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Benchmarking pluripotent stem cell-derived organoid models.
Cerebral organoids are stem cell-derived, self-organizing three-dimensional cultures. Owing to the remarkable degree to which they recreate the cellular diversity observed in the human brain, they have attracted significant interest as a novel model system for research and drug development, as well as capturing the public imagination. However, many questions remain about the extent to which these cultures recapitulate neurodevelopment and the defining features of the human brain. To clarify the fidelity of human organoid models, Bhaduri and colleagues compared the molecular profile of brain organoid cells with that of primary cells from fetal brain. They observed that, whilst brain organoids broadly recapitulate the cellular profile of human brain, they lack the subtypes of cell classes seen in human brain. In addition, they showed marked expression of cellular stress markers, which could be reversed by transplanting organoid cells into neonatal mouse brain. The authors hypothesise that in vitro culture induces a cellular stress response and that it is this that impairs maturation. Thus, whilst their findings strike a note of caution in the use of organoids as a model for early human brain development, they lay a foundation for improving the accuracy of organoid models in the future.
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Early Unguided Human Brain Organoid Neurovascular Niche Modeling into the Permissive Chick Embryo Chorioallantoic Membrane.
Engrafting organoids into vascularized tissues in model animals, such as the immunodeficient mouse or chick embryo chorioallantoic membrane (CAM), has proven efficient for neovascularization modeling. The CAM is a richly vascularized extraembryonic membrane, which shows limited immunoreactivity, thus becoming an excellent hosting model for human origin cell transplants. This paper describes the strategy to engraft human brain organoids differentiated at multiple maturation stages into the CAM. The cellular composition of brain organoids changes with time, reflecting the milestones of human brain development. We grafted brain organoids at relevant maturation stages: neuroepithelial expansion (18 DIV), early neurogenesis (60 DIV), and early gliogenesis (180 DIV) into the CAM of embryonic day (E)7 chicken embryos. Engrafted brain organoids were harvested 5 days later and their histological features were analyzed. No histological signs of neovascularization in the grafted organoids or abnormal blood vessels adjacent to the graftings were detected. Moreover, remarkable changes were observed in the cellular composition of the grafted organoids, namely, an increase in the number of glial fibrillary acidic protein-positive-reactive astrocytes. However, the cytoarchitectural changes were dependent on the organoid maturation stage. Altogether, these results suggest that brain organoids can grow in the CAM, and they show differences in the cytoarchitecture depending on their maturation stage at grafting.
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Dysregulation of the BRD2-FGF17 Signal Pathway Induces Abnormal Forebrain Development Associated with Schizophrenia.
Schizophrenia (SCZ) is a severe and hereditary neurodevelopmental disorder with unknown etiology. Here, we found that the SCZ risk gene BRD2, as an epigenetic reader, is consistently expressed in developing mouse and human cortical astrocytes. Astrocyte-specific Brd2 knockout in mice leads to dysregulation of immune responses and reduces Fgf17 expression, resulting in SCZ-like behaviors, including impaired sensorimotor gating, memory, and cognitive deficits. Moreover, BRD2 inhibition using JQ1 in forebrain organoids leads to FGF17 reduction, inducing developmental deficits involved in neural patterning and gliogenesis. The decrease of FGF17 expression was also found in SCZ patient-derived forebrain organoids, similar to BRD2-inhibited forebrain organoids. FGF17 treatment partially rescued the disrupted gene expression in BRD2-inhibited human forebrain organoids. Taken together, these findings suggest that disrupting the BRD2-FGF17 signaling pathway in early brain development may contribute to the pathogenesis of schizophrenia and may represent a potential therapeutic target for SCZ.
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Imaging three-dimensional brain organoid architecture from meso- to nanoscale across development.
Organoids are stem cell-derived three-dimensional cultures offering a new avenue to model human development and disease. Brain organoids allow the study of various aspects of human brain development in the finest details in vitro in a tissue-like context. However, spatial relationships of subcellular structures, such as synaptic contacts between distant neurons, are hardly accessible by conventional light microscopy. This limitation can be overcome by systems that quickly image the entire organoid in three dimensions and in super-resolution. To that end we have developed a system combining tissue expansion and light-sheet fluorescence microscopy for imaging and quantifying diverse spatial parameters during organoid development. This technique enables zooming from a mesoscopic perspective into super-resolution within a single imaging session, thus revealing cellular and subcellular structural details in three spatial dimensions, including unequivocal delineation of mitotic cleavage planes as well as the alignment of pre- and postsynaptic proteins. We expect light-sheet fluorescence expansion microscopy to facilitate qualitative and quantitative assessment of organoids in developmental and disease-related studies.
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Deciphering potential vascularization factors of on-chip co-cultured hiPSC-derived cerebral organoids.
The lack of functional vascular system in stem cell-derived cerebral organoids (COs) limits their utility in modeling developmental processes and disease pathologies. Unlike other organs, brain vascularization is poorly understood, which makes it particularly difficult to mimic in vitro. Although several attempts have been made to vascularize COs, complete vascularization leading to functional capillary network development has only been achieved via transplantation into a mouse brain. Understanding the cues governing neurovascular communication is therefore imperative for establishing an efficient in vitro system for vascularized cerebral organoids that can emulate human brain development. Here, we used a multidisciplinary approach combining microfluidics, organoids, and transcriptomics to identify molecular changes in angiogenic programs that impede the successful in vitro vascularization of human induced pluripotent stem cell (iPSC)-derived COs. First, we established a microfluidic cerebral organoid (CO)-vascular bed (VB) co-culture system and conducted transcriptome analysis on the outermost cell layer of COs cultured on the preformed VB. Results revealed coordinated regulation of multiple pro-angiogenic factors and their downstream targets. The VEGF-HIF1A-AKT network was identified as a central pathway involved in the angiogenic response of cerebral organoids to the preformed VB. Among the 324 regulated genes associated with angiogenesis, six transcripts represented significantly regulated growth factors with the capacity to influence angiogenic activity during co-culture. Subsequent on-chip experiments demonstrated the angiogenic and vasculogenic potential of cysteine-rich angiogenic inducer 61 (CYR61) and hepatoma-derived growth factor (HDGF) as potential enhancers of organoid vascularization. Our study provides the first global analysis of cerebral organoid response to three-dimensional microvasculature for in vitro vascularization.
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Protocol for the generation of cultured cortical brain organoid slices.
Three-dimensional brain organoids from human pluripotent stem cells are a powerful tool for studying human neural networks. Here, we present a protocol for generating cortical brain organoid slices (cBOS) derived from regionalized cortical organoids and grown at the air-liquid interphase. We provide steps for slicing organoids and maintaining them in long-term culture. We then detail approaches for quality control including the evaluation of cell death and cellular identity. Finally, we describe procedures for the expression of a genetically encoded nanosensor for ATP. For complete details on the use and execution of this protocol, please refer to Petersilie et al.1.
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Modular 3D printed platform for fluidically connected human brain organoid culture.
Brain organoid technology has transformed both basic and applied biomedical research and paved the way for novel insights into developmental processes and disease states of the human brain. While the use of brain organoids has been rapidly growing in the past decade, the accompanying bioengineering and biofabrication solutions have remained scarce. As a result, most brain organoid protocols still rely on commercially available tools and culturing platforms that had previously been established for different purposes, thus entailing suboptimal culturing conditions and excessive use of plasticware. To address these issues, we developed a 3D printing pipeline for the fabrication of tailor-made culturing platforms for fluidically connected but spatially separated brain organoid array culture. This all-in-one platform allows all culturing steps-from cellular aggregation, spheroid growth, hydrogel embedding, and organoid maturation-to be performed in a single well plate without the need for organoid manipulation or transfer. Importantly, the approach relies on accessible materials and widely available 3D printing equipment. Furthermore, the developed design principles are modular and highly customizable. As such, we believe that the presented technology can be easily adapted by other research groups and fuel further development of culturing tools and platforms for brain organoids and other 3D cellular systems.
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Meta-analysis of single-cell RNA sequencing co-expression in human neural organoids reveals their high variability in recapitulating primary tissue.
Human neural organoids offer an exciting opportunity for studying inaccessible human-specific brain development; however, it remains unclear how precisely organoids recapitulate fetal/primary tissue biology. We characterize field-wide replicability and biological fidelity through a meta-analysis of single-cell RNA-sequencing data for first and second trimester human primary brain (2.95 million cells, 51 data sets) and neural organoids (1.59 million cells, 173 data sets). We quantify the degree primary tissue cell type marker expression and co-expression are recapitulated in organoids across 10 different protocol types. By quantifying gene-level preservation of primary tissue co-expression, we show neural organoids lie on a spectrum ranging from virtually no signal to co-expression indistinguishable from primary tissue, demonstrating a high degree of variability in biological fidelity among organoid systems. Our preserved co-expression framework provides cell type-specific measures of fidelity applicable to diverse neural organoids, offering a powerful tool for uncovering unifying axes of variation across heterogeneous neural organoid experiments.
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A refined protocol for the large-scale production of high-quality cerebral organoids.
Human pluripotent stem cell-derived cerebral organoids have emerged as a valuable tool for investigating early brain development and modeling neurodevelopmental disorders. However, considerable variation in both organoid size and cellular composition has been widely reported. Moreover, the large-scale production of organoids using conventional methods is a significant challenge, particularly considering that some stem cell lines exhibit variable efficiency in neuroepithelial formation. This heterogeneity of cerebral organoids can complicate experimental reproducibility and comparability in studies of brain development and disease mechanisms. Therefore, we developed a protocol using CERO 3D bioreactors, which enables scalable organoid production and improves size homogeneity compared to conventional approaches. In addition, we conducted a pilot study, utilizing MS-based proteomic analysis of single organoids from two different lines, aiming to explore whether size variability is associated with differences in protein expression within cerebral organoids. The organoids produced in the CERO 3D bioreactors reproducibly form neuroepithelial structures and express key neurodevelopmental markers. The presented protocol provides an efficient way of producing cerebral organoids, supporting reproducibility and high-throughput applications in neurodevelopmental research.
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Derivation of a Human Brain Organoid with Microglia Development.
Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool for studying human brain development and pathogenesis of neurological diseases. However, the lack of incorporation of microglia in the human brain organoids is still a major hurdle for 3D models of neuroinflammation. Current approaches include either the incorporation of fully differentiated microglia into mature brain organoids or the induction of microglial differentiation from the early stage of iPSC-derived embryoid bodies (EBs). The first approach misses the stage when microglial differentiation interacts with the adjacent neural environment, and the later approach is technically challenging, resulting in inconsistency among the final organoids in terms of the quantity and quality of microglia. To model brain organoids with microglia to study the early interactions between microglial and neuronal development, highly pure hematopoietic progenitor cells (HPC) differentiated from human iPSCs were incorporated into iPSC-derived EBs to make brain organoids. Using immunostaining and single-cell RNA sequencing (sc-RNA-seq) analysis, we confirmed that HPCs were incorporated into the 3D organoids, which eventually developed into brain organoids with both microglia and neurons. Compared to brain organoids without HPCs, this approach produces significant microglial incorporation in the brain organoids. This novel 3D organoid model, which consists of both microglial and neural development properties, can be used to study the early interactions between innate immune and nervous system development and potentially as a model for neuroinflammation and neuroinfectious disorders.