Artículos de revistas sobre el tema "Mouse Brain Organoids"
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1
Roosen, Mieke, Chris Meulenbroeks, Phylicia Stathi, Joris Maas, Julie Morscio, Jens Bunt y Marcel Kool. "BIOL-11. PRECLINICAL MODELLING OF PEDIATRIC BRAIN TUMORS USING ORGANOID TECHNOLOGY". Neuro-Oncology 25, Supplement_1 (1 de junio de 2023): i8. http://dx.doi.org/10.1093/neuonc/noad073.030.
Resumen
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.
2
Simsa, Robin, Theresa Rothenbücher, Hakan Gürbüz, Nidal Ghosheh, Jenny Emneus, Lachmi Jenndahl, David L. Kaplan, Niklas Bergh, Alberto Martinez Serrano y Per Fogelstrand. "Brain organoid formation on decellularized porcine brain ECM hydrogels". PLOS ONE 16, n.º 1 (28 de enero de 2021): e0245685. http://dx.doi.org/10.1371/journal.pone.0245685.
Resumen
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.
3
Sukhinich, K. K., K. M. Shakirova, E. B. Dashinimaev y M. A. Aleksandrova. "Development of 3D Cerebral Aggregates in the Brain Ventricles of Adult Mice". Russian Journal of Developmental Biology 52, n.º 3 (mayo de 2021): 164–75. http://dx.doi.org/10.1134/s1062360421030061.
Resumen
Abstract The cerebral organoids are three-dimensional cell cultures formed from brain-specific cell types arising from embryonic or pluripotent stem cells. Organoids provide an opportunity to study the early stages of brain development and diseases of the central nervous system. However, the modeling of organoids is associated with a number of unsolved problems. Organoid production techniques involve a complex cell culture process that requires special media, growth factors, and often the use of a bioreactor. Even under standardized conditions, structures of different morphology are formed: from disorganized cell aggregates to structured minibrains, which are selected for study. For natural reasons, organoids grown in vitro do not have a blood supply, which limits their development. We tried to obtain cerebral aggregates similar to organoids in an in vivo model, where vascular growth and tissue blood supply are provided, for which we transplanted a cell suspension from the mouse embryonic neocortex into the lateral ventricles of the brain of adult mice. Therefore, the medium for cultivation was the cerebrospinal fluid, and the lateral ventricles of the brain, where it circulates, served as a bioreactor. The results showed that the neocortex from E14.5 is a suitable source of stem/progenitor cells that self-assemble into three-dimensional aggregates and vascularized in vivo. The aggregates consisted of a central layer of mature neurons, the marginal zone free of cells and a glia limitans, which resembled cerebral organoids. Thus, the lateral ventricles of the adult mouse brain can be used to obtain vascularized cell aggregates resembling cerebral organoids.
4
Bao, Zhongyuan, Kaiheng Fang, Zong Miao, Chong Li, Chaojuan Yang, Qiang Yu, Chen Zhang, Zengli Miao, Yan Liu y Jing Ji. "Human Cerebral Organoid Implantation Alleviated the Neurological Deficits of Traumatic Brain Injury in Mice". Oxidative Medicine and Cellular Longevity 2021 (22 de noviembre de 2021): 1–16. http://dx.doi.org/10.1155/2021/6338722.
Resumen
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.
5
Ferdaos, Nurfarhana, Sally Lowell y John O. Mason. "Pax6 mutant cerebral organoids partially recapitulate phenotypes of Pax6 mutant mouse strains". PLOS ONE 17, n.º 11 (28 de noviembre de 2022): e0278147. http://dx.doi.org/10.1371/journal.pone.0278147.
Resumen
Cerebral organoids show great promise as tools to unravel the complex mechanisms by which the mammalian brain develops during embryogenesis. We generated mouse cerebral organoids harbouring constitutive or conditional mutations in Pax6, which encodes a transcription factor with multiple important roles in brain development. By comparing the phenotypes of mutant organoids with the well-described phenotypes of Pax6 mutant mouse embryos, we evaluated the extent to which cerebral organoids reproduce phenotypes previously described in vivo. Organoids lacking Pax6 showed multiple phenotypes associated with its activity in mice, including precocious neural differentiation, altered cell cycle and an increase in abventricular mitoses. Neural progenitors in both Pax6 mutant and wild type control organoids cycled more slowly than their in vivo counterparts, but nonetheless we were able to identify clear changes to cell cycle attributable to the absence of Pax6. Our findings support the value of cerebral organoids as tools to explore mechanisms of brain development, complementing the use of mouse models.
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García-Delgado, Ana Belén, Rafael Campos-Cuerva, Cristina Rosell-Valle, María Martin-López, Carlos Casado, Daniela Ferrari, Javier Márquez-Rivas, Rosario Sánchez-Pernaute y Beatriz Fernández-Muñoz. "Brain Organoids to Evaluate Cellular Therapies". Animals 12, n.º 22 (15 de noviembre de 2022): 3150. http://dx.doi.org/10.3390/ani12223150.
Resumen
Animal models currently used to test the efficacy and safety of cell therapies, mainly murine models, have limitations as molecular, cellular, and physiological mechanisms are often inherently different between species, especially in the brain. Therefore, for clinical translation of cell-based medicinal products, the development of alternative models based on human neural cells may be crucial. We have developed an in vitro model of transplantation into human brain organoids to study the potential of neural stem cells as cell therapeutics and compared these data with standard xenograft studies in the brain of immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Neural stem cells showed similar differentiation and proliferation potentials in both human brain organoids and mouse brains. Our results suggest that brain organoids can be informative in the evaluation of cell therapies, helping to reduce the number of animals used for regulatory studies.
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Yakoub, Abraam M. y Mark Sadek. "Analysis of Synapses in Cerebral Organoids". Cell Transplantation 28, n.º 9-10 (4 de junio de 2019): 1173–82. http://dx.doi.org/10.1177/0963689718822811.
Resumen
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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Estridge, R. Chris, Jennifer E. O’Neill y Albert J. Keung. "Matrigel Tunes H9 Stem Cell-Derived Human Cerebral Organoid Development". Organoids 2, n.º 4 (5 de octubre de 2023): 165–76. http://dx.doi.org/10.3390/organoids2040013.
Resumen
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.
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Antonica, Francesco, Lucia Santomaso, Davide Pernici, Linda Petrucci, Giuseppe Aiello, Alessandro Cutarelli, Luciano Conti et al. "MODL-22. Establishment of a novel system to specifically trace and ablate quiescent/slow cycling cells in high-grade glioma". Neuro-Oncology 24, Supplement_1 (1 de junio de 2022): i173. http://dx.doi.org/10.1093/neuonc/noac079.645.
Resumen
Abstract Pediatric and adult high-grade gliomas are the most common malignant brain tumors, with poor prognosis due to recurrence and tumor infiltration after surgical removal and chemotherapy. Quiescent/slow cycling stem cells have been proposed to be one of the main players of tumor relapse but their involvement in in the infiltration remain unclear. Despite they have been described in mouse models or after transcriptional profiling of human tumor samples, their direct visualization, targeting and ablation remains a challenge. Here, we identified a population of malignant cells expressing Prominin-1 in a non-proliferating state in pediatric high-grade glioma patients. We next used a fluorescent cell cycle sensor to visualize quiescence tumor cells in mouse brain cancer and human cancer organoids. In particular, we characterized them within the tumor revealing the invasiveness capacity of slow cycling tumor cells. Furthermore, we generated a new system to specifically trace and ablate such cells. Indeed, lineage tracing experiments allowed to trace quiescent Prom1 progeny in the tumors after temozolomide treatment. In addition, the selective ablation of quiescent Prom1+ cells in mouse brain cancer reduced tumor infiltration and improved survival. Furthermore, time-lapse experiments showed that slow cycling cells are also able to infiltrate co-cultured human brain cancer organoids. Finally, using our new cancer organoid- and GBM spheroids-based models we identified a drug acting on quiescent cells leading to a reduction in cell invasion. Overall, our data show that quiescence/slow cycling cells are key driver of tumor invasiveness, the major malignant feature of high-grade brain cancer.
10
Antonica, F., L. Santomaso, G. Aiello, D. Pernici, E. Miele y L. Tiberi. "OS13.3.A Establishment of a novel system to specifically trace and ablate quiescent/slow cycling cells in high-grade glioma". Neuro-Oncology 23, Supplement_2 (1 de septiembre de 2021): ii16. http://dx.doi.org/10.1093/neuonc/noab180.051.
Resumen
Abstract BACKGROUND High-grade gliomas are the most common malignant brain tumors, with poor prognosis due to recurrence and tumor infiltration after surgical removal and chemotherapy. Quiescent/slow cycling stem cells have been proposed to be one of the main players of tumor relapse but their involvement in in the infiltration remain unclear. Despite they have been described in mouse models or after transcriptional profiling of human tumor samples, their direct visualization, targeting and ablation remains a challenge. MATERIAL AND METHODS Tumors were induced over-expressing oncogenic forms of MET and p53 in the subventricular zone (SVZ) of P2 mouse brain as well as human forebrain organoids. The co-expression with specific cell cycle sensors as well as lineage specific CreERT2 under control of stem cells promoters allowed to visualize and target glioma stem cells. RESULTS Here, we used a fluorescent cell cycle sensor to visualize quiescent tumor cells in mouse brain cancer and human cancer organoids. In particular, we characterized them within the tumor revealing the invasiveness capacity of slow cycling tumor cells. Furthermore, we generated a new system to specifically trace and ablate such cells. Indeed, lineage tracing experiments allowed to trace quiescent Prom1 progeny in the tumors after temozolomide treatment. In addition, the selective ablation of qProm1 in mouse brain cancer reduced tumor infiltration. Finally, time-lapse experiments showed that slow cycling cells are also able to infiltrate co-cultured human brain cancer organoids. CONCLUSION Overall, our data show that quiescent/slow cycling cells are key driver of tumor invasiveness, the major malignant feature of high-grade brain cancer.
11
Lee, Won Ji, Jeong Eon Lee, Yean Ju Hong, Sang Hoon Yoon, Hyuk Song, Chankyu Park, Kwonho Hong, Youngsok Choi y Jeong Tae Do. "Generation of brain organoids from mouse ESCs via teratoma formation". Stem Cell Research 49 (diciembre de 2020): 102100. http://dx.doi.org/10.1016/j.scr.2020.102100.
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Golebiewska, Anna, Ann-Christin Hau, Anais Oudin, Daniel Stieber, Yahaya A. Yabo, Yong-Jun Kwon, Barbara Klink et al. "TMOD-08. PRIMARY AND RECURRENT GLIOMA PATIENT-DERIVED ORTHOTOPIC XENOGRAFTS (PDOX) REPRESENT RELEVANT PATIENT AVATARS FOR PRECISION MEDICINE". Neuro-Oncology 22, Supplement_2 (noviembre de 2020): ii229. http://dx.doi.org/10.1093/neuonc/noaa215.959.
Resumen
Abstract Patient-derived cancer models are essential tools for studying tumor biology and for preclinical interventions. Although numerous clinical cancer trials are being conducted, many fail due to inappropriate selection of compounds at the preclinical stage. Therefore, better preclinical models are crucial for predicting successful clinical impact. Orthotropic patient-derived xenograft (PDOX) models are of particular importance for brain cancers, as they allow to better recapitulate the brain tumor environment and the blood brain barrier. We created a large collection of PDOXs from primary and recurrent gliomas with and without mutations in IDH1. PDOX models were based on 3D organoids, derived from mechanically minced patient material. Organoids were implanted in the brain of immunodeficient mice and further propagated by serial intracranial transplantations. High grade glioma PDOX models, starting with viable patient-derived organoids, have generally a high tumor take rate, a reproducible phenotype and tumor development time. PDOXs retain histopathological, genetic, epigenetic and transcriptomic features of patient tumors with no mouse-specific clonal evolution. Longitudinal PDOX models confirmed limited evolution of gliomas upon treatment observed in patient tumors. PDOX-derived standardized tumor organoid cultures enabled assessment of drug responses, which were validated in mice. PDOXs showed clinically relevant responses to Temozolomide and to targeted treatments such as EGFR and CDK4/6 inhibitors in (epi)genetically defined groups, according to MGMT promoter and EGFR/CDK status respectively. These data indicate that glioma PDOXs represent clinically relevant avatars for personalized treatment. The use of these models should lead to a more realistic evaluation of the efficacy of novel drugs, thereby increasing the success of clinical studies.
13
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 (28 de noviembre de 2023): 1407. http://dx.doi.org/10.1182/blood-2023-178613.
Resumen
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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Posadas, Inmaculada, Laura Romero-Castillo, Rosa-Anna Ronca, Andrii Karpus, Serge Mignani, Jean-Pierre Majoral, Mariángeles Muñoz-Fernández y Valentín Ceña. "Engineered Neutral Phosphorous Dendrimers Protect Mouse Cortical Neurons and Brain Organoids from Excitotoxic Death". International Journal of Molecular Sciences 23, n.º 8 (15 de abril de 2022): 4391. http://dx.doi.org/10.3390/ijms23084391.
Resumen
Nanoparticles are playing an increasing role in biomedical applications. Excitotoxicity plays a significant role in the pathophysiology of neurodegenerative diseases, such as Alzheimer’s or Parkinson’s disease. Glutamate ionotropic receptors, mainly those activated by N-methyl-D-aspartate (NMDA), play a key role in excitotoxic death by increasing intraneuronal calcium levels; triggering mitochondrial potential collapse; increasing free radicals; activating caspases 3, 9, and 12; and inducing endoplasmic reticulum stress. Neutral phosphorous dendrimers, acting intracellularly, have neuroprotective actions by interfering with NMDA-mediated excitotoxic mechanisms in rat cortical neurons. In addition, phosphorous dendrimers can access neurons inside human brain organoids, complex tridimensional structures that replicate a significant number of properties of the human brain, to interfere with NMDA-induced mechanisms of neuronal death. Phosphorous dendrimers are one of the few nanoparticles able to gain access to the inside of neurons, both in primary cultures and in brain organoids, and to exert pharmacological actions by themselves.
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Yokoi, Remi, Nami Nagafuku, Yuto Ishibashi, Naoki Matsuda y Ikuro Suzuki. "Contraindicated Drug Responses in Dravet Syndrome Brain Organoids Utilizing Micro Electrode Array Assessment Methods". Organoids 2, n.º 4 (26 de octubre de 2023): 177–91. http://dx.doi.org/10.3390/organoids2040014.
Resumen
Ensuring drug safety for patients with specific neurological disorders is of paramount importance. For instance, certain antiepileptic drugs (AEDs) are contraindicated in Dravet Syndrome (DS), which is characterized by a deficiency in Na+ channel function. Constructing in vitro assessment methods capable of detecting contraindicated drug responses and medication effects on neurons derived from DS patients is highly anticipated for drug safety assessment and therapeutic innovation. This study used micro electrode array (MEA) measurements with low-frequency analysis on human iPSC-derived DS organoids to investigate AED responses. When exposed to the contraindicated drugs carbamazepine and phenytoin, the number of network oscillations increased in DS organoids while maintaining oscillation intensity. Furthermore, carbamazepine administration appeared to enhance activities beyond oscillations which is partially consistent with findings in the DS mouse model. Conversely, treatment with the therapeutic drug sodium valproate resulted in a similar decrease in activity both in healthy and DS organoids. The frequency characteristics of spontaneous firings and AEDs responsiveness in DS organoids demonstrated partial correlation with typical electroencephalography patterns observed in vivo. In conclusion, this study, employing MEA measurements with low-frequency analysis, revealed contraindicated drug responses and disease-specific functional characteristics in DS organoids, effective for DS patient safety assessment, precision medicine, and antiepileptic drug screening.
16
Islam, Rehnuma, Humna Noman, Ashkan Azimi, Ricky Siu, Vorapin Chinchalongporn, Carol Schuurmans y Cindi M. Morshead. "Primitive and Definitive Neural Precursor Cells Are Present in Human Cerebral Organoids". International Journal of Molecular Sciences 25, n.º 12 (14 de junio de 2024): 6549. http://dx.doi.org/10.3390/ijms25126549.
Resumen
Activation of neural stem cells (NSCs) correlates with improved functional outcomes in mouse models of injury. In the murine brain, NSCs have been extensively characterized and comprise (1) primitive NSCs (pNSCs) and (2) definitive NSCs (dNSCs). pNSCs are the earliest cells in the NSC lineage giving rise to dNSCs in the embryonic and adult mouse brain. pNSCs are quiescent under baseline conditions and can be activated upon injury. Herein, we asked whether human pNSCs and dNSCs can be isolated during the maturation of human cerebral organoids (COs) and activated by drugs known to regulate mouse NSC behavior. We demonstrate that self-renewing, multipotent pNSC and dNSC populations are present in human COs and express genes previously characterized in mouse NSCs. The drug NWL283, an inhibitor of apoptosis, reduced cell death in COs but did not improve NSC survival. Metformin, a drug used to treat type II diabetes that is known to promote NSC activation in mice, was found to expand human NSC pools. Together, these findings are the first to identify and characterize human pNSCs, advancing our understanding of the human NSC lineage and highlighting drugs that enhance their activity.
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Ramirez, Santiago, Abhisek Mukherjee, Sofia Sepulveda, Andrea Becerra-Calixto, Nicolas Bravo-Vasquez, Camila Gherardelli, Melissa Chavez y Claudio Soto. "Modeling Traumatic Brain Injury in Human Cerebral Organoids". Cells 10, n.º 10 (7 de octubre de 2021): 2683. http://dx.doi.org/10.3390/cells10102683.
Resumen
Traumatic brain injury (TBI) is a head injury that disrupts the normal brain structure and function. TBI has been extensively studied using various in vitro and in vivo models. Most of the studies have been done with rodent models, which may respond differently to TBI than human nerve cells. Taking advantage of the recent development of cerebral organoids (COs) derived from human induced pluripotent stem cells (iPSCs), which resemble the architecture of specific human brain regions, here, we adapted the controlled cortical impact (CCI) model to induce TBI in human COs as a novel in vitro platform. To adapt the CCI procedure into COs, we have developed a phantom brain matrix, matching the mechanical characteristics of the brain, altogether with an empty mouse skull as a platform to allow the use of the stereotactic CCI equipment on COs. After the CCI procedure, COs were histologically prepared to evaluate neurons and astrocyte populations using the microtubule-associated protein 2 (MAP2) and the glial fibrillary acidic protein (GFAP). Moreover, a marker of metabolic response, the neuron-specific enolase (NSE), and cellular death using cleaved caspase 3 were also analyzed. Our results show that human COs recapitulate the primary pathological changes of TBI, including metabolic alterations related to neuronal damage, neuronal loss, and astrogliosis. This novel approach using human COs to model TBI in vitro holds great potential and opens new alternatives for understanding brain abnormalities produced by TBI, and for the development and testing of new therapeutic approaches.
18
Zhou, Qinjie, Diego F. Niño, Yukihiro Yamaguchi, Sanxia Wang, William B. Fulton, Hongpeng Jia, Peng Lu et al. "Necrotizing enterocolitis induces T lymphocyte–mediated injury in the developing mammalian brain". Science Translational Medicine 13, n.º 575 (6 de enero de 2021): eaay6621. http://dx.doi.org/10.1126/scitranslmed.aay6621.
Resumen
Necrotizing enterocolitis (NEC) causes acute intestinal necrosis in premature infants and is associated with severe neurological impairment. In NEC, Toll-like receptor 4 is activated in the intestinal epithelium, and NEC-associated brain injury is characterized by microglial activation and white matter loss through mechanisms that remain unclear. We now show that the brains of mice and humans with NEC contained CD4+ T lymphocytes that were required for the development of brain injury. Inhibition of T lymphocyte influx into the brains of neonatal mice with NEC reduced inflammation and prevented myelin loss. Adoptive intracerebroventricular delivery of gut T lymphocytes from mice with NEC into Rag1−/− recipient mice lacking CD4+ T cells resulted in brain injury. Brain organoids derived from mice with or without NEC and from human neuronal progenitor cells revealed that IFN-γ release by CD4+ T lymphocytes induced microglial activation and myelin loss in the organoids. IFN-γ knockdown in CD4+ T cells derived from mice with NEC abrogated the induction of NEC-associated brain injury after adoptive transfer to naïve Rag1−/− recipient mice. T cell receptor sequencing revealed that NEC mouse brain-derived T lymphocytes shared homology with gut T lymphocytes from NEC mice. Intraperitoneal injection of NEC gut-derived CD4+ T lymphocytes into naïve Rag1−/− recipient mice induced brain injury, suggesting that gut-derived T lymphocytes could mediate neuroinflammation in NEC. These findings indicate that NEC-associated brain injury may be induced by gut-derived IFN-γ–releasing CD4+ T cells, suggesting that early management of intestinal inflammation in children with NEC could improve neurological outcomes.
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Ciarpella, Francesca, Raluca Georgiana Zamfir, Alessandra Campanelli, Giulia Pedrotti, Marzia Di Chio, Emanuela Bottani y Ilaria Decimo. "Generation of mouse hippocampal brain organoids from primary embryonic neural stem cells". STAR Protocols 4, n.º 3 (septiembre de 2023): 102413. http://dx.doi.org/10.1016/j.xpro.2023.102413.
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Alaali, Lujain, Jinchong Xu, Jeff Mumm, Ming Yuan, Charles Eberhart y Eric Raabe. "BIOL-13. DEPLOYING NEW MODELS OF NF-1 MUTANT LOW GRADE GLIOMA TO ACCELERATE THERAPEUTIC DEVELOPMENT". Neuro-Oncology 25, Supplement_1 (1 de junio de 2023): i8. http://dx.doi.org/10.1093/neuonc/noad073.032.
Resumen
Abstract Pediatric low grade glioma (pLGG) research is hampered by a paucity of genetically accurate cell line models and xenografts. To address this need, we have developed and characterized multiple in vivo and organoid models. We used cell reprogramming conditions (CRC) to establish and propagate JHH-NF1-PA1, a NF1-mutant line derived from a low grade optic pathway glioma of a patient with type 1 neurofibromatosis. JHH-NF1-PA1 cells persist and take up midline positions when xenografted into the brains of zebrafish, allowing testing of therapeutics in vivo. We developed a human iPSC-brain organoid system comprising of midline neurons, glia, and microglia, largely recapitulating the microenvironment of midline low grade glioma. Our JHH-NF1-PA1 cells incorporate into these brain organoids, allowing us to test potential therapeutics in an in vitro setting in which the tumor cells grow in the appropriate microenvironment. We have also further defined the mouse BL6 NF1 null glioma cell line 1810, which forms orthotopic xenografts in mice. This last model will be useful for therapeutic testing in a syngeneic BL6 background, allowing assessment of immune infiltration and signaling downstream of NF1 loss and potential immunotherapeutics. Together, these in vitro and in vivo models of NF1-driven low grade glioma make an important contribution to our understanding of the biology of low grade glioma.
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Song, Chenyun, Xinyu Chen, Jixin Ma, Hada Buhe, Yang Liu, Hexige Saiyin y Lixiang Ma. "Construction of a pancreatic cancer nerve invasion system using brain and pancreatic cancer organoids". Journal of Tissue Engineering 14 (enero de 2023): 204173142211471. http://dx.doi.org/10.1177/20417314221147113.
Resumen
Pancreatic cancer (PC) is a fatal malignancy in the human abdominal cavity that prefers to invade the surrounding nerve/nerve plexus and even the spine, causing devastating and unbearable pain. The limitation of available in vitro models restricts revealing the molecular mechanism of pain and screening pain-relieving strategies to improve the quality of life of end-stage PC patients. Here, we report a PC nerve invasion model that merged human brain organoids (hBrO) with mouse PC organoids (mPCO). After merging hBrOs with mPCOs, we monitored the structural crosstalk, growth patterns, and mutual interaction dynamics of hBrO with mPCOs for 7 days. After 7 days, we also analyzed the pathophysiological statuses, including proliferation, apoptosis and inflammation. The results showed that mPCOs tend to approximate and intrude into the hBrOs, merge entirely into the hBrOs, and induce the retraction/shrinking of neuronal projections that protrude from the margin of the hBrOs. The approximating of mPCOs to hBrOs accelerated the proliferation of neuronal progenitor cells, intensified the apoptosis of neurons in the hBrOs, and increased the expression of inflammatory molecules in hBrOs, including NLRP3, IL-8, and IL-1β. Our system pathophysiologically replicated the nerve invasions in mouse GEMM (genetically engineered mouse model) primary and human PCs and might have the potential to be applied to reveal the molecular mechanism of nerve invasion and screen therapeutic strategies in PCs.
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Kan, Ryan, Weihong Ge, Can Yilgor, Nicholas Bayley, Christopher Tse, Andrew Tum, Kunal Patel, David Nathanson y Aparna Bhaduri. "CSIG-15. PTN-PTPRZ1 SIGNALING MEDIATES TUMOR-NORMAL CROSSTALK IN GLIOBLASTOMA". Neuro-Oncology 25, Supplement_5 (1 de noviembre de 2023): v43. http://dx.doi.org/10.1093/neuonc/noad179.0171.
Resumen
Abstract Glioblastoma (GBM) is the most devastating form of brain cancer with poor patient prognosis and high recurrence. An increasing body of literature suggests crosstalk between tumor and its surroundings, both molding the immune microenvironment and forming functional synapses with neighboring normal cells. Despite the high intra-tumoral and inter-patient heterogeneity, we have discovered PTN-PTPRZ1 signaling as the most significant and preserved communication pathway between GBM cells and their immediate neighboring cells. Through a novel tumor transplantation protocol onto cortical organoids, which are human embryonic stem cell-derived aggregates that faithfully mimic the human brain, we have seen evidence of tumor-normal crosstalk where PTN-PTPRZ1 is a key signaling axis. We hypothesize that PTN-PTPRZ1 signaling is pivotal to tumor growth and invasion, which ultimately leads to recurrence. Modulating PTN and/or PTPRZ1 levels on the cortical organoids via shRNA affects PTN and/or PTPRZ1 expression on the tumors, suggesting dynamic communication between tumor and normal cells via PTN-PTPRZ1 signaling. During the process of generating PTN knockdown cortical organoids, we have additionally discovered a pivotal role for PTN in the very early stages of cortical development. PTN and PTPRZ1 are signature markers of the outer radial glia in the developing human brain, which are absent in mouse models where the majority of PTN biology has been studied. By supplementing exogenous PTN to PTN knockdown organoids and withdrawing it at various timepoints, we have identified drastic phenotypes of neuron and radial glia survival and cell fate specification. With cortical organoids, we can interrogate the role of PTN in the early stages of neurodevelopment via a human-derived system.
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Tiberi, Luca. "PDTM-35. MODELLING MEDULLOBLASTOMA WITH MOUSE MODELS AND HUMAN CEREBELLAR ORGANOIDS". Neuro-Oncology 21, Supplement_6 (noviembre de 2019): vi195. http://dx.doi.org/10.1093/neuonc/noz175.811.
Resumen
Abstract Malignant medulloblastoma (MB) is the most common brain tumor affecting children and it remains responsible for a high percentage of morbidity and mortality among cancer patients. During the past few years, studies on human MB and mouse models have uncovered the existence of four major MB groups, with specific pathological and molecular features: WNT, SHH, Group 3 and Group 4. Of the four groups, patients with Group 3 MB have the worst outcome with nearly 50% of the tumors metastatic at the time of diagnosis. Therefore, developing “humanized “ mouse model of Group3 Medulloblastoma would be of paramount importance for the identification and testing of new drugs for pediatric patients, tailored on the genetic condition of the patient itself. We performed an in-vivo screen to identify new cancer-inducing genes starting from the published genome-wide analyses of MB patients. This screen led to the identification of novel driver gene combinations required for tumorigenesis. To study Medulloblastoma in human cells, we generated human cerebellar organoids. Organoids are cellular structures that resemble whole organs and can be generated from pluripotent stem cells or isolated organ progenitors. We were able to induce cerebellar progenitors to self-form neuro-epithelial structures that mimic early human cerebellar plate, composed by cerebellar progenitors, cerebellar neurons (interneurons, Purkinje cells, and granule neurons) and glial cells. Notably, we have generated human putative iPSC-derived cancer organoids with gene combinations mimicking human Group3 MB. Indeed, organoids that mimic cancer can be used as an alternative system for drug testing that better recapitulate effects in human patients as compared to other in-vitro and in-vivo systems.
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Slonchak, Andrii, Leon E. Hugo, Morgan Freney, Alberto A. Amarilla, Sonja Hall-Mendelin, Kexin Yan, Francisco Torres et al. "Zika Virus sfRNA Plays an Essential Role in the Infection of Insects and Mammals". Proceedings 50, n.º 1 (1 de julio de 2020): 112. http://dx.doi.org/10.3390/proceedings2020050112.
Resumen
Similar to other flaviviruses, Zika virus (ZIKV) produces abundant subgenomic flavivirus RNA (sfRNA) derived from the 3’ untranslated region. The molecular mechanisms that determine the functions of sfRNA are currently not completely understood. Here, we created ZIKV mutants deficient in sfRNA production and employed them to investigate the role of this RNA in virus interactions with mammalian and insect hosts. We found that in mosquitoes, sfRNA facilitates virus replication and is required for ZIKV dissemination into saliva and virus transmission. The production of sfRNA was found to have no effect on the RNAi pathway, but instead downregulated the expression of genes involved in the regulation of apoptosis. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) of histological sections from infected mosquitoes confirmed that sfRNA prevents the apoptotic death of infected cells, thus identifying inhibition of apoptosis as a novel mechanism of sfRNA action in mosquitoes. We also found that sfRNA facilitates ZIKV replication in mammalian cells, mice, and human brain organoids. Moreover, ZIKV mutants deficient in sfRNA production were unable to form plaques, cause the death of human brain organoids, or establish infection in the mouse foetal brain. We then found that the proviral activity of sfRNA in mammalian cells relies on its ability to suppress type I interferon signalling. We showed that this is achieved via the inhibition of phosphorylation and the nuclear translocation of STAT1. In addition, we found that the production of sfRNA in the ZIKV infection of human brain organoids is associated with the suppression of multiple genes involved in brain development, indicating that sfRNA can be involved in the disruption of brain development associated with ZIKV infection.
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Gautam, Shailendra K., Ranjana K. Kanchan, Jawed A. Siddiqui, Shailendra K. Maurya, Sanchita Rauth, Naveenkumar Perumal, Pranita Atri et al. "Blocking c-MET/ERBB1 Axis Prevents Brain Metastasis in ERBB2+ Breast Cancer". Cancers 12, n.º 10 (1 de octubre de 2020): 2838. http://dx.doi.org/10.3390/cancers12102838.
Resumen
Brain metastasis (BrM) remains a significant cause of cancer-related mortality in epidermal growth factor receptor 2-positive (ERBB2+) breast cancer (BC) patients. We proposed here that a combination treatment of irreversible tyrosine kinase inhibitor neratinib (NER) and the c-MET inhibitor cabozantinib (CBZ) could prevent brain metastasis. To address this, we first tested the combination treatment of NER and CBZ in the brain-seeking ERBB2+ cell lines SKBrM3 and JIMT-1-BR3, and in ERBB2+ organoids that expressed the c-MET/ERBB1 axis. Next, we developed and characterized an orthotopic mouse model of spontaneous BrM and evaluated the therapeutic effect of CBZ and NER in vivo. The combination treatment of NER and CBZ significantly inhibited proliferation and migration in ERBB2+ cell lines and reduced the organoid growth in vitro. Mechanistically, the combination treatment of NER and CBZ substantially inhibited ERK activation downstream of the c-MET/ERBB1 axis. Orthotopically implanted SKBrM3+ cells formed primary tumor in the mammary fat pad and spontaneously metastasized to the brain and other distant organs. Combination treatment with NER and CBZ inhibited primary tumor growth and predominantly prevented BrM. In conclusion, the orthotopic model of spontaneous BrM is clinically relevant, and the combination therapy of NER and CBZ might be a useful approach to prevent BrM in BC.
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Gamboa, Christian Moya, Kelly Jara, Sahithi Pamarthy, Liqiong Liu, Robert Aiken, Zhenggang Xiong, Shabbar Danish y Hatem E. Sabaawy. "Generation of glioblastoma patient-derived organoids and mouse brain orthotopic xenografts for drug screening". STAR Protocols 2, n.º 1 (marzo de 2021): 100345. http://dx.doi.org/10.1016/j.xpro.2021.100345.
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Drucker, Kristen, Thomas Kollmeyer, Connor Yanchus, Silvana De Lorenzo, Jeanette Eckel-Passow, Daniel Honore Lachance, Daniel Schramek y Robert Jenkins. "MODL-03. IDH-MUTANT GLIOMA GWAS ALLELE RS55705857 ON 8Q24 IS CHANGING THE DYNAMICS OF UNPATTERNED CEREBRAL ORGANOID DEVELOPMENT". Neuro-Oncology 24, Supplement_7 (1 de noviembre de 2022): vii291. http://dx.doi.org/10.1093/neuonc/noac209.1131.
Resumen
Abstract BACKGROUND The topologically associated domain (TAD) on 8q24 surrounding MYC is important for many types of cancer. The region is also important in the formation of IDH-mutant glioma; the risk allele rs55705857 being associated with the development of these tumors. The rs55705857 risk allele significantly increased the formation of tumors in an IDH-mutant mouse model. Cerebral organoids have been found to recapitulate the early development of the brain. We hypothesized that the rs55705857 risk allele may alter the phenotype of cerebral organoids. METHODS Isogenic induced pluripotent stem cells (iPSCs) were developed with (n=3) and without (n=2) the rs55705857 risk allele from the parental cell line PGP-1 (GM23338). These cells were cultured and differentiated into unpatterned cerebral organoids and observed throughout differentiation. Organoids were fixed, sectioned, and stained by H&E and immunofluorescence. RESULTS Organoids containing the risk allele were smaller throughout development, starting at the embryoid body stage and continuing up to over 75 days in culture, compared to two isogenic non-risk lines. At 4 weeks the risk allele containing organoids also contained a larger number of small rosette-like neuroepithelia, instead of the more continuous neuroepithelia observed in the non-risk allele organoids. At 75 days, the organoids with the risk allele maintained a larger proportion of Sox2 positive cells, which are enriched in the tight clusters of small cells with round nuclei. CONCLUSIONS The risk allele rs55705857 in the MYC TAD at 8q24 is altering the development of unpatterned cerebral organoids. This difference is visible as early as embryoid body development and persists through neuroepithelial development. Further studies of these differences may help our understanding of how rs55705857 accelerates IDH-mutant glioma development.
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Kishida, Kunihiro, Sarah C. Pearce, Shiyan Yu, Nan Gao y Ronaldo P. Ferraris. "Nutrient sensing by absorptive and secretory progenies of small intestinal stem cells". American Journal of Physiology-Gastrointestinal and Liver Physiology 312, n.º 6 (1 de junio de 2017): G592—G605. http://dx.doi.org/10.1152/ajpgi.00416.2016.
Resumen
Nutrient sensing triggers responses by the gut-brain axis modulating hormone release, feeding behavior and metabolism that become dysregulated in metabolic syndrome and some cancers. Except for absorptive enterocytes and secretory enteroendocrine cells, the ability of many intestinal cell types to sense nutrients is still unknown; hence we hypothesized that progenitor stem cells (intestinal stem cells, ISC) possess nutrient sensing ability inherited by progenies during differentiation. We directed via modulators of Wnt and Notch signaling differentiation of precursor mouse intestinal crypts into specialized organoids each containing ISC, enterocyte, goblet, or Paneth cells at relative proportions much higher than in situ as determined by mRNA expression and immunocytochemistry of cell type biomarkers. We identified nutrient sensing cell type(s) by increased expression of fructolytic genes in response to a fructose challenge. Organoids comprised primarily of enterocytes, Paneth, or goblet, but not ISC, cells responded specifically to fructose without affecting nonfructolytic genes. Sensing was independent of Wnt and Notch modulators and of glucose concentrations in the medium but required fructose absorption and metabolism. More mature enterocyte- and goblet-enriched organoids exhibited stronger fructose responses. Remarkably, enterocyte organoids, upon forced dedifferentiation to reacquire ISC characteristics, exhibited a markedly extended lifespan and retained fructose sensing ability, mimicking responses of some dedifferentiated cancer cells. Using an innovative approach, we discovered that nutrient sensing is likely repressed in progenitor ISCs then irreversibly derepressed during specification into sensing-competent absorptive or secretory lineages, the surprising capacity of Paneth and goblet cells to detect fructose, and the important role of differentiation in modulating nutrient sensing. NEW & NOTEWORTHY Small intestinal stem cells differentiate into several cell types transiently populating the villi. We used specialized organoid cultures each comprised of a single cell type to demonstrate that 1) differentiation seems required for nutrient sensing, 2) secretory goblet and Paneth cells along with enterocytes sense fructose, suggesting that sensing is acquired after differentiation is triggered but before divergence between absorptive and secretory lineages, and 3) forcibly dedifferentiated enterocytes exhibit fructose sensing and lifespan extension.
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Ge, Weihong, Ryan Kan, Elisa Fazzari, Daria Azizad, Joyce Ito, Can Yilgor, Christopher Tse et al. "TMIC-05. UNVEILING THE IMPACT OF PTN-PTPRZ1 SIGNALING ON GLIOBLASTOMA PROGRESSION THROUGH TUMOR MICROENVIRONMENT COMMUNICATION". Neuro-Oncology 25, Supplement_5 (1 de noviembre de 2023): v278—v279. http://dx.doi.org/10.1093/neuonc/noad179.1071.
Resumen
Abstract Glioblastoma (GBM) is the most lethal primary brain tumor with high rate of recurrence. GBM’s high inter- and intra-heterogeneity present a huge challenge to both disease modeling and treatment. Recent progress in the field has highlighted the role of signals associated with the microenvironment factor in driving tumor progression, suggesting that more detailed characterizations of the GBM microenvironment may provide novel therapeutic targets. In this study, we used a novel stem cell derived cortical organoid transplantation model, where we transplanted patient IDH wild type GBM cells onto organoids and provided multiple culture conditions to explore the impact of the environment on tumor cell identity. After several weeks of culture, single cell RNA-sequencing of tumor and non-neoplastic organoid cells was used to profile molecular features, demonstrating not only cellular composition of GBM and its surrounding normal brain cell, but also the bidirectional communication between the two. We observed functional heterogeneity where individual tumors responded differently to environmental perturbations. However, the most significant cell-cell interaction between tumors and the microenvironment was found to be PTN-PTPRZ1, which is well-maintained across all transplantation models we tested, as well as in re-analysis of published patient data that included tumor and normal cells. Encouraged by our previous works as well as others, which have revealed PTPRZ1 as an outer radial glia marker gene driving tumor invasion, we further verified PTN/PTPRZ1 expression not only in our organoid transplantation models but also on FFPE sections from GBM patients and mouse xenograft samples. These data suggest that this signaling axis promotes tumor progression through tumor – microenvironmental crosstalk, and using the organoid model we show a functional role for this communication. Together, this study presents a GBM organoid transplantation modeling system, which we leverage to detect a promising therapeutically target existing between GBM and its microenvironment.
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Cerrizuela, Santiago, Oguzhan Kaya, Lukas P. M. Kremer, Andrea Sarvari, Tobias Ellinger, Jannes Straub, Jan Brunken, Andrés Sanz-Morejón, Aylin Korkmaz y Ana Martín-Villalba. "High-throughput scNMT protocol for multiomics profiling of single cells from mouse brain and pancreatic organoids". STAR Protocols 3, n.º 3 (septiembre de 2022): 101555. http://dx.doi.org/10.1016/j.xpro.2022.101555.
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Sun, Alfred Xuyang, Qiang Yuan, Masahiro Fukuda, Weonjin Yu, Haidun Yan, Grace Gui Yin Lim, Mui Hoon Nai et al. "Potassium channel dysfunction in human neuronal models of Angelman syndrome". Science 366, n.º 6472 (19 de diciembre de 2019): 1486–92. http://dx.doi.org/10.1126/science.aav5386.
Resumen
Disruptions in the ubiquitin protein ligase E3A (UBE3A) gene cause Angelman syndrome (AS). Whereas AS model mice have associated synaptic dysfunction and altered plasticity with abnormal behavior, whether similar or other mechanisms contribute to network hyperactivity and epilepsy susceptibility in AS patients remains unclear. Using human neurons and brain organoids, we demonstrate that UBE3A suppresses neuronal hyperexcitability via ubiquitin-mediated degradation of calcium- and voltage-dependent big potassium (BK) channels. We provide evidence that augmented BK channel activity manifests as increased intrinsic excitability in individual neurons and subsequent network synchronization. BK antagonists normalized neuronal excitability in both human and mouse neurons and ameliorated seizure susceptibility in an AS mouse model. Our findings suggest that BK channelopathy underlies epilepsy in AS and support the use of human cells to model human developmental diseases.
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Nguyen, Phuong, Fadi Jacob, Ryan Salinas, Daniel Zhang, Hongjun Song y Guo-li Ming. "TMOD-26. MODELING GLIOBLASTOMA BY IMPLANTATION OF INTACT PATIENT-DERIVED ORGANOIDS INTO RODENT BRAINS". Neuro-Oncology 21, Supplement_6 (noviembre de 2019): vi268. http://dx.doi.org/10.1093/neuonc/noz175.1125.
Resumen
Abstract Glioblastoma multiforme (GBM) is the most common primary and aggressive brain tumors in adults with extremely poor prognosis and limited treatment options. A major hallmark of GBM is the rapid and diffused infiltration of tumor cells into the surrounding healthy tissue that contribute to tumor recurrence and therapeutic resistance. However, existing in vitro cell culture or in vivo xenograft models inadequately recapitulate the inter-tumoral and intra-tumoral heterogeneity which are key features of GBM. For example, common oncogenic drivers of GBM such as epidermal growth factor receptor (EGFR) amplification and EGFRvIII mutation do not persist in traditional in vitro models due to selection pressures, thus requires exogenous overexpression. Alternatively, EGFR statuses can be maintained in xenografted mice, but implantation of the primary GBM cells into the flank is required to first establish the tumor prior to secondary injection into the brains. Recently, we have established a novel protocol for culturing GBM tissue as organoids (GBOs) directly from patient tumor resection that retain many distinct cell populations in vitro with high fidelity evidenced by histological, whole-exome, bulk and single cell RNA analyses. Compared to prolonged generation time of previously established in vitro and xenograft models, our methodology is robust for generating GBOs within 1–2 weeks from initial resection. In addition, these GBOs can be readily xenografted into the adult mouse brains as an intact organoid, exhibit rapid and aggressive infiltration phenotypes, and maintains driver mutation EGFRviii within as little as one month. Consequently, they can be used to test in vivo treatment efficacies in a timely fashion. The presence of diverse cell types in this GBO model offers a promising platform for not only understanding of tumor biology, but also more strategic development of new therapies.
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Moreno-Sanchez, P. M., A. Oudin, Y. A. Yabo, E. Klein, V. Baus, A. Poli, A. Michelucci, S. P. Niclou y A. Golebiewska. "OS10.5.A Modeling immunocompetent tumor microenvironment in glioblastoma patient-derived orthotopic xenografts". Neuro-Oncology 24, Supplement_2 (1 de septiembre de 2022): ii21—ii22. http://dx.doi.org/10.1093/neuonc/noac174.068.
Resumen
Abstract Background To date, glioblastoma (GBM) remains a fatal disease, with a median overall survival of roughly over a year. There is a crucial need of new treatment options, yet most clinical trials have failed partly due to the lack of predictive preclinical model systems. Currently, most patient-derived preclinical models suffer from the reduction or absence of immune system components, which represents a bottleneck for adequate immunotherapy testing. Humanized mice offer new opportunities here, since they rebuild an adaptive human immune system in a NSG mouse. Derivation of glioblastoma patient-derived orthotopic xenografts (PDOXs) in humanized mice appears thus as a promising tool for testing new treatment strategies targeting the tumor microenvironment (TME). Material and Methods We derived PDOXs through intracranial implantation of GBM primary organoids in different immunocompromised mouse strains (Nude, NOD/SCID, NSG). To introduce back the adaptive human immune system, GBM PDOXs were further derived in human CD34+ hematopoietic stem cell-engrafted NSG (HU-CD34+) mice. We applied single-cell RNA-sequencing, multicolor flow cytometry, immunohistochemical analyses and functional studies to examine the heterogeneous TME in a cohort of GBM PDOX models. We further interrogated the contribution and crosstalk between the human and mouse components constituting the brain TME in HU-CD34+ PDOXs. Results We show that glioma PDOXs can be derived in mice of different background including Nude, NOD-SCID, NSG and HU-CD34+ mice. Mouse-derived TME created in PDOX models contains tumor-associated macrophages (TAMs) known as major immuno-suppressive components of human GBM tumors. We further show that PDOXs derived in HU-CD34+ NSG mice present human CD45+ immune cells in the bone marrow and blood. Interestingly, we detect an influx of human immune cells in tumors developed in the mouse brain, which interact with the brain-derived immunosuppressive TME of mouse origin. Conclusion We here provide a thorough characterization of the heterogeneous brain TME created in GBM PDOX models. We show that human GBM can instruct mouse-derived brain cells towards immune-suppressive TME. The missing adaptive immune component can be introduced by derivation of GBM PDOXs in humanized mice. Such immunocompetent in vivo models will be important for testing novel therapies targeting different immune components in GBM.
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Amado, Beatriz, Lúcia Melo, Raquel Pinto, Andrea Lobo, Pedro Barros y João R. Gomes. "Ischemic Stroke, Lessons from the Past towards Effective Preclinical Models". Biomedicines 10, n.º 10 (13 de octubre de 2022): 2561. http://dx.doi.org/10.3390/biomedicines10102561.
Resumen
Ischemic stroke is a leading cause of death worldwide, mainly in western countries. So far, approved therapies rely on reperfusion of the affected brain area, by intravenous thrombolysis or mechanical thrombectomy. The last approach constitutes a breakthrough in the field, by extending the therapeutic window to 16–24 h after stroke onset and reducing stroke mortality. The combination of pharmacological brain-protective strategies with reperfusion is the future of stroke therapy, aiming to reduce brain cell death and decrease patients’ disabilities. Recently, a brain-protective drug—nerinetide—reduced brain infarct and stroke mortality, and improved patients’ functional outcomes in clinical trials. The success of new therapies relies on bringing preclinical studies and clinical practice close together, by including a functional outcome assessment similar to clinical reality. In this review, we focused on recent upgrades of in vitro and in vivo stroke models for more accurate and effective evaluation of therapeutic strategies: from spheroids to organoids, in vitro models that include all brain cell types and allow high throughput drug screening, to advancements in in vivo preclinical mouse stroke models to mimic the clinical reality in surgical procedures, postsurgical care, and functional assessment.
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Collot, Raphael, Cristian Ruiz Moreno, Amber Wezenaar, Anoek Zomer, Hannah Johnson, Henk Stunnenberg y Anne Rios. "DIPG-11. DISSECTING THE DIFFUSE MIDLINE GLIOMA TUMOR MICROENVIRONMENT COMMUNICATIONS USING MULTI-OMICS APPROACHES". Neuro-Oncology 25, Supplement_1 (1 de junio de 2023): i15. http://dx.doi.org/10.1093/neuonc/noad073.058.
Resumen
Abstract Diffuse midline glioma (DMG) represents a highly aggressive pediatric brain tumor with no chance of survival. Treatment options are urgently needed, and efforts have been put towards developing T-cell immunotherapy. However, this should consider DMG’s unique tumor microenvironment (TME), both in terms of anatomical location and developmental stage. To gain knowledge on this unique tumor niche, we implemented an immuno-competent, syngeneic mouse model based on in-utero electroporation (IUE) of the key driver mutations to induce tumor formation at an embryonic stage. We characterized DMG’s spatial and molecular landscape by combining snRNAseq, spatial transcriptomics, and state-of-the-art imaging. We observed a high intratumoral heterogeneity with spatially restricted cell populations. Importantly, their molecular profiles correlate with patient data, illustrating the clinical relevance of the IUE model. Analysis of cell-cell communications in the TME revealed novel signaling between DMG and immunosuppressive myeloid cells that could impact T-cell response. Using an advanced patient-derived DMG organoid co-culture model with macrophages and engineered T-cells, we are currently investigating the role of this pathway on T-cells mediated killing of DMG organoids. Thus, this project fills a critical need to delineate and overcome the immune-suppressive environment in DMG and potentiate T cell targeting.
36
Mao, Rui, Xiaoyun Zhang, Youyong Kong, Shanshan Wu, Hai-qin Huo, Yue Kong, Zhen Wang, Yan Liu, Zhengping Jia y Zikai Zhou. "Transcriptome Regulation by Oncogenic ALK Pathway in Mammalian Cortical Development Revealed by Single-Cell RNA Sequencing". Cerebral Cortex 31, n.º 8 (1 de abril de 2021): 3911–24. http://dx.doi.org/10.1093/cercor/bhab058.
Resumen
Abstract Precise regulation of embryonic neurodevelopment is crucial for proper structural organization and functioning of the adult brain. The key molecular machinery orchestrating this process remains unclear. Anaplastic lymphoma kinase (ALK) is an oncogenic receptor-type protein tyrosine kinase that is specifically and transiently expressed in developing nervous system. However, its role in the mammalian brain development is unknown. We found that transient embryonic ALK inactivation caused long-lasting abnormalities in the adult mouse brain, including impaired neuronal connectivity and cognition, along with delayed neuronal migration and decreased neuronal proliferation during neurodevelopment. scRNA-seq on human cerebral organoids revealed a delayed transition of cell-type composition. Molecular characterization identified a group of differentially expressed genes (DEGs) that were temporally regulated by ALK at distinct developmental stages. In addition to oncogenes, many DEGs found by scRNA-seq are associated with neurological or neuropsychiatric disorders. Our study demonstrates a pivotal role of oncogenic ALK pathway in neurodevelopment and characterized cell-type-specific transcriptome regulated by ALK for better understanding mammalian cortical development.
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Antonica, Francesco, Francesca Garilli, Maria Del Mar Gardeazabal Bataller, Lucia Santomaso y Luca Tiberi. "TMOD-06. MODELLING ADULT AND PAEDIATRIC GLIOBLASTOMA MULTIFORME (GBM) USING A GENE SCREEN-BASED APPROACH IN MICE AND HUMAN IPSC-DERIVED CEREBRAL ORGANOIDS". Neuro-Oncology 21, Supplement_6 (noviembre de 2019): vi263. http://dx.doi.org/10.1093/neuonc/noz175.1105.
Resumen
Abstract Glioblastoma multiforme (GBM) represent the most devastating form of high-grade glioma (HGG) affecting adults and children. Despite a multi-therapeutic approach consisting in surgery, radio- and chemo-therapy the prognosis remains poor. Several models such as xenograft, animal models and recently organoids, have been developed in order to investigate the physiopathology of GBM. Although several mouse models (where either gain- or loss-of function of genes/pathway found altered in patients induce tumour formation) have been generated many aspects of how the tumour is formed, evolves, infiltrates and recurs after treatments remain unclear. Another challenge is the creation of proper GBM models showing intratumor heterogeneity found in the patient tumour but missing in the animal model generated so far. To overcome the lack of that human tumour characteristic, we decided to generate new model of GBM using a gene screen-based approach in mice. Firstly, we analysed the genes found amplified or mutated in GBM patients; secondly, we over-expressed the candidate genes (combination of multiple genes found to be mutated or amplified in specific GBM patients) in the subventricular zone (SVZ) of P2 mouse brain. Thirdly, we analysed the formation of tumour after 2 months. We over-expressed roughly 50 combinations in newborn mice and found that only 3 successfully led to the formation of lesions positive for proliferation and brain tumour marker (i.e. GFAP). We are currently characterising the tumours by DNA methylation analysis and RNA-seq (for further confirmation of the cancer subtype and intratumor heterogeneity) or investigating the cell of origin of the tumour. Moreover, we are testing the tumorigenicity of specific in GBM-amplified/mutated gene combinations in hiPSC-derived cerebal organoids. Our data suggest that a gene screen-based approach can be used for quickly and easily assaying the tumorigenicity of genes found amplified/mutated in GBM patients as well as the biology behind such complex process.
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Gallardo, Amador, Aldara Molina, Helena G. Asenjo, Jordi Martorell-Marugán, Rosa Montes, Verónica Ramos-Mejia, Antonio Sanchez-Pozo, Pedro Carmona-Sáez, Lourdes Lopez-Onieva y David Landeira. "The molecular clock protein Bmal1 regulates cell differentiation in mouse embryonic stem cells". Life Science Alliance 3, n.º 5 (13 de abril de 2020): e201900535. http://dx.doi.org/10.26508/lsa.201900535.
Resumen
Mammals optimize their physiology to the light–dark cycle by synchronization of the master circadian clock in the brain with peripheral clocks in the rest of the tissues of the body. Circadian oscillations rely on a negative feedback loop exerted by the molecular clock that is composed by transcriptional activators Bmal1 and Clock, and their negative regulators Period and Cryptochrome. Components of the molecular clock are expressed during early development, but onset of robust circadian oscillations is only detected later during embryogenesis. Here, we have used naïve pluripotent mouse embryonic stem cells (mESCs) to study the role of Bmal1 during early development. We found that, compared to wild-type cells, Bmal1−/− mESCs express higher levels of Nanog protein and altered expression of pluripotency-associated signalling pathways. Importantly, Bmal1−/− mESCs display deficient multi-lineage cell differentiation capacity during the formation of teratomas and gastrula-like organoids. Overall, we reveal that Bmal1 regulates pluripotent cell differentiation and propose that the molecular clock is an hitherto unrecognized regulator of mammalian development.
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Collot, R., C. Ruiz Moreno, A. Wezenaar, A. Zomer, H. Johnson, H. Stunnenberg y A. Rios. "P02.05.A DISSECTING THE DIFFUSE MIDLINE GLIOMA TUMOUR MICROENVIRONMENT COMMUNICATIONS USING MULTI-OMICS APPROACHES". Neuro-Oncology 25, Supplement_2 (1 de septiembre de 2023): ii30. http://dx.doi.org/10.1093/neuonc/noad137.090.
Resumen
Abstract BACKGROUND Diffuse midline glioma (DMG) represents a highly aggressive pediatric brain tumour with no chance of survival. Treatment options are urgently needed, and efforts have been put towards the development of T cell immunotherapy. However, this should consider the unique tumour microenvironment (TME) of DMG, both in terms of anatomical location and developmental stage. MATERIAL AND METHODS To gain knowledge on this unique tumour niche, we implemented an immuno-competent, syngeneic mouse model based on in-utero electroporation (IUE) of the key driver-mutations to induce tumour formation at embryonic stage. By combining snRNAseq, Spatial Transcriptomics and state-of-the-art imaging, we characterized the spatial and molecular landscape of DMG. RESULTS AND CONCLUSION We observed a high intratumoral heterogeneity with spatially restricted cell populations. Importantly, their molecular profiles correlate with patient data, illustrating the clinical relevance of the IUE model. Analysis of cell-cell communications in the TME revealed a novel signalling between DMG and immunosuppressive myeloid cells, that could impact T-cell response. Using an advanced patient-derived DMG organoid co-culture model with macrophages and engineered T-cells, we are currently investigating the role of this pathway on T-cells mediated killing of DMG organoids. Thus, this project fills a critical need to delineate and overcome the immune-suppressive environment in DMG and potentiate T cell targeting.
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Zhu, Zhe, Matthew J. Gorman, Lisa D. McKenzie, Jiani N. Chai, Christopher G. Hubert, Briana C. Prager, Estefania Fernandez et al. "Zika virus has oncolytic activity against glioblastoma stem cells". Journal of Experimental Medicine 214, n.º 10 (5 de septiembre de 2017): 2843–57. http://dx.doi.org/10.1084/jem.20171093.
Resumen
Glioblastoma is a highly lethal brain cancer that frequently recurs in proximity to the original resection cavity. We explored the use of oncolytic virus therapy against glioblastoma with Zika virus (ZIKV), a flavivirus that induces cell death and differentiation of neural precursor cells in the developing fetus. ZIKV preferentially infected and killed glioblastoma stem cells (GSCs) relative to differentiated tumor progeny or normal neuronal cells. The effects against GSCs were not a general property of neurotropic flaviviruses, as West Nile virus indiscriminately killed both tumor and normal neural cells. ZIKV potently depleted patient-derived GSCs grown in culture and in organoids. Moreover, mice with glioblastoma survived substantially longer and at greater rates when the tumor was inoculated with a mouse-adapted strain of ZIKV. Our results suggest that ZIKV is an oncolytic virus that can preferentially target GSCs; thus, genetically modified strains that further optimize safety could have therapeutic efficacy for adult glioblastoma patients.
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Lim, Jung Yeon, Jung Eun Lee, Soon A. Park, Sang In Park, Jung-Min Yon, Jeong-Ah Park, Sin-Soo Jeun et al. "Protective Effect of Human-Neural-Crest-Derived Nasal Turbinate Stem Cells against Amyloid-β Neurotoxicity through Inhibition of Osteopontin in a Human Cerebral Organoid Model of Alzheimer’s Disease". Cells 11, n.º 6 (18 de marzo de 2022): 1029. http://dx.doi.org/10.3390/cells11061029.
Resumen
The aim of this study was to validate the use of human brain organoids (hBOs) to investigate the therapeutic potential and mechanism of human-neural-crest-derived nasal turbinate stem cells (hNTSCs) in models of Alzheimer’s disease (AD). We generated hBOs from human induced pluripotent stem cells, investigated their characteristics according to neuronal markers and electrophysiological features, and then evaluated the protective effect of hNTSCs against amyloid-β peptide (Aβ1–42) neurotoxic activity in vitro in hBOs and in vivo in a mouse model of AD. Treatment of hBOs with Aβ1–42 induced neuronal cell death concomitant with decreased expression of neuronal markers, which was suppressed by hNTSCs cocultured under Aβ1–42 exposure. Cytokine array showed a significantly decreased level of osteopontin (OPN) in hBOs with hNTSC coculture compared with hBOs only in the presence of Aβ1–42. Silencing OPN via siRNA suppressed Aβ-induced neuronal cell death in cell culture. Notably, compared with PBS, hNTSC transplantation significantly enhanced performance on the Morris water maze, with reduced levels of OPN after transplantation in a mouse model of AD. These findings reveal that hBO models are useful to evaluate the therapeutic effect and mechanism of stem cells for application in treating AD.
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Karakaya, Melike y Pınar Obakan Yerlikaya. "Recent in vitro models and tissue engineering strategies to study glioblastoma". Biotech Studies 33, n.º 1 (2 de abril de 2024): 52–68. http://dx.doi.org/10.38042/biotechstudies.1463814.
Resumen
Glioblastoma is a highly malignant brain tumor classified as grade IV with a poor prognosis and approximately a year of survival rate. The molecular changes that trigger primary glioblastoma are usually epidermal growth factor receptor mutations and amplifications, Mouse Double Minute and TP53 mutations, p16 deletion, phosphatase and tensin homolog and telomerase promoter mutations. In the vast majority of glioblastomas, altered signaling pathways were identified as receptor tyrosine kinase/Ras/PI3K, p53. Isocitrate dehydrogenase 1/2 mutations have also been associated with poor prognosis in glioblastoma The treatment options are very limited and complicated because of the diverse composition and heterogeneity of the tumors and unresponsiveness to the treatments with the existence of barriers reaching the brain tissue. Despite new trials, drug candidates that appeared effective in cell culture or mouse models failed in the clinic. Recently, new sophisticated experimental systems, including the those that mimic the tumor microenvironment, have started being used by several research groups, which will allow accurate prediction of drug efficacy. Tissue engineering strategies are also being combined with innovative cancer models, including spheroids, tumorspheres, organotypic slices, explants, tumoroids, and organoids. Such 3D systems provide powerful tools for studying glioblastoma biology by representing the dynamic evolution of the disease from the early to the metastatic stages and enabling interaction with the microenvironment. In this review, we both enlighten the molecular mechanisms that lead to glioblastoma development and detailed information on the tissue engineering approaches that have been used to model glioblastoma and the tumor microenvironment with the advantages and disadvantages. We anticipate that these novel approaches could improve the reliability of preclinical data by reducing the need for animal models.
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Berg, Anastasia L., Ashley Rowson-Hodel, Michelle Hu, Michael Keeling, Hao Wu, Kacey VanderVorst, Jenny J. Chen et al. "The Cationic Amphiphilic Drug Hexamethylene Amiloride Eradicates Bulk Breast Cancer Cells and Therapy-Resistant Subpopulations with Similar Efficiencies". Cancers 14, n.º 4 (14 de febrero de 2022): 949. http://dx.doi.org/10.3390/cancers14040949.
Resumen
The resistance of cancer cell subpopulations, including cancer stem cell (CSC) populations, to apoptosis-inducing chemotherapeutic agents is a key barrier to improved outcomes for cancer patients. The cationic amphiphilic drug hexamethylene amiloride (HMA) has been previously demonstrated to efficiently kill bulk breast cancer cells independent of tumor subtype or species but acts poorly toward non-transformed cells derived from multiple tissues. Here, we demonstrate that HMA is similarly cytotoxic toward breast CSC-related subpopulations that are resistant to conventional chemotherapeutic agents, but poorly cytotoxic toward normal mammary stem cells. HMA inhibits the sphere-forming capacity of FACS-sorted human and mouse mammary CSC-related cells in vitro, specifically kills tumor but not normal mammary organoids ex vivo, and inhibits metastatic outgrowth in vivo, consistent with CSC suppression. Moreover, HMA inhibits viability and sphere formation by lung, colon, pancreatic, brain, liver, prostate, and bladder tumor cell lines, suggesting that its effects may be applicable to multiple malignancies. Our observations expose a key vulnerability intrinsic to cancer stem cells and point to novel strategies for the exploitation of cationic amphiphilic drugs in cancer treatment.
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Furst, Liam M., Enola M. Roussel, Ryan F. Leung, Ankita M. George, Sarah A. Best, James R. Whittle, Ron Firestein, Maree C. Faux y David D. Eisenstat. "The Landscape of Pediatric High-Grade Gliomas: The Virtues and Pitfalls of Pre-Clinical Models". Biology 13, n.º 6 (7 de junio de 2024): 424. http://dx.doi.org/10.3390/biology13060424.
Resumen
Pediatric high-grade gliomas (pHGG) are malignant and usually fatal central nervous system (CNS) WHO Grade 4 tumors. The majority of pHGG consist of diffuse midline gliomas (DMG), H3.3 or H3.1 K27 altered, or diffuse hemispheric gliomas (DHG) (H3.3 G34-mutant). Due to diffuse tumor infiltration of eloquent brain areas, especially for DMG, surgery has often been limited and chemotherapy has not been effective, leaving fractionated radiation to the involved field as the current standard of care. pHGG has only been classified as molecularly distinct from adult HGG since 2012 through Next-Generation sequencing approaches, which have shown pHGG to be epigenetically regulated and specific tumor sub-types to be representative of dysregulated differentiating cells. To translate discovery research into novel therapies, improved pre-clinical models that more adequately represent the tumor biology of pHGG are required. This review will summarize the molecular characteristics of different pHGG sub-types, with a specific focus on histone K27M mutations and the dysregulated gene expression profiles arising from these mutations. Current and emerging pre-clinical models for pHGG will be discussed, including commonly used patient-derived cell lines and in vivo modeling techniques, encompassing patient-derived xenograft murine models and genetically engineered mouse models (GEMMs). Lastly, emerging techniques to model CNS tumors within a human brain environment using brain organoids through co-culture will be explored. As models that more reliably represent pHGG continue to be developed, targetable biological and genetic vulnerabilities in the disease will be more rapidly identified, leading to better treatments and improved clinical outcomes.
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Daniel, Andy, Saritha Krishna y Shawn Hervey-Jumper. "CNSC-26. GLIOBLASTOMA-NEURONAL CIRCUIT INTEGRATION IS MODULATED BY INTERLEUKIN-6". Neuro-Oncology 25, Supplement_5 (1 de noviembre de 2023): v28. http://dx.doi.org/10.1093/neuonc/noad179.0110.
Resumen
Abstract The pleiotropic cytokine interleukin-6 (IL-6) is known to be involved in both pro- and anti-inflammatory signaling cascades. In glioblastoma studies using animal and human models, IL-6 promotes immunosuppression and tumor progression and is negatively associated with survival. Intriguingly, IL-6 also contributes to increased synaptogenesis during development and following focal brain injury. Glioblastoma remodeling of neuronal circuits influences patient survival; therefore, it is possible that IL-6 has mechanistic significance. Here, we investigated IL-6 as a driver of activity-dependent glioblastoma proliferation using patient-derived xenograft (PDX) mouse and human glioblastoma models. We assessed neuronal activity by microelectrode arrays and in vitro calcium imaging using weighted mean firing rate (WMFR), network burst frequency (NBF), and synchrony index. Next, we used bulk and single-cell RNA sequencing on 13,731 cells from ten tumors including pair-matched samples to identify differentially expressed genes in intratumoral regions with elevated functional connectivity and found IL6 to be highly upregulated. Mouse embryonic cortical neurons and cerebral organoids co-cultured with primary patient-derived glioblastoma cells demonstrated concentration-dependent neuronal hyperexcitability (increased WMFR and NBF) in IL-6-overexpressing conditions compared to the neuron-only condition. Pharmacological inhibition using the humanized IL-6 receptor antibody tocilizumab reduced network synchrony across models. These findings suggest that IL-6-induced glioma-neuronal hyperexcitability may be inhibited by the FDA-approved tocilizumab, thereby providing preclinical support for its use to treat activity-dependent mechanisms of glioblastoma proliferation. Future studies are needed to uncover mechanisms and clinical efficacy.
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Wilson, Madison N., Martin Thunemann, Xin Liu, Yichen Lu, Francesca Puppo, Jason W. Adams, Jeong-Hoon Kim et al. "Multimodal monitoring of human cortical organoids implanted in mice reveal functional connection with visual cortex". Nature Communications 13, n.º 1 (26 de diciembre de 2022). http://dx.doi.org/10.1038/s41467-022-35536-3.
Resumen
AbstractHuman cortical organoids, three-dimensional neuronal cultures, are emerging as powerful tools to study brain development and dysfunction. However, whether organoids can functionally connect to a sensory network in vivo has yet to be demonstrated. Here, we combine transparent microelectrode arrays and two-photon imaging for longitudinal, multimodal monitoring of human cortical organoids transplanted into the retrosplenial cortex of adult mice. Two-photon imaging shows vascularization of the transplanted organoid. Visual stimuli evoke electrophysiological responses in the organoid, matching the responses from the surrounding cortex. Increases in multi-unit activity (MUA) and gamma power and phase locking of stimulus-evoked MUA with slow oscillations indicate functional integration between the organoid and the host brain. Immunostaining confirms the presence of human-mouse synapses. Implantation of transparent microelectrodes with organoids serves as a versatile in vivo platform for comprehensive evaluation of the development, maturation, and functional integration of human neuronal networks within the mouse brain.
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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, 13 de octubre de 2020. http://dx.doi.org/10.1038/s41380-020-00910-4.
Resumen
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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Li, Yang, Peng-Ming Zeng, Jian Wu y Zhen-Ge Luo. "Advances and Applications of Brain Organoids". Neuroscience Bulletin, 24 de mayo de 2023. http://dx.doi.org/10.1007/s12264-023-01065-2.
Resumen
AbstractUnderstanding the fundamental processes of human brain development and diseases is of great importance for our health. However, existing research models such as non-human primate and mouse models remain limited due to their developmental discrepancies compared with humans. Over the past years, an emerging model, the “brain organoid” integrated from human pluripotent stem cells, has been developed to mimic developmental processes of the human brain and disease-associated phenotypes to some extent, making it possible to better understand the complex structures and functions of the human brain. In this review, we summarize recent advances in brain organoid technologies and their applications in brain development and diseases, including neurodevelopmental, neurodegenerative, psychiatric diseases, and brain tumors. Finally, we also discuss current limitations and the potential of brain organoids.
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Chen, Wenyi, Qigu Yao, Ruo Wang, Bing Fen, Junyao Chen, Yanping Xu, Jiong Yu, Lanjuan Li y Hongcui Cao. "Highly Efficient Methods to Culture Mouse Cholangiocytes and Small Intestine Organoids". Frontiers in Microbiology 13 (20 de mayo de 2022). http://dx.doi.org/10.3389/fmicb.2022.907901.
Resumen
BackgroundOrganoids, which enable disease modeling and drug screening closer to an in vivo environment, can be isolated and grown from organs such as the brain, small intestine, kidney, lungs, and liver. To facilitate the establishment of liver and small intestinal organoids, we developed efficient protocols for cholangiocytes and intestine crypts collecting and organoid culturing.MethodsCholangiocytes were collected from intrahepatic bile ducts, the gallbladder, and small intestine crypts by gravity settling and multistep centrifugation methods. The cells isolated were embedded with Matrigel and grew in three-dimensional spheroids in a suitable culture medium. The stability of organoid cells was assessed by subculture, cryopreservation, and thawing. RNA and DNA extraction of organoids, as well as immunostaining procedure, were also optimized. Hand-picking procedures were developed and performed to ensure similar growth characteristics of organoids.ResultsA large number of cholangiocytes and small intestine crypts were collected under these protocols. Cholangiocytes developed into cyst-like structures after 3–4 days in Matrigel. After 1–2 weeks of cultivation, small intestinal organoids (in-orgs) developed buds and formed a mature structure. Compared to organoids derived from the gallbladder, cholangiocyte organoids (Cho-orgs) from intrahepatic the bile ducts grew more slowly but had a longer culture term, expressed the cholangiocytes markers Krt19 and Krt7, and recapitulated in vivo tissue organization.ConclusionsOur protocols simplified the cell collection procedure and avoided the possibility of exposing tissue-derived stem cells to mechanical damage or chemical injury by gravity settling and multistep centrifugation. In addition, our approach allowed similar growth characteristics of organoids from different mammalian tissue sources. The protocol requires 2–4 weeks to establish a stable organoid growth system. Organoids could be stably passaged, cryopreserved, and recovered under protocol guidance. Besides, the organoids of cholangiocytes and small intestines retained their original tissue characteristics, such as tissue-specific marker expression, which prepares them for further experiments such as preclinical in vitro trials and mechanism research studies.
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Barnhart, Andrew J. y Kris Dierickx. "A Tale of Two Chimeras: Applying the Six Principles to Human Brain Organoid Xenotransplantation". Cambridge Quarterly of Healthcare Ethics, 27 de febrero de 2023, 1–17. http://dx.doi.org/10.1017/s0963180123000051.
Resumen
Abstract Cerebral organoid models in-of-themselves are considered as an alternative to research animal models. But their developmental and biological limitations currently inhibit the probability that organoids can fully replace animal models. Furthermore, these organoid limitations have, somewhat ironically, brought researchers back to the animal model via xenotransplantation, thus creating hybrids and chimeras. In addition to attempting to study and overcome cerebral organoid limitations, transplanting cerebral organoids into animal models brings an opportunity to observe behavioral changes in the animal itself. Traditional animal ethics frameworks, such as the well-known three Rs (reduce, refine, and replace), have previously addressed chimeras and xenotransplantation of tissue. But these frameworks have yet to completely assess the neural-chimeric possibilities. And while the three Rs framework was a historical landmark in animal ethics, there are identifiable gaps in the framework that require attention. The authors propose to utilize an expanded three Rs framework initially developed by David DeGrazia and Tom L. Beauchamp, known as the Six Principles (6Ps). This framework aims to expand upon the three Rs, fill in the gaps, and be a practical means for assessing animal ethical issues like that of neural-chimeras and cerebral organoid xenotransplantation. The scope of this 6Ps application will focus on two separate but recent studies, which were published in 2019 and 2020. First, they consider a study wherein cerebral organoids were grown from donors with Down syndrome and from neurotypical donors. After these organoids were grown and studied, they were then surgically implanted into mouse models to observe the physiological effects and any behavioral change in the chimera. Second, they consider a separate study wherein neurotypical human embryonic stem cell-derived cerebral organoids were grown and transplanted into mouse and macaque models. The aim was to observe if such a transplantation method would contribute to therapies for brain injury or stroke. The authors place both studies under the lens of the 6Ps framework, assess the relevant contexts of each case, and provide relevant normative conclusions. In this way, they demonstrate how the 6Ps could be applied in future cases of neural-chimeras and cerebral organoid xenotransplantation.