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Humanized cerebral organoids-based ischemic stroke model for discovering of potential anti-stroke agents.
Establishing a stoke experimental model, which is better in line with the physiology and function of human brain, is the bottleneck for the development of effective anti-stroke drugs. A three-dimensional cerebral organoids (COs) from human pluripotent stem cells can mimic cell composition, cortical structure, brain neural connectivity and epigenetic genomics of in-vivo human brain, which provides a promising application in establishing humanized ischemic stroke model. COs have been used for modeling low oxygen condition-induced hypoxic injury, but there is no report on the changes of COs in response to in vitro oxygen-glucose deprivation (OGD)-induced damage of ischemic stroke as well as its application in testing anti-stroke drugs. In this study we compared the cell composition of COs at different culture time and explored the cell types, cell ratios and volume size of COs at 85 days (85 d-CO). The 85 d-CO with diameter more than 2 mm was chosen for establishing humanized ischemic stroke model of OGD. By determining the time-injury relationship of the model, we observed aggravated ischemic injury of COs with OGD exposure time, obtaining first-hand evidence for the damage degree of COs under different OGD condition. The sensitivity of the model to ischemic injury and related treatment was validated by the proven pan-Caspase inhibitor Z-VAD-FMK (20 μM) and Bcl-2 inhibitor navitoclax (0.5 μM). Neuroprotective agents edaravone, butylphthalide, P7C3-A20 and ZL006 (10 μM for each) exerted similar beneficial effects in this model. Taken together, this study establishes a humanized ischemic stroke model based on COs, and provides evidence as a new research platform for anti-stroke drug development.
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p53 controls genomic stability and temporal differentiation of human neural stem cells and affects neural organization in human brain organoids.
In this study, we take advantage of human induced pluripotent stem (iPS) cell-derived neural stem cells and brain organoids to study the role of p53 during human brain development. We knocked down (KD) p53 in human neuroepithelial stem (NES) cells derived from iPS cells. Upon p53KD, NES cells rapidly show centrosome amplification and genomic instability. Furthermore, a reduced proliferation rate, downregulation of genes involved in oxidative phosphorylation (OXPHOS), and an upregulation of glycolytic capacity was apparent upon loss of p53. In addition, p53KD neural stem cells display an increased pace of differentiating into neurons and exhibit a phenotype corresponding to more mature neurons compared to control neurons. Using brain organoids, we modeled more specifically cortical neurogenesis. Here we found that p53 loss resulted in brain organoids with disorganized stem cell layer and reduced cortical progenitor cells and neurons. Similar to NES cells, neural progenitors isolated from brain organoids also show a downregulation in several OXPHOS genes. Taken together, this demonstrates an important role for p53 in controlling genomic stability of neural stem cells and regulation of neuronal differentiation, as well as maintaining structural organization and proper metabolic gene profile of neural progenitors in human brain organoids.
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[Ultrastructure of the capillary cellular elements of the human brain in prenatal ontogeny].
Peculiarities of ultrastructural organization of cellular elements of the brain capillaries have been studied for human embryos and fetuses during prenatal ontogenesis (10-32 weeks). Redistribution of organoids, alterations of amounts and size of microvilli and vesicles in the cytoplasm of endothelyocytes were observed during intrauterine development. The character and extent of correlations among the endothelyocytes and pericytes were found to alter. The differences revealed in the structural organization of capillaries are presumably related to the changeable hemodynamic conditions of functioning of the brain capillaries during the intrauterine development.
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Protocol for generating embedding-free brain organoids enriched with oligodendrocytes.
In response to the scarcity of advanced in vitro models dedicated to human CNS white matter research, we present a protocol to generate neuroectoderm-derived embedding-free human brain organoids enriched with oligodendrocytes. We describe steps for neuroectoderm differentiation, development of neural spheroids, and their transferal to Matrigel. We then detail procedures for the development, maturation, and application of oligodendrocyte-enriched brain organoids. The presence of myelin-producing cells makes these organoids useful for studying human white matter diseases, such as leukodystrophy.
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Comparison of human cerebral organoids infected with wild-type Zika versus attenuated DN-2 virus strains uncovers differences in host immune responses.
Zika virus (ZIKV), a neurotropic virus, poses significant global health challenges because of its ability to cause severe neurological complications and congenital brain abnormalities. Prenatal ZIKV infection impairs early brain development. However, details of the molecular mechanisms driving their virulence remain incompletely understood. Here, we performed comparatively analyses of infection outcomes caused by 2 different ZIKV strains - wild-type (WT) ZIKV versus an attenuated strain (DN-2) - in human cerebral organoids (hCOs) to dissect host responses that could contribute to pathogenicity. Although both viral strains productively infected hCOs with indiscernible gross pathological changes, differences in host responses and subtypes of cells infected discriminate WT ZIKV from DN-2 infections. Single cell RNA sequencing analyses uncovered differently expressed genes (DEGs) that were common to both virus strains, as well as DEGs that were specific to either WT ZIKV or DN-2. For common DEGs, WT ZIKV infections elicited stronger expression of key genes involved in host immune response pathways, such as IFIT2, DDX60, OAS1 and XAF1. Moreover, analyses of WT ZIKV-specific upregulated DEGs uncovered mobilisation of additional innate immune response pathways not found in DN-2. Additionally, while WT ZIKV infected a broad range of cell types in hCOs, DN-2 infections were predominantly limited to radial glial, which are neuroprogenitors essential for neurogenesis. These results highlight that differences in host immune responses and tropism distinguish neurotropic WT ZIKV and attenuated DN-2 infections, underscoring the mechanistic differences involved in pathogenicity. Our study provides further insights into mechanisms used by neurotrophic ZIKV to drive neuropathogenesis during infection of the developing brain.
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Schizophrenia endothelial cells exhibit higher permeability and altered angiogenesis patterns in patient-derived organoids.
Schizophrenia (SCZ) is a complex neurodevelopmental disorder characterized by the manifestation of psychiatric symptoms in early adulthood. While many research avenues into the origins of SCZ during brain development have been explored, the contribution of endothelial/vascular dysfunction to the disease remains largely elusive. To model the neuropathology of SCZ during early critical periods of brain development, we utilized patient-derived induced pluripotent stem cells (iPSCs) to generate 3D cerebral organoids and define cell-specific signatures of disease. Single-cell RNA sequencing revealed that while SCZ organoids were similar in their macromolecular diversity to organoids generated from healthy controls (CTRL), SCZ organoids exhibited a higher percentage of endothelial cells when normalized to total cell numbers. Additionally, when compared to CTRL, differential gene expression analysis revealed a significant enrichment in genes that function in vessel formation, vascular regulation, and inflammatory response in SCZ endothelial cells. In line with these findings, data from 23 donors demonstrated that PECAM1+ microvascular vessel-like structures were increased in length and number in SCZ organoids in comparison to CTRL organoids. Furthermore, we report that patient-derived endothelial cells displayed higher paracellular permeability, implicating elevated vascular activity. Collectively, our data identified altered gene expression patterns, vessel-like structural changes, and enhanced permeability of endothelial cells in patient-derived models of SCZ. Hence, brain microvascular cells could play a role in the etiology of SCZ by modulating the permeability of the developing blood brain barrier (BBB).
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Patient-derived brain organoids reveal divergent neuronal activity across subpopulations of autism spectrum disorder.
Patient-derived brain organoids have emerged as a powerful model for investigating the mechanisms underlying neurological and psychiatric disorders. They provide novel insights into autism spectrum disorder (ASD), a heterogeneous neurodevelopmental condition whose underlying mechanisms remain poorly understood. Recent advancements in generating electrophysiological functional 3D brain organoids enable the study of molecular and network-level neuronal activity. Here, we aimed to characterize the neurophysiological underpinnings of ASD by comparing electrophysiological properties of brain organoids derived from eleven individuals diagnosed with autism spectrum disorder, 10 with monogenic syndromic ASD across five genetic subtypes, and 1 with idiopathic ASD, to organoids derived from 4 neurotypical control individuals. We identified distinct differences in baseline activity (resting state) and evoked responses (synaptic plasticity and network dynamics) across ASD subgroups. To comprehensively assess these differences, we applied dimensionality reduction (principal component analysis, PCA) to integrate multiple electrophysiological features into a unified framework. Our findings reveal subtype-specific neurophysiological alterations in ASD brain organoids, offering mechanistic insights into ASD heterogeneity and potential applications for early diagnostics, drug screening, and therapeutic development.
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Suspension Culture With Uniform Shear Stress in Brain Organoids-on-a-Chip for Modelling Alzheimer's Disease.
Research on Alzheimer's disease (AD) has been hindered by the absence of customizable and physiologically relevant in vitro brain models. Although human induced pluripotent stem cell-derived brain organoids (BOs) are beginning to shift the way it is study neurodegenerative disease, BOs notoriously suffer from the limited maturation and 'batch effect'. Due to the lack of vascular systems, suspension cultures often maintained with spinning bioreactors allow for the growth of BOs with large volumes. But spinning bioreactors are limited in size and throughput, as well as in the uneven distribution of shear stress. Here, through computer simulation, suspension culture is achieved by constructing a brain-organoids-on-a-chip (BOoC), which allows adequate oxygenation and nutrient diffusion, facilitating the long-term culture and high maturity of BOs. The uniformly distributed and precisely controllable fluid shear stress in the chip, accompanied by the uniform microstructural units, provided bionic physiological clues for the homogeneous development of organoids. Then the suspension culture design physiologically mimics serum exposure, induced the elevated amyloid β aggregation and tau phosphorylation in BOs, as well as neuronal reduction and synaptic loss, which recapitulate the key pathological features of AD. This BOoC platform provides a microphysiological system for generating highly mature and homogeneous BOs and simulating the pathological process of AD in vitro.
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Human Brain In Vitro Model for Pathogen Infection-Related Neurodegeneration Study.
Recent advancements in stem cell biology and tissue engineering have revolutionized the field of neurodegeneration research by enabling the development of sophisticated in vitro human brain models. These models, including 2D monolayer cultures, 3D organoids, organ-on-chips, and bioengineered 3D tissue models, aim to recapitulate the cellular diversity, structural organization, and functional properties of the native human brain. This review highlights how these in vitro brain models have been used to investigate the effects of various pathogens, including viruses, bacteria, fungi, and parasites infection, particularly in the human brain cand their subsequent impacts on neurodegenerative diseases. Traditional studies have demonstrated the susceptibility of different 2D brain cell types to infection, elucidated the mechanisms underlying pathogen-induced neuroinflammation, and identified potential therapeutic targets. Therefore, current methodological improvement brought the technology of 3D models to overcome the challenges of 2D cells, such as the limited cellular diversity, incomplete microenvironment, and lack of morphological structures by highlighting the need for further technological advancements. This review underscored the significance of in vitro human brain cell from 2D monolayer to bioengineered 3D tissue model for elucidating the intricate dynamics for pathogen infection modeling. These in vitro human brain cell enabled researchers to unravel human specific mechanisms underlying various pathogen infections such as SARS-CoV-2 to alter blood-brain-barrier function and Toxoplasma gondii impacting neural cell morphology and its function. Ultimately, these in vitro human brain models hold promise as personalized platforms for development of drug compound, gene therapy, and vaccine. Overall, we discussed the recent progress in in vitro human brain models, their applications in studying pathogen infection-related neurodegeneration, and future directions.
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For Difficult-to-Model Brain Diseases, Brain Organoids Come to the Rescue.