Developing human pluripotent stem cell-based cerebral organoids with a controllable microglia ratio for modeling brain development and pathology.
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ABSTRACT:
SUBMITTER: Xu R
PROVIDER: S-EPMC8365109 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Microglia play an important role in neuroinflammation and neurodegeneration. Here, we report an approach for generating microglia-containing cerebral organoids derived from human pluripotent stem cells involving the supplementation of growth factors (FGF, EGF, heparin) and 10% CO2 culture conditions. Using this platform, Western Pacific Amyotrophic Lateral Sclerosis and Parkinsonism-Dementia Complex (ALS-PDC) cerebral organoids were generated from patient-derived induced pluripotent stem cells (iPSCs). These ALS-PDC-affected organoids had more reactive astrocytes and M1 microglia, and had fewer M2 microglia than their unaffected counterparts, leading to impaired microglia-mediated phagocytosis. RNA-seq analysis of ALS-PDC and control organoids indicated that the most significant changes were microglia- and astrocyte-related genes (IFITM1/2, TGF-β, and GFAP). The most significantly downregulated pathway was type I interferon signaling. Interferon-gamma supplementation increased IFITM expression, enhanced microglia-mediated phagocytosis, and reduced beta-amyloid accumulation in ALS-PDC-affected network. The results demonstrated the feasibility of using microglia-containing organoids for the study of neurodegenerative diseases.
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Project description:Human cerebral organoid (CO) is a three-dimensional (3D) cell culture system that recapitulates the developing human brain. While CO has proved an invaluable tool for studying neurological disorders in a more clinically relevant matter, there have still been several shortcomings including CO variability and reproducibility as well as lack of or underrepresentation of certain cell types typically found in the brain. As the technology to generate COs has continued to improve, more efficient and streamlined protocols have addressed some of these issues. Here we present a novel scalable and simplified system to generate microglia-containing CO (MCO). We characterize the cell types and dynamic development of MCOs and validate that these MCOs harbor microglia, astrocytes, neurons, and neural stem/progenitor cells, maturing in a manner that reflects human brain development. We introduce a novel technique for the generation of embryoid bodies (EBs) directly from induced pluripotent stem cells (iPSCs) that involves simplified steps of transitioning directly from 3D cultures as well as orbital shaking culture in a standard 6-well culture plate. This allows for the generation of MCOs with an easy-to-use system that is affordable and accessible by any general lab.
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Project description:Maternal alcohol exposure during pregnancy can substantially impact the development of the fetus, causing a range of symptoms, known as fetal alcohol spectrum disorders (FASDs), such as cognitive dysfunction and psychiatric disorders, with the pathophysiology and mechanisms largely unknown. Recently developed human cerebral organoids from induced pluripotent stem cells are similar to fetal brains in the aspects of development and structure. These models allow more relevant in vitro systems to be developed for studying FASDs than animal models. Modeling binge drinking using human cerebral organoids, we sought to quantify the downstream toxic effects of alcohol (ethanol) on neural pathology phenotypes and signaling pathways within the organoids. The results revealed that alcohol exposure resulted in unhealthy organoids at cellular, subcellular, bioenergetic metabolism, and gene expression levels. Alcohol induced apoptosis on organoids. The apoptotic effects of alcohol on the organoids depended on the alcohol concentration and varied between cell types. Specifically, neurons were more vulnerable to alcohol-induced apoptosis than astrocytes. The alcohol-treated organoids exhibit ultrastructural changes such as disruption of mitochondria cristae, decreased intensity of mitochondrial matrix, and disorganized cytoskeleton. Alcohol exposure also resulted in mitochondrial dysfunction and metabolic stress in the organoids as evidenced by (1) decreased mitochondrial oxygen consumption rates being linked to basal respiration, ATP production, proton leak, maximal respiration and spare respiratory capacity, and (2) increase of non-mitochondrial respiration in alcohol-treated organoids compared with control groups. Furthermore, we found that alcohol treatment affected the expression of 199 genes out of 17,195 genes analyzed. Bioinformatic analyses showed the association of these dysregulated genes with 37 pathways related to clinically relevant pathologies such as psychiatric disorders, behavior, nervous system development and function, organismal injury and abnormalities, and cellular development. Notably, 187 of these genes are critically involved in neurodevelopment, and/or implicated in nervous system physiology and neurodegeneration. Furthermore, the identified genes are key regulators of multiple pathways linked in networks. This study extends for the first time animal models of binge drinking-related FASDs to a human model, allowing in-depth analyses of neurotoxicity at tissue, cellular, subcellular, metabolism, and gene levels. Hereby, we provide novel insights into alcohol-induced pathologic phenotypes, cell type-specific vulnerability, and affected signaling pathways and molecular networks, that can contribute to a better understanding of the developmental neurotoxic effects of binge drinking during pregnancy.
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Project description:The typical abnormalities observed in the brain of Alzheimer's disease (AD) patients include synaptic alterations, neuronal death, brain inflammation, and the accumulation of protein aggregates in the form of amyloid plaques and neurofibrillary tangles. Despite the development of many animal and in vitro models for AD, there is a lack of an experimental approach that fully recapitulates essential aspects of the disease in human cells. Here, we report the generation of a new model to study AD, consisting of cerebral organoids (COs) produced from human-induced pluripotent stem cells (iPSCs). Under our experimental conditions, COs grow to form three-dimensional (3D) structures containing neural areas with cortical-like organization. Analysis of COs by histological and biochemical methods revealed that organoids produced from iPSCs derived from patients affected by familial AD or Down syndrome (DS) spontaneously develop over time pathological features of AD, including accumulation of structures highly reminiscent to amyloid plaques and neurofibrillary tangles. These pathological abnormalities were not observed in COs generated from various controls, including human iPSCs from healthy individuals, human iPSCs from patients affected by Creutzfeldt-Jakob disease, mouse embryonic stem cells (ESCs), or mouse iPSCs. These findings enable modeling genetic AD in a human cellular context in a 3D cortical-like tissue developed in vitro from patient-specific stem cells. This system provides a more relevant disease model compared to pre-existing methods and offers a new platform for discovery of novel targets and screening of drugs for therapeutic intervention.
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Project description:Human organoid systems recapitulate in vivo organ architecture yet fail to capture complex pathologies such as inflammation and fibrosis. Here, using 11 different healthy and diseased pluripotent stem cell lines, we developed a reproducible method to derive multi-cellular human liver organoids composed of hepatocyte-, stellate-, and Kupffer-like cells that exhibit transcriptomic resemblance to in vivo-derived tissues. Under free fatty acid treatment, organoids, but not reaggregated cocultured spheroids, recapitulated key features of steatohepatitis, including steatosis, inflammation, and fibrosis phenotypes in a successive manner. Interestingly, an organoid-level biophysical readout with atomic force microscopy demonstrated that organoid stiffening reflects the fibrosis severity. Furthermore, organoids from patients with genetic dysfunction of lysosomal acid lipase phenocopied severe steatohepatitis, rescued by FXR agonism-mediated reactive oxygen species suppression. The presented key methodology and preliminary results offer a new approach for studying a personalized basis for inflammation and fibrosis in humans, thus facilitating the discovery of effective treatments.
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Project description:The brain is a genomic mosaic. Cell-to-cell genomic differences, which are the result of somatic mutations during development and aging, contribute to cellular diversity in the nervous system. This genomic diversity has important implications for nervous system development, function, and disease. Brain somatic mosaicism might contribute to individualized behavioral phenotypes and has been associated with several neuropsychiatric and neurodegenerative disorders. Therefore, understanding the causes and consequences of somatic mosaicism in neural circuits is of great interest. Recent advances in 3D cell culture technology have provided new means to study human organ development and various human pathologies in vitro. Cerebral organoids ("mini-brains") are pluripotent stem cell-derived 3D culture systems that recapitulate, to some extent, the developmental processes and organization of the developing human brain. Here, I discuss the application of these neural organoids for modeling brain somatic mosaicism in a lab dish. Special emphasis is given to the potential role of microglial mutations in the pathogenesis of neurodegenerative diseases.
| S-EPMC6868038 | biostudies-literature
Project description:Human cerebral organoid (hCO) models offer the opportunity to understand fundamental processes underlying human-specific cortical development and pathophysiology in an experimentally tractable system. Although diverse methods to generate brain organoids have been developed, a major challenge has been the production of organoids with reproducible cell type heterogeneity and macroscopic morphology. Here, we have directly addressed this problem by establishing a robust production pipeline to generate morphologically consistent hCOs and achieve a success rate of >80%. These hCOs include both a radial glial stem cell compartment and electrophysiologically competent mature neurons. Moreover, we show using immunofluorescence microscopy and single-cell profiling that individual organoids display reproducible cell type compositions that are conserved upon extended culture. We expect that application of this method will provide new insights into brain development and disease processes.
| S-EPMC7167289 | biostudies-literature
Project description:Cerebral organoids are 3D stem cell-derived models that can be utilized to study the human brain. The current consensus is that cerebral organoids consist of cells derived from the neuroectodermal lineage. This limits their value and applicability, as mesodermal-derived microglia are important players in neural development and disease. Remarkably, here we show that microglia can innately develop within a cerebral organoid model and display their characteristic ramified morphology. The transcriptome and response to inflammatory stimulation of these organoid-grown microglia closely mimic the transcriptome and response of adult microglia acutely isolated from post mortem human brain tissue. In addition, organoid-grown microglia mediate phagocytosis and synaptic material is detected inside them. In all, our study characterizes a microglia-containing organoid model that represents a valuable tool for studying the interplay between microglia, macroglia, and neurons in human brain development and disease.
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Project description:Microglia are the primary resident immune cells in the retina. They regulate neuronal survival and synaptic pruning making them essential for normal development. Following injury, they mediate adaptive responses and under pathological conditions they can trigger neurodegeneration exacerbating the effect of a disease. Retinal organoids derived from human induced pluripotent stem cells (hiPSCs) are increasingly being used for a range of applications, including disease modelling, development of new therapies and in the study of retinogenesis. Despite many similarities to the retinas developed in vivo, they lack some key physiological features, including immune cells. We engineered an hiPSC co-culture system containing retinal organoids and microglia-like (iMG) cells and tested their retinal invasion capacity and function. We incorporated iMG into retinal organoids at 13 weeks and tested their effect on function and development at 15 and 22 weeks of differentiation. Our key findings showed that iMG cells were able to respond to endotoxin challenge in monocultures and when co-cultured with the organoids. We show that retinal organoids developed normally and retained their ability to generate spiking activity in response to light. Thus, this new co-culture immunocompetent in vitro retinal model provides a platform with greater relevance to the in vivo human retina.
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Project description:Human cerebral organoids (COs) are self-organizing three-dimensional (3D) neural structures that provide a human-specific platform to study the cellular and molecular processes that underlie different neurological events. The first step of CO generation from human pluripotent stem cells (hPSCs) is neural induction, which is an in vitro simulation of neural ectoderm development. Several signaling pathways cooperate during neural ectoderm development and in vitro differentiation of hPSCs toward neural cell lineages is also affected by them. In this study, we considered some of the known sources of these variable signaling cues arising from cell culture media components and sought to modulate their effects by applying a comprehensive combination of small molecules and growth factors for CO generation. Histological analysis demonstrated that these COs recapitulate the neural progenitor zone and early cortical layer organization, containing different types of neuronal and glial cells which was in accordance with single-nucleus transcriptome profiling results. Moreover, patch clamp and intracellular Ca2+ dynamic studies demonstrated that the COs behave as a functional neural network. Thus, this method serves as a facile protocol for generating hPSC-derived COs that faithfully mimic the features of their in vivo counterparts in the developing human brain. See also Figure 1(Fig. 1).
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