/10.1093/neuonc/noad073.030.
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Abstract Molecular characterization has resulted in improved classification of pediatric brain tumors, leading to many novel (sub)types with distinct oncodriving events. To study tumor biology and to perform translational research on each of these tumors, preclinical models are essential. However, we are currently lacking sufficient models, especially in vitro, to represent each (sub)type and their heterogeneity. To generate large series of preclinical in vitro models for pediatric brain tumors, we are using organoid technology. Cells from patient samples and patient-derived xenograft samples have been taken into culture to establish 3D organoids using tumor type specific culture conditions. These organoid lines retain the molecular characteristics of the original tumor tissue. They can be used to perform high-throughput drug screens, genetic manipulations, and co-cultures with, for instance, immune cells. Viable tissue is not always available for all tumor (sub)types and specific oncodrivers. To circumvent this lack of tissue, we can also induce tumors in vitro. Therefore, we generate cerebral and cerebellar brain organoids from human pluripotent stem cells. These organoids mimic human developing brain cells and can be genetically manipulated to model different brain tumor types. These genetically engineered brain tumor models allow us to study the cellular origins of pediatric brain tumors and the different tumor driving mechanisms. Tumors induced in the brain organoids histologically and molecularly resemble human patient samples based on (single cell) transcriptomic analyses. Moreover, the tumor cells are able to establish xenografts in mouse brains. In summary, organoid technology provides a novel avenue to establish in vitro models for pediatric brain tumors. At the meeting we will present data for various new ependymoma, medulloblastoma and embryonal brain tumor organoid models.
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Li, Chong, Jonas Simon Fleck, Catarina Martins-Costa, Thomas R. Burkard, Jan Themann, Marlene Stuempflen, Angela Maria Peer, et al. "Single-cell brain organoid screening identifies developmental defects in autism." Nature 621, no. 7978 (September 13, 2023): 373–80. http://dx.doi.org/10.1038/s41586-023-06473-y.
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AbstractThe development of the human brain involves unique processes (not observed in many other species) that can contribute to neurodevelopmental disorders1–4. Cerebral organoids enable the study of neurodevelopmental disorders in a human context. We have developed the CRISPR–human organoids–single-cell RNA sequencing (CHOOSE) system, which uses verified pairs of guide RNAs, inducible CRISPR–Cas9-based genetic disruption and single-cell transcriptomics for pooled loss-of-function screening in mosaic organoids. Here we show that perturbation of 36 high-risk autism spectrum disorder genes related to transcriptional regulation uncovers their effects on cell fate determination. We find that dorsal intermediate progenitors, ventral progenitors and upper-layer excitatory neurons are among the most vulnerable cell types. We construct a developmental gene regulatory network of cerebral organoids from single-cell transcriptomes and chromatin modalities and identify autism spectrum disorder-associated and perturbation-enriched regulatory modules. Perturbing members of the BRG1/BRM-associated factor (BAF) chromatin remodelling complex leads to enrichment of ventral telencephalon progenitors. Specifically, mutating the BAF subunit ARID1B affects the fate transition of progenitors to oligodendrocyte and interneuron precursor cells, a phenotype that we confirmed in patient-specific induced pluripotent stem cell-derived organoids. Our study paves the way for high-throughput phenotypic characterization of disease susceptibility genes in organoid models with cell state, molecular pathway and gene regulatory network readouts.
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Yakoub, Abraam M., and Mark Sadek. "Analysis of Synapses in Cerebral Organoids." Cell Transplantation 28, no. 9-10 (June 4, 2019): 1173–82. http://dx.doi.org/10.1177/0963689718822811.
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Cerebral organoids are an emerging cutting-edge technology to model human brain development and neurodevelopmental disorders, for which mouse models exhibit significant limitations. In the human brain, synaptic connections define neural circuits, and synaptic deficits account for various neurodevelopmental disorders. Thus, harnessing the full power of cerebral organoids for human brain modeling requires the ability to visualize and analyze synapses in cerebral organoids. Previously, we devised an optimized method to generate human cerebral organoids, and showed that optimal organoids express mature-neuron markers, including synaptic proteins and neurotransmitter receptors and transporters. Here, we give evidence for synaptogenesis in cerebral organoids, via microscopical visualization of synapses. We also describe multiple approaches to quantitatively analyze synapses in cerebral organoids. Collectively, our work provides sufficient evidence for the possibility of modeling synaptogenesis and synaptic disorders in cerebral organoids, and may help advance the use of cerebral organoids in molecular neuroscience and studies of neurodevelopmental disorders such as autism.
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Ferreira, Rodolfo Sanches, Bruno H. S. Araujo, and Oswaldo Okamoto. "MODL-06. ASSESSMENT OF ONCOLYTIC VIRUS SPECIFICITY AND CYTOTOXICITY IN A HYBRID GLIOBLASTOMA-CEREBRAL ORGANOID MODEL." Neuro-Oncology 24, Supplement_7 (November 1, 2022): vii292. http://dx.doi.org/10.1093/neuonc/noac209.1134.
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Abstract Recent studies have demonstrated potent oncolytic effects of wild-type Zika virus (ZIKV) against primary central nervous system (CNS) tumors, including Medulloblastoma, Atypical Teratoid/Rhabdoid Tumor (AT/RT), and Glioblastoma (GBM). However, the neurotropism of ZIKV urges further evaluation of specific tumor-targeting properties and comparative toxicity to non-neoplastic neural cells in order to address its therapeutic potential. We have developed a hybrid organoid model by co-culturing GBM cells with mature human cerebral organoids and assessed cytotoxicity of the Brazilian ZIKV isolate (ZIKVBR) towards normal brain organoid cells and GBM cells. Human induced pluripotent stem cells (hiPSC)-derived cerebral organoids were co-cultured with 105 GFP-expressing cells of three different GBM cell lines. A total of 20.000 PFU of ZIKVBR or mock condition was added per co-cultured model. Viable cells were analyzed by flow cytometry (FC) and fluorescence microscopy at different time points. ZIKVBR presence was assessed by PFU assay. Although ZIKVBR infection did not cause a pronounced reduction of GFP+ cell proportion over time in all GBM cell lines, cell viability analysis showed a greater amount of non-viable GFP+ cells over GFP- cells in all ZIKVBR-treated groups, compared to their corresponding mock groups. PFU assays of cell culture supernatants confirmed the presence of infectious viral particles in treated groups and absence in mock groups. Our data reveal that ZIKVBR has a potential oncolytic effect characterized by preferential killing of GBM tumor cells over normal cerebral cells, in a hybrid organoid model. These findings support the development of an oncolytic virus therapy platform based on ZIKV.
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da Silva, Bárbara, Ryan K. Mathew, Euan S. Polson, Jennifer Williams, and Heiko Wurdak. "Spontaneous Glioblastoma Spheroid Infiltration of Early-Stage Cerebral Organoids Models Brain Tumor Invasion." SLAS DISCOVERY: Advancing the Science of Drug Discovery 23, no. 8 (March 15, 2018): 862–68. http://dx.doi.org/10.1177/2472555218764623.
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Organoid methodology provides a platform for the ex vivo investigation of the cellular and molecular mechanisms underlying brain development and disease. The high-grade brain tumor glioblastoma multiforme (GBM) is considered a cancer of unmet clinical need, in part due to GBM cell infiltration into healthy brain parenchyma, making complete surgical resection improbable. Modeling the process of GBM invasion in real time is challenging as it requires both tumor and neural tissue compartments. Here, we demonstrate that human GBM spheroids possess the ability to spontaneously infiltrate early-stage cerebral organoids (eCOs). The resulting formation of hybrid organoids demonstrated an invasive tumor phenotype that was distinct from noncancerous adult neural progenitor (NP) spheroid incorporation into eCOs. These findings provide a basis for the modeling and quantification of the GBM infiltration process using a stem-cell-based organoid approach, and may be used for the identification of anti-GBM invasion strategies.
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Sapir, Gal, Daniel J. Steinberg, Rami I. Aqeilan, and Rachel Katz-Brull. "Real-Time Non-Invasive and Direct Determination of Lactate Dehydrogenase Activity in Cerebral Organoids—A New Method to Characterize the Metabolism of Brain Organoids?" Pharmaceuticals 14, no. 9 (August 30, 2021): 878. http://dx.doi.org/10.3390/ph14090878.
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Organoids are a powerful tool in the quest to understand human diseases. As the developing brain is extremely inaccessible in mammals, cerebral organoids (COs) provide a unique way to investigate neural development and related disorders. The aim of this study was to utilize hyperpolarized 13C NMR to investigate the metabolism of COs in real-time, in a non-destructive manner. The enzymatic activity of lactate dehydrogenase (LDH) was determined by quantifying the rate of [1-13C]lactate production from hyperpolarized [1-13C]pyruvate. Organoid development was assessed by immunofluorescence imaging. Organoid viability was confirmed using 31P NMR spectroscopy. A total of 15 organoids collated into 3 groups with a group total weight of 20–77 mg were used in this study. Two groups were at the age of 10 weeks and one was at the age of 33 weeks. The feasibility of this approach was demonstrated in both age groups, and the LDH activity rate was found to be 1.32 ± 0.75 nmol/s (n = 3 organoid batches). These results suggest that hyperpolarized NMR can be used to characterize the metabolism of brain organoids with a total tissue wet weight of as low as 20 mg (<3 mm3) and a diameter ranging from 3 to 6 mm.
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Bunt, Jens, Mieke Roosen, Evie Egelmeers, Joris Maas, Zelda Ode, and Marcel Kool. "TMOD-02. GEBTO: GENETICALLY ENGINEERED BRAIN TUMOR ORGANOIDS AS A NOVEL PRECLINICAL MODEL." Neuro-Oncology 23, Supplement_1 (June 1, 2021): i35—i36. http://dx.doi.org/10.1093/neuonc/noab090.143.
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Abstract Background One of the bottlenecks in basic and translational research on pediatric brain tumors, is the lack of suitable and representative preclinical models to study tumor biology and drug sensitivity. Over the last decades, extensive molecular characterization has uncovered many entities and subgroups with their unique oncodriving events. However, this heterogeneity is currently not reflected in the models available, especially not for in vitro models. Objectives We aim to generate genetically engineered brain tumor organoids (GEBTO) to represent the molecular variety of embryonal brain tumors and ependymomas. Method Human brain organoids derived from embryonic stem cells are generated to represent the region of tumor origin. To mimic oncodriving events, DNA plasmids are introduced via electroporation in the organoid cells to knockout tumor suppressor genes or overexpress oncogenes. Results Cerebellar and cerebral forebrain organoids were generated as the tissue of origin for medulloblastoma and supratentorial ependymoma (ST-EPN), respectively. Based on the detection of GFP protein encoded by DNA plasmids, the organoid cells can be manipulated within a wide developmental window, which corresponds with the presence of the proposed cells of origin. Different oncodrivers and combinations thereof are now being tested to see whether they result in ectopic growth in cerebral or cerebellar organoids. When successful, the GEBTOs are histologically and molecularly characterized using (single cell) transcriptomic and epigenomic analyses to see how well they resemble human tumors. Discussion Although further development is required, GEBTOs provide a novel avenue to model especially rare pediatric brain tumors, for which tissue and therefore patient-derived models are limited. It also allows for in-depth analyses of the potential cells of origin and the contribution of different mutations to tumor biology.
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Krieger, Teresa G., Stephan M. Tirier, Jeongbin Park, Katharina Jechow, Tanja Eisemann, Heike Peterziel, Peter Angel, Roland Eils, and Christian Conrad. "Modeling glioblastoma invasion using human brain organoids and single-cell transcriptomics." Neuro-Oncology 22, no. 8 (April 16, 2020): 1138–49. http://dx.doi.org/10.1093/neuonc/noaa091.
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Abstract Background Glioblastoma (GBM) consists of devastating neoplasms with high invasive capacity, which have been difficult to study in vitro in a human-derived model system. Therapeutic progress is also limited by cellular heterogeneity within and between tumors, among other factors such as therapy resistance. To address these challenges, we present an experimental model using human cerebral organoids as a scaffold for patient-derived GBM cell invasion. Methods This study combined tissue clearing and confocal microscopy with single-cell RNA sequencing of GBM cells before and after co-culture with organoid cells. Results We show that tumor cells within organoids extend a network of long microtubes, recapitulating the in vivo behavior of GBM. Transcriptional changes implicated in the invasion process are coherent across patient samples, indicating that GBM cells reactively upregulate genes required for their dispersion. Potential interactions between GBM and organoid cells identified by an in silico receptor–ligand pairing screen suggest functional therapeutic targets. Conclusions Taken together, our model has proven useful for studying GBM invasion and transcriptional heterogeneity in vitro, with applications for both pharmacological screens and patient-specific treatment selection on a time scale amenable to clinical practice.
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Roosen, Mieke, Julie Morscio, Phylicia Stathi, Norman Mack, Benjamin Schwalm, Panagiotis A. Polychronopoulos, Mariëtte E. G. Kranendonk, Eelco Hoving, Jens Bunt, and Marcel Kool. "EPEN-17.IN VITRO MODELLING OF PEDIATRIC SUPRATENTORIAL EPENDYMOMAS USING CEREBRAL ORGANOIDS." Neuro-Oncology 26, Supplement_4 (June 18, 2024): 0. http://dx.doi.org/10.1093/neuonc/noae064.219.
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Abstract BACKGROUND Ependymomas (EPN) are glial tumors of the central nervous system occurring in children and adults. ZFTA-fusion positive and YAP1-fusion positive EPN are the main supratentorial (ST) subgroups in children. While most ST-EPN-YAP1 patients survive, only 50% of ZFTA positive patients survive longer than 5 years. Improving survival of ST-EPN patients requires a better understanding of tumor biology. Human embryonal stem cell-derived (hESC) brain organoids provide a novel opportunity to model ST-EPN and study the impact of oncogenic fusions on tumor development within a healthy brain environment. METHODS hESC-derived cerebral organoids were genetically modified with YAP1 or ZFTA fusion genes and histologically and molecularly analyzed using antibody stainings, bulk and single cell RNA sequencing. RESULTS scRNA-seq analyses showed that our cerebral organoids mimic embryonal brain development and that radial glia, the presumed cell-of-origin of ST-EPN are abundant in 11-day old organoids. At this timepoint, electroporation of oncogenic ZFTA-RELA or YAP1-MAMLD1/YAP1-FAM118B fusions led to ectopic tumor outgrowth. Histological and molecular analyses showed that ZFTA and YAP1 EPN tumor organoids displayed different phenotypes and fusion-specific gene signatures, closely resembling human ZFTA and YAP1 EPN patient samples. ScRNA-seq data of organoid tumors showed a skewed differentiation compared to normal development with ZFTA tumors being more neuronal and YAP1 tumors having a more extracellular matrix-like phenotype. Analysis of the healthy compartment showed that YAP1 tumors influence the differentiation of the healthy cells and in both subtypes a new cell cluster was identified with high expression of tumor associated markers. Intercellular interactions revealed potential targetable tumor(specific) interactions such as YBX1-NOTCH1 in ZFTA or GNAI2-CAV1 in YAP1 tumor organoid models, as well as CD99-CD81 in both. CONCLUSIONS These models contribute to a better molecular and biological understanding of ependymomas and can be used to identify targeted therapies, especially those targeting the tumor microenvironment.
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Ahn, Yujin, Ju-Hyun An, Hae-Jun Yang, Dong Gil Lee, Jieun Kim, Hyebin Koh, Young-Ho Park, et al. "Human Blood Vessel Organoids Penetrate Human Cerebral Organoids and Form a Vessel-Like System." Cells 10, no. 8 (August 9, 2021): 2036. http://dx.doi.org/10.3390/cells10082036.
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Vascularization of tissues, organoids and organ-on-chip models has been attempted using endothelial cells. However, the cultured endothelial cells lack the capacity to interact with other somatic cell types, which is distinct from developing vascular cells in vivo. Recently, it was demonstrated that blood vessel organoids (BVOs) recreate the structure and functions of developing human blood vessels. However, the tissue-specific adaptability of BVOs had not been assessed in somatic tissues. Herein, we investigated whether BVOs infiltrate human cerebral organoids and form a blood–brain barrier. As a result, vascular cells arising from BVOs penetrated the cerebral organoids and developed a vessel-like architecture composed of CD31+ endothelial tubes coated with SMA+ or PDGFR+ mural cells. Molecular markers of the blood-brain barrier were detected in the vascularized cerebral organoids. We revealed that BVOs can form neural-specific blood-vessel networks that can be maintained for over 50 days.
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Tongkrajang, Nongnat, Porntida Kobpornchai, Pratima Dubey, Urai Chaisri, and Kasem Kulkeaw. "Modelling amoebic brain infection caused by Balamuthia mandrillaris using a human cerebral organoid." PLOS Neglected Tropical Diseases 18, no. 6 (June 20, 2024): e0012274. http://dx.doi.org/10.1371/journal.pntd.0012274.
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The lack of disease models adequately resembling human tissue has hindered our understanding of amoebic brain infection. Three-dimensional structured organoids provide a microenvironment similar to human tissue. This study demonstrates the use of cerebral organoids to model a rare brain infection caused by the highly lethal amoeba Balamuthia mandrillaris. Cerebral organoids were generated from human pluripotent stem cells and infected with clinically isolated B. mandrillaris trophozoites. Histological examination showed amoebic invasion and neuron damage following coculture with the trophozoites. The transcript profile suggested an alteration in neuron growth and a proinflammatory response. The release of intracellular proteins specific to neuronal bodies and astrocytes was detected at higher levels postinfection. The amoebicidal effect of the repurposed drug nitroxoline was examined using the human cerebral organoids. Overall, the use of human cerebral organoids was important for understanding the mechanism of amoeba pathogenicity, identify biomarkers for brain injury, and in the testing of a potential amoebicidal drug in a context similar to the human brain.
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Gumbs, Stephanie B. H., Amber Berdenis van Berlekom, Raphael Kübler, Pauline J. Schipper, Lavina Gharu, Marco P. Boks, Paul R. Ormel, Annemarie M. J. Wensing, Lot D. de Witte, and Monique Nijhuis. "Characterization of HIV-1 Infection in Microglia-Containing Human Cerebral Organoids." Viruses 14, no. 4 (April 16, 2022): 829. http://dx.doi.org/10.3390/v14040829.
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The achievement of an HIV cure is dependent on the eradication or permanent silencing of HIV-latent viral reservoirs, including the understudied central nervous system (CNS) reservoir. This requires a deep understanding of the molecular mechanisms of HIV’s entry into the CNS, latency establishment, persistence, and reversal. Therefore, representative CNS culture models that reflect the intercellular dynamics and pathophysiology of the human brain are urgently needed in order to study the CNS viral reservoir and HIV-induced neuropathogenesis. In this study, we characterized a human cerebral organoid model in which microglia grow intrinsically as a CNS culture model to study HIV infection in the CNS. We demonstrated that both cerebral organoids and isolated organoid-derived microglia (oMG), infected with replication-competent HIVbal reporter viruses, support productive HIV infection via the CCR5 co-receptor. Productive HIV infection was only observed in microglial cells. Fluorescence analysis revealed microglia as the only HIV target cell. Susceptibility to HIV infection was dependent on the co-expression of microglia-specific markers and the CD4 and CCR5 HIV receptors. Altogether, this model will be a valuable tool within the HIV research community to study HIV–CNS interactions, the underlying mechanisms of HIV-associated neurological disorders (HAND), and the efficacy of new therapeutic and curative strategies on the CNS viral reservoir.
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Robles, Denise, Andrew Boreland, Zhiping Pang, and Jeffrey Zahn. "A Cerebral Organoid Connectivity Apparatus to Model Neuronal Tract Circuitry." Micromachines 12, no. 12 (December 17, 2021): 1574. http://dx.doi.org/10.3390/mi12121574.
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Mental disorders have high prevalence, but the efficacy of existing therapeutics is limited, in part, because the pathogenic mechanisms remain enigmatic. Current models of neural circuitry include animal models and post-mortem brain tissue, which have allowed enormous progress in understanding the pathophysiology of mental disorders. However, these models limit the ability to assess the functional alterations in short-range and long-range network connectivity between brain regions that are implicated in many mental disorders, e.g., schizophrenia and autism spectrum disorders. This work addresses these limitations by developing an in vitro model of the human brain that models the in vivo cerebral tract environment. In this study, microfabrication and stem cell differentiation techniques were combined to develop an in vitro cerebral tract model that anchors human induced pluripotent stem cell-derived cerebral organoids (COs) and provides a scaffold to promote the formation of a functional connecting neuronal tract. Two designs of a Cerebral Organoid Connectivity Apparatus (COCA) were fabricated using SU-8 photoresist. The first design contains a series of spikes which anchor the CO to the COCA (spiked design), whereas the second design contains flat supporting structures with open holes in a grid pattern to anchor the organoids (grid design); both designs allow effective media exchange. Morphological and functional analyses reveal the expression of key neuronal markers as well as functional activity and signal propagation along cerebral tracts connecting CO pairs. The reported in vitro models enable the investigation of critical neural circuitry involved in neurodevelopmental processes and has the potential to help devise personalized and targeted therapeutic strategies.
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Ogawa, Junko, Gerald M. Pao, Maxim N. Shokhirev, and Inder M. Verma. "Glioblastoma Model Using Human Cerebral Organoids." Cell Reports 23, no. 4 (April 2018): 1220–29. http://dx.doi.org/10.1016/j.celrep.2018.03.105.
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Browning, Heather, and Walter Veit. "Regulating Possibly Sentient Human Cerebral Organoids." AJOB Neuroscience 14, no. 2 (April 3, 2023): 197–99. http://dx.doi.org/10.1080/21507740.2023.2188293.
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Fagerlund, Ilkka, Antonios Dougalis, Anastasia Shakirzyanova, Mireia Gómez-Budia, Anssi Pelkonen, Henna Konttinen, Sohvi Ohtonen, et al. "Microglia-like Cells Promote Neuronal Functions in Cerebral Organoids." Cells 11, no. 1 (December 30, 2021): 124. http://dx.doi.org/10.3390/cells11010124.
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Human cerebral organoids, derived from induced pluripotent stem cells, offer a unique in vitro research window to the development of the cerebral cortex. However, a key player in the developing brain, the microglia, do not natively emerge in cerebral organoids. Here we show that erythromyeloid progenitors (EMPs), differentiated from induced pluripotent stem cells, migrate to cerebral organoids, and mature into microglia-like cells and interact with synaptic material. Patch-clamp electrophysiological recordings show that the microglia-like population supported the emergence of more mature and diversified neuronal phenotypes displaying repetitive firing of action potentials, low-threshold spikes and synaptic activity, while multielectrode array recordings revealed spontaneous bursting activity and increased power of gamma-band oscillations upon pharmacological challenge with NMDA. To conclude, microglia-like cells within the organoids promote neuronal and network maturation and recapitulate some aspects of microglia-neuron co-development in vivo, indicating that cerebral organoids could be a useful biorealistic human in vitro platform for studying microglia-neuron interactions.
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Yin, He. "Human brain organoids combined with CRISPR technology to gain insight into neurological diseases." Highlights in Science, Engineering and Technology 102 (July 11, 2024): 75–79. http://dx.doi.org/10.54097/m3grdg15.
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As the most complex organ of human beings, the brain's functions cover various high-level intellectual activities such as cognition, language and thinking. These functions are closely related to the developmental processes in the cerebral cortex granted the composition structure of the nervous system in the cortex. Therefore, scientists have long been exploring the developmental processes of the cerebral cortex and the constituent structures of the nervous system, the purpose of the system is to deepen comprehend how the brain works. The study of neurological diseases by improving three-dimensional brain models is a hot issue at present, because the brain is very complex and fragile, and many therapies are weak and still theoretical. Brain organoids are a kind of three-dimensional tissue culture derived from human pluripotent stem cells (hPSCs), which can simulate the type composition, spatial structure, physiological function and other characteristics of human brain cells by culture in vitro. The use of brain organoid technology can simulate the development process of the brain in vitro, providing an unprecedented opportunity to study the occurrence of brain function and nervous system diseases, and combining CRISPR technology for screening, so that it can be further applied efficiently. This research will summarize the development process, progress and deficiency of brain organoids and the research situation of neurological diseases, and make a summary of the current development and prospect of the future development.
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Gebing, Philip, Stefanos Loizou, Sebastian Hänsch, Julian Schliehe-Diecks, Lea Spory, Pawel Stachura, Aleksandra Pandyra, et al. "CNS Invasion of TCF3::PBX1+ Leukemia Cells Requires Upregulation of AP-1 Signaling As Revealed By Brain Organoid Model." Blood 142, Supplement 1 (November 28, 2023): 1407. http://dx.doi.org/10.1182/blood-2023-178613.
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Introduction: Involvement of the central nervous system (CNS) remains a challenge in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Currently, the investigation of CNS leukemia mechanisms relies heavily on 2D cell culture and mouse models. However, given the differences between human and murine CNS in cellular identity and architecture, it becomes crucial to explore alternative models to study CNS leukemia. Moreover, novel targets for diagnosis and treatment of CNS ALL are critically needed. Methods: In this study, we established a pioneering 3D co-culture model that combines human induced pluripotent stem cell (iPSC)-derived cerebral organoids with BCP-ALL cells. We developed new methods to extract data from our invasion assays by enhancing the visualization of 3D organoid images, enabling us to accurately measure the invasion depth of leukemia cells compared to healthy controls within the organoids relative to their surface. To gain further insights on leukemia cells invading the organoids compared to those in the non-invaded fraction, we conducted RNA sequencing and immunofluorescence staining. Subsequently, we validated these results in a BCP-ALL in vivo mouse model and in patients initially diagnosed with CNS-positive BCP-ALL compared to CNS-negative cases within a cohort of 100 BCP-ALL patients. Results: Our experiments demonstrated robust and deep engraftment of TCF3::PBX1+ leukemia cell lines and patient-derived xenograft (PDX) cells into cerebral organoids (within a 14-day of co-culture). In contrast, the engraftment of healthy human CD34+ hematopoietic stem and progenitor cells (HSPCs) was limited ( p < 0.05) as compared to leukemia cells. Utilizing the co-culture model, we successfully validated the targeting of CNS leukemia-relevant pathways, such as CD79a/Igα or CXCR4-SDF1, via genetic knockdown or blocking experiments using inhibitor, respectively, which reduced the invasion of BCP-ALL cells into the organoids. Of note, RNA sequencing and immunofluorescence staining analysis revealed a significant upregulation of members of the AP-1 transcription factor complex, namely FOS, FOSB, and JUN, in the organoid-invading cells. Furthermore, we found an enrichment of AP-1 pathway genes in PDX cells recovered from the CNS compared to spleen blasts of mice transplanted with TCF3::PBX1+ PDX BCP-ALL cells (n = 5), thereby supporting the critical role of AP-1 signaling in CNS disease (Figure 1A). In line with these findings, we observed significantly higher levels of the AP-1 gene JUN in patients initially diagnosed with CNS-positive BCP-ALL compared to CNS-negative cases (mean JUN expression: 633.4 ± 78.51 in CNS-negative vs 1142 ± 296.5 in CNS-positive) within a cohort of 100 BCP-ALL patients (Figure 1B). Summary: In summary, we present ALL co-culture systems with iPSC-derived cerebral organoids as a promising complementary model to investigate CNS involvement in BCP-ALL including therapeutic targeting approaches, and identified the AP-1 pathway as a marker of CNS disease in TCF3::PBX1+ BCP-ALL.
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Fu, Yingying, Zhen Qi, Zhanguan Zuo, Spencer Chiang, An Ouyang, Glory Gao, Shuge Guan, Jin-Qiu (Jessie) Chen, Rosanna Zhang, and Cheng Wang. "Abstract 4245: Selection of AAV capsids by evaluating transgene delivery using human organoid models." Cancer Research 84, no. 6_Supplement (March 22, 2024): 4245. http://dx.doi.org/10.1158/1538-7445.am2024-4245.
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Abstract Recombinant adeno-associated viruses (AAVs) play a pivotal role in gene therapy, a promising approach aimed at treating various genetic disorders by introducing modified genetic material into cells or tissues. These AAV capsids are utilized as a vector in transferring genetic material into host cells. In discovery research, many AAV serotypes are developed in parallel to identify the optimal subtype for subsequent preclinical and clinical use. However, gene therapies have found limited success in clinical translation, in part, due to variable transgene delivery efficacy between traditional in vitro and in vivo models and human models. This study presents an organoid-based approach that mimics human physiology, as well as organ-specific features and cell diversity within an organ. Organoids are 3D structures derived from stem cells that resemble the cellular architecture and functionality of a specific organ. Compared to conventional 2D cell cultures, organoids provide a more comprehensive modeling of complex cell signaling, cell-cell and cell-extracellular matrix interactions, simulating the complex interplay between various cell types and functions. Within this study, several human-based organoid models were built, cerebral and cardiac, and verified by identifying the expression level of known marker genes through RNA sequencing. Five different AAV serotypes were utilized to evaluate transgene efficacy using each type of organoid model. Expression of delivered GFP transgene within each organoid was then compared to evaluate transduction and select the optimal AAV serotype in comparison to commercial wild-type AAVs. We outline the use of human organoid as an improved preclinical model for selecting AAV serotypes and evaluating transgene delivery efficacy to help provide better guidance for gene therapy development when moving into clinical research. Citation Format: Yingying Fu, Zhen Qi, Zhanguan Zuo, Spencer Chiang, An Ouyang, Glory Gao, Shuge Guan, Jin-Qiu (Jessie) Chen, Rosanna Zhang, Cheng Wang. Selection of AAV capsids by evaluating transgene delivery using human organoid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4245.
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Nowakowski, Tomasz J., and Sofie R. Salama. "Cerebral Organoids as an Experimental Platform for Human Neurogenomics." Cells 11, no. 18 (September 8, 2022): 2803. http://dx.doi.org/10.3390/cells11182803.
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The cerebral cortex forms early in development according to a series of heritable neurodevelopmental instructions. Despite deep evolutionary conservation of the cerebral cortex and its foundational six-layered architecture, significant variations in cortical size and folding can be found across mammals, including a disproportionate expansion of the prefrontal cortex in humans. Yet our mechanistic understanding of neurodevelopmental processes is derived overwhelmingly from rodent models, which fail to capture many human-enriched features of cortical development. With the advent of pluripotent stem cells and technologies for differentiating three-dimensional cultures of neural tissue in vitro, cerebral organoids have emerged as an experimental platform that recapitulates several hallmarks of human brain development. In this review, we discuss the merits and limitations of cerebral organoids as experimental models of the developing human brain. We highlight innovations in technology development that seek to increase its fidelity to brain development in vivo and discuss recent efforts to use cerebral organoids to study regeneration and brain evolution as well as to develop neurological and neuropsychiatric disease models.
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Amiri, Anahita, Gianfilippo Coppola, Soraya Scuderi, Feinan Wu, Tanmoy Roychowdhury, Fuchen Liu, Sirisha Pochareddy, et al. "Transcriptome and epigenome landscape of human cortical development modeled in organoids." Science 362, no. 6420 (December 13, 2018): eaat6720. http://dx.doi.org/10.1126/science.aat6720.
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Genes implicated in neuropsychiatric disorders are active in human fetal brain, yet difficult to study in a longitudinal fashion. We demonstrate that organoids from human pluripotent cells model cerebral cortical development on the molecular level before 16 weeks postconception. A multiomics analysis revealed differentially active genes and enhancers, with the greatest changes occurring at the transition from stem cells to progenitors. Networks of converging gene and enhancer modules were assembled into six and four global patterns of expression and activity across time. A pattern with progressive down-regulation was enriched with human-gained enhancers, suggesting their importance in early human brain development. A few convergent gene and enhancer modules were enriched in autism-associated genes and genomic variants in autistic children. The organoid model helps identify functional elements that may drive disease onset.
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Graham, Maya, Paolo Codega, Carl Campos, Subhiksha Nandakumar, Marc Rosenblum, Cristina Antonescu, Meaghan Grogan, et al. "MODL-37. MODELING REVERSIBLE TUMORIGENESIS IN CEREBRAL ORGANOIDS." Neuro-Oncology 25, Supplement_5 (November 1, 2023): v307. http://dx.doi.org/10.1093/neuonc/noad179.1188.
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Abstract Human cerebral organoids have emerged as a new approach to modeling the role of cancer-associated genes in tumorigenesis ex vivo. Here, we asked whether high-grade tumorigenesis in cerebral organoids is a reversible process. Using inducible cassettes carrying key oncogenic mutations histone H3K27M, dominant negative p53 and activated PDGFRA, we show that their expression in cerebral organoids reproducibly drives high grade malignant transformation in vitro and in vivo, including in orthotopic xenografts. We characterize this transformation as a fusion-negative rhabdomyosarcoma (FN-RMS) phenotype and identify a putative FN-RMS cell-of-origin in cerebral organoids. Finally, we show this transformation is dependent on persistent transgenic oncogene expression, as oncogene withdrawal causes complete tumor regression. These findings demonstrate that cerebral organoids may be used to pinpoint previously unappreciated and distinct roles of different mutations in tumor development and maintenance.
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Lavazza, Andrea, and Marcello Massimini. "Cerebral organoids: ethical issues and consciousness assessment." Journal of Medical Ethics 44, no. 9 (February 28, 2018): 606–10. http://dx.doi.org/10.1136/medethics-2017-104555.
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Organoids are three-dimensional biological structures grown in vitro from different kinds of stem cells that self-organise mimicking real organs with organ-specific cell types. Recently, researchers have managed to produce human organoids which have structural and functional properties very similar to those of different organs, such as the retina, the intestines, the kidneys, the pancreas, the liver and the inner ear. Organoids are considered a great resource for biomedical research, as they allow for a detailed study of the development and pathologies of human cells; they also make it possible to test new molecules on human tissue. Furthermore, organoids have helped research take a step forward in the field of personalised medicine and transplants. However, some ethical issues have arisen concerning the origin of the cells that are used to produce organoids (ie, human embryos) and their properties. In particular, there are new, relevant and so-far overlooked ethical questions concerning cerebral organoids. Scientists have created so-called mini-brains as developed as a few-months-old fetus, albeit smaller and with many structural and functional differences. However, cerebral organoids exhibit neural connections and electrical activity, raising the question whether they are or (which is more likely) will one day be somewhat sentient. In principle, this can be measured with some techniques that are already available (the Perturbational Complexity Index, a metric that is directly inspired by the main postulate of the Integrated Information Theory of consciousness), which are used for brain-injured non-communicating patients. If brain organoids were to show a glimpse of sensibility, an ethical discussion on their use in clinical research and practice would be necessary.
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Biunno, Ida, Emanuela Paiola, and Pasquale De Blasio. "The Application of the Tissue Microarray (TMA) Technology to Analyze Cerebral Organoids." Journal of Histochemistry & Cytochemistry 69, no. 7 (June 18, 2021): 451–60. http://dx.doi.org/10.1369/00221554211025327.
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“Multi-Omics” technologies have contributed greatly to the understanding of various diseases by enabling researchers to accurately and rapidly investigate the molecular circuitry that connects cellular systems. The tissue-engineered, three-dimensional (3D), in vitro disease model “organoid” integrates the “omics” results in a model system, elucidating the complex links between genotype and phenotype. These 3D structures have been used to model cancer, infectious disease, toxicity, and neurological disorders. Here, we describe the advantage of using the tissue microarray (TMA) technology to analyze human-induced pluripotent stem cell–derived cerebral organoids. Compared with the conventional processing of individual samples, sectioning and staining of TMA slides are faster and can be automated, decreasing labor and reagent costs. The TMA technology faithfully captures cell morphology variations and detects specific biomarkers. The use of this technology can scale up organoid research results in at least two ways: (1) in the number of specimens that can be analyzed simultaneously and (2) in the number of consecutive sections that can be produced for analysis with different probes and antibodies.
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Silva-Pedrosa, Rita, Jonas Campos, Aline Marie Fernandes, Miguel Silva, Carla Calçada, Ana Marote, Olga Martinho, et al. "Cerebral Malaria Model Applying Human Brain Organoids." Cells 12, no. 7 (March 23, 2023): 984. http://dx.doi.org/10.3390/cells12070984.
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Neural injuries in cerebral malaria patients are a significant cause of morbidity and mortality. Nevertheless, a comprehensive research approach to study this issue is lacking, so herein we propose an in vitro system to study human cerebral malaria using cellular approaches. Our first goal was to establish a cellular system to identify the molecular alterations in human brain vasculature cells that resemble the blood–brain barrier (BBB) in cerebral malaria (CM). Through transcriptomic analysis, we characterized specific gene expression profiles in human brain microvascular endothelial cells (HBMEC) activated by the Plasmodium falciparum parasites. We also suggest potential new genes related to parasitic activation. Then, we studied its impact at brain level after Plasmodium falciparum endothelial activation to gain a deeper understanding of the physiological mechanisms underlying CM. For that, the impact of HBMEC-P. falciparum-activated secretomes was evaluated in human brain organoids. Our results support the reliability of in vitro cellular models developed to mimic CM in several aspects. These systems can be of extreme importance to investigate the factors (parasitological and host) influencing CM, contributing to a molecular understanding of pathogenesis, brain injury, and dysfunction.
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Albanese, Alexandre, Justin M. Swaney, Dae Hee Yun, Nicholas B. Evans, Jenna M. Antonucci, Silvia Velasco, Chang Ho Sohn, Paola Arlotta, Lee Gehrke, and Kwanghun Chung. "Multiscale 3D phenotyping of human cerebral organoids." Scientific Reports 10, no. 1 (December 2020). http://dx.doi.org/10.1038/s41598-020-78130-7.
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AbstractBrain organoids grown from human pluripotent stem cells self-organize into cytoarchitectures resembling the developing human brain. These three-dimensional models offer an unprecedented opportunity to study human brain development and dysfunction. Characterization currently sacrifices spatial information for single-cell or histological analysis leaving whole-tissue analysis mostly unexplored. Here, we present the SCOUT pipeline for automated multiscale comparative analysis of intact cerebral organoids. Our integrated technology platform can rapidly clear, label, and image intact organoids. Algorithmic- and convolutional neural network-based image analysis extract hundreds of features characterizing molecular, cellular, spatial, cytoarchitectural, and organoid-wide properties from fluorescence microscopy datasets. Comprehensive analysis of 46 intact organoids and ~ 100 million cells reveals quantitative multiscale “phenotypes" for organoid development, culture protocols and Zika virus infection. SCOUT provides a much-needed framework for comparative analysis of emerging 3D in vitro models using fluorescence microscopy.
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Singh, Sanjay K., Yan Wang, Ahmed Habib, Mamindla Priyadarshini, Chowdari V. Kodavali, Apeng Chen, Wencai Ma, et al. "TP53-PTEN-NF1 depletion in human brain organoids produces a glioma phenotype in vitro." Frontiers in Oncology 13 (October 10, 2023). http://dx.doi.org/10.3389/fonc.2023.1279806.
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Glioblastoma (GBM) is fatal and the study of therapeutic resistance, disease progression, and drug discovery in GBM or glioma stem cells is often hindered by limited resources. This limitation slows down progress in both drug discovery and patient survival. Here we present a genetically engineered human cerebral organoid model with a cancer-like phenotype that could provide a basis for GBM-like models. Specifically, we engineered a doxycycline-inducible vector encoding shRNAs enabling depletion of the TP53, PTEN, and NF1 tumor suppressors in human cerebral organoids. Designated as inducible short hairpin-TP53-PTEN-NF1 (ish-TPN), doxycycline treatment resulted in human cancer-like cerebral organoids that effaced the entire organoid cytoarchitecture, while uninduced ish-TPN cerebral organoids recapitulated the normal cytoarchitecture of the brain. Transcriptomic analysis revealed a proneural GBM subtype. This proof-of-concept study offers a valuable resource for directly investigating the emergence and progression of gliomas within the context of specific genetic alterations in normal cerebral organoids.
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Dong, Xin, Shi-Bo Xu, Xin Chen, Mengdan Tao, Xiao-Yan Tang, Kai-Heng Fang, Min Xu, et al. "Human cerebral organoids establish subcortical projections in the mouse brain after transplantation." Molecular Psychiatry, October 13, 2020. http://dx.doi.org/10.1038/s41380-020-00910-4.
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Abstract Numerous studies have used human pluripotent stem cell-derived cerebral organoids to elucidate the mystery of human brain development and model neurological diseases in vitro, but the potential for grafted organoid-based therapy in vivo remains unknown. Here, we optimized a culturing protocol capable of efficiently generating small human cerebral organoids. After transplantation into the mouse medial prefrontal cortex, the grafted human cerebral organoids survived and extended projections over 4.5 mm in length to basal brain regions within 1 month. The transplanted cerebral organoids generated human glutamatergic neurons that acquired electrophysiological maturity in the mouse brain. Importantly, the grafted human cerebral organoids functionally integrated into pre-existing neural circuits by forming bidirectional synaptic connections with the mouse host neurons. Furthermore, compared to control mice, the mice transplanted with cerebral organoids showed an increase in freezing time in response to auditory conditioned stimuli, suggesting the potentiation of the startle fear response. Our study showed that subcortical projections can be established by microtransplantation and may provide crucial insights into the therapeutic potential of human cerebral organoids for neurological diseases.
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Sozzi, Edoardo, Janko Kajtez, Andreas Bruzelius, Milan Finn Wesseler, Fredrik Nilsson, Marcella Birtele, Niels B. Larsen, et al. "Silk scaffolding drives self-assembly of functional and mature human brain organoids." Frontiers in Cell and Developmental Biology 10 (October 14, 2022). http://dx.doi.org/10.3389/fcell.2022.1023279.
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Human pluripotent stem cells (hPSCs) are intrinsically able to self-organize into cerebral organoids that mimic features of developing human brain tissue. These three-dimensional structures provide a unique opportunity to generate cytoarchitecture and cell-cell interactions reminiscent of human brain complexity in a dish. However, current in vitro brain organoid methodologies often result in intra-organoid variability, limiting their use in recapitulating later developmental stages as well as in disease modeling and drug discovery. In addition, cell stress and hypoxia resulting from long-term culture lead to incomplete maturation and cell death within the inner core. Here, we used a recombinant silk microfiber network as a scaffold to drive hPSCs to self-arrange into engineered cerebral organoids. Silk scaffolding promoted neuroectoderm formation and reduced heterogeneity of cellular organization within individual organoids. Bulk and single cell transcriptomics confirmed that silk cerebral organoids display more homogeneous and functionally mature neuronal properties than organoids grown in the absence of silk scaffold. Furthermore, oxygen sensing analysis showed that silk scaffolds create more favorable growth and differentiation conditions by facilitating the delivery of oxygen and nutrients. The silk scaffolding strategy appears to reduce intra-organoid variability and enhances self-organization into functionally mature human brain organoids.