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A mouse organoid platform for modeling cerebral cortex development and cis-regulatory evolution in vitro.
Natural selection has shaped the gene regulatory networks that orchestrate cortical development, leading to structural and functional variation across mammals, but the molecular and cellular mechanisms underpinning these changes have only begun to be characterized. Here, we develop a reproducible protocol for cerebral cortex organoid generation from mouse epiblast stem cells (EpiSCs), which recapitulates the timing and cellular differentiation programs of the embryonic cortex. We generated cortical organoids from F1 hybrid EpiSCs derived from crosses between laboratory mice (C57BL/6J) and four wild-derived inbred strains spanning ∼1 M years of evolutionary divergence to comprehensively map cis-acting transcriptional regulatory variation across developing cortical cell types, using single-cell RNA sequencing (scRNA-seq). We identify hundreds of genes that exhibit dynamic allelic imbalances, providing the first insight into the developmental mechanisms underpinning changes in cortical structure and function between subspecies. These experimental methods and cellular resources represent a powerful platform for investigating gene regulation in the developing cerebral cortex.
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Identifying the Pathogenicity of a Novel NPRL3 Missense Mutation Using Personalized Cortical Organoid Model of Focal Cortical Dysplasia.
Focal cortical dysplasia (FCD) II is a cortical malformation characterized by cortical architectural abnormalities, dysmorphic neurons, with or without balloon cells. Here, we systematically explored the pathophysiological role of the GATOR1 subunit NPRL3 variants including a novel mutation from iPSCs derived from one FCD II patient. Three FCD II children aged 0.5-7 years who underwent cerebral lesion resection in our hospital from March 2019 to October 2019 were included in this study. We generated patient-derived iPSCs and performed whole-exome sequencing to accurately identify somatic cells with mutations. The effect of the newly identified NPRL3 mutation found in one of our FCD II patients was evaluated using the personalized cortical organoid model and the NPRL3 knockout HEK293T cells. Whole-exome sequencing of iPSCs derived from FCD II patients revealed a novel NPRL3 C.767G > C (p.R256P) heterozygous mutation. Cortical organoids generated from iPSCs of FCD II patients were larger than control iPSCs, with increased number of p-S6+ cells and NeuN+ neurons. In NPRL3 knockout HEK293T cells, overexpression of NPRL3 together with NPRL2 protein is necessary to reduce p-S6 level upon amino acid starvation. The reduced binding between NPRL3 Arg256Pro and NPRL2 protein leads to downregulation of the relative total protein amount of both proteins in the cell. Our study describes a novel cortical organoid model generated from iPSCs of the FCD patients to investigate the underlying mechanism of NPRL3-related epilepsy. The mutation of NPRL3 Arg256Pro impaired the function of NPRL3 protein via affecting the binding with NPRL2 protein, which resulted in unstable protein monomer.
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Human brain organoid model of maternal immune activation identifies radial glia cells as selectively vulnerable.
Maternal immune activation (MIA) during critical windows of gestation is correlated with long-term neurodevelopmental deficits in the offspring, including increased risk for autism spectrum disorder (ASD) in humans. Interleukin 6 (IL-6) derived from the gestational parent is one of the major molecular mediators by which MIA alters the developing brain. In this study, we establish a human three-dimensional (3D) in vitro model of MIA by treating induced pluripotent stem cell-derived dorsal forebrain organoids with a constitutively active form of IL-6, Hyper-IL-6. We validate our model by showing that dorsal forebrain organoids express the molecular machinery necessary for responding to Hyper-IL-6 and activate STAT signaling upon Hyper-IL-6 treatment. RNA sequencing analysis reveals the upregulation of major histocompatibility complex class I (MHCI) genes in response to Hyper-IL-6 exposure, which have been implicated with ASD. We find a small increase in the proportion of radial glia cells after Hyper-IL-6 treatment through immunohistochemistry and single-cell RNA-sequencing. We further show that radial glia cells are the cell type with the highest number of differentially expressed genes, and Hyper-IL-6 treatment leads to the downregulation of genes related to protein translation in line with a mouse model of MIA. Additionally, we identify differentially expressed genes not found in mouse models of MIA, which might drive species-specific responses to MIA. Finally, we show abnormal cortical layering as a long-term consequence of Hyper-IL-6 treatment. In summary, we establish a human 3D model of MIA, which can be used to study the cellular and molecular mechanisms underlying the increased risk for developing disorders such as ASD.
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Engineered Vascularization: Neurovascular Niche Remodeling Enables Biomimetic Brain Organoids for Central Nervous System Research.
Preclinical central nervous system (CNS) disease models face a persistent translational gap arising from interspecies differences and limited representation of the human neurovascular microenvironment. Human induced pluripotent stem cell-derived brain organoids provide a human-relevant platform for modeling brain development and disease, but limited vascularization restricts tissue growth, maturation, and neurovascular function. Engineering approaches, including genetic engineering, biomaterials, 3D bioprinting, and organ-on-a-chip technologies, enable greater control over vascular specification, spatial organization, extracellular matrix properties, and perfusion, facilitating more controlled remodeling of the neurovascular niche. This Review summarizes recent advances in engineered vascularization of brain organoids, focusing on engineering strategies, biological outcomes, and applications in neurodevelopmental research, CNS disease modeling, and preclinical drug screening. Particular emphasis is placed on evaluating vascularization using complementary evidence rather than individual vascular markers. Based on quantitative data from 13 published studies, we propose the Multidimensional Quality Assessment Framework for Vascularized Brain Organoids (MQAF-VBO) and highlight relationships between engineering parameters and biological outcomes. Emerging opportunities in nanoengineered biomaterials, programmable platforms, spatial profiling, integrated biosensing, and adaptive bioelectronic systems are also discussed. Collectively, these advances may facilitate the development of more reproducible and physiologically relevant vascularized brain organoids for CNS disease modeling and preclinical drug evaluation.
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iPSC toolbox for understanding and repairing disrupted brain circuits in autism.
Over the past decade, tremendous progress has been made in defining autism spectrum disorder (ASD) as a disorder of brain connectivity. Indeed, whole-brain imaging studies revealed altered connectivity in the brains of individuals with ASD, and genetic studies identified rare ASD-associated mutations in genes that regulate synaptic development and function. However, it remains unclear how specific mutations alter the development of neuronal connections in different brain regions and whether altered connections can be restored therapeutically. The main challenge is the lack of preclinical models that recapitulate important aspects of human development for studying connectivity. Through recent technological innovations, it is now possible to generate patient- or mutation-specific human neurons or organoids from induced pluripotent stem cells (iPSCs) and to study altered connectivity in vitro or in vivo upon xenotransplantation into an intact rodent brain. Here, we discuss how deficits in neurodevelopmental processes may lead to abnormal brain connectivity and how iPSC-based models can be used to identify abnormal connections and to gain insights into underlying cellular and molecular mechanisms to develop novel therapeutics.
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Probing disrupted neurodevelopment in autism using human stem cell-derived neurons and organoids: An outlook into future diagnostics and drug development.
Autism spectrum disorders (ASDs) represent a spectrum of neurodevelopmental disorders characterized by impaired social interaction, repetitive or restrictive behaviors, and problems with speech. According to a recent report by the Centers for Disease Control and Prevention, one in 68 children in the US is diagnosed with ASDs. Although ASD-related diagnostics and the knowledge of ASD-associated genetic abnormalities have improved in recent years, our understanding of the cellular and molecular pathways disrupted in ASD remains very limited. As a result, no specific therapies or medications are available for individuals with ASDs. In this review, we describe the neurodevelopmental processes that are likely affected in the brains of individuals with ASDs and discuss how patient-specific stem cell-derived neurons and organoids can be used for investigating these processes at the cellular and molecular levels. Finally, we propose a discovery pipeline to be used in the future for identifying the cellular and molecular deficits and developing novel personalized therapies for individuals with idiopathic ASDs.
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Human CNS barrier-forming organoids with cerebrospinal fluid production.
Cerebrospinal fluid (CSF) is a vital liquid, providing nutrients and signaling molecules and clearing out toxic by-products from the brain. The CSF is produced by the choroid plexus (ChP), a protective epithelial barrier that also prevents free entry of toxic molecules or drugs from the blood. Here, we establish human ChP organoids with a selective barrier and CSF-like fluid secretion in self-contained compartments. We show that this in vitro barrier exhibits the same selectivity to small molecules as the ChP in vivo and that ChP-CSF organoids can predict central nervous system (CNS) permeability of new compounds. The transcriptomic and proteomic signatures of ChP-CSF organoids reveal a high degree of similarity to the ChP in vivo. Finally, the intersection of single-cell transcriptomics and proteomic analysis uncovers key human CSF components produced by previously unidentified specialized epithelial subtypes.
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An in vivo and in vitro spatiotemporal profile of human midbrain development.
The dopaminergic system has key roles in human physiology and is implicated in a broad range of neurological and neuropsychiatric conditions that are increasingly investigated using induced pluripotent stem cell-derived midbrain models. To determine similarities of such models to human systems, here we undertake single-cell and spatial profiling of first and second trimester fetal midbrain and compare it to in vitro midbrain models. Histological examination reveals that, by the second trimester, fetal midbrain tissue exhibits structural complexity comparable to that of adults. At the molecular level, single-cell profiling uncovers differences in cellular composition across models, with brain organoids most closely resembling late first trimester tissue - an observation supported by meta-integration of existing midbrain datasets. By reconstructing developmental trajectories of neuronal and astrocytic lineages, we map gene expression dynamics associated with maturation. Importantly, integration of spatial transcriptomics provides critical context for aligning organoid models, revealing that their spatial organization and intercellular signaling resemble the architecture and microenvironment of the second trimester midbrain. Ultimately, we leverage our findings to study Dopamine Transporter Deficiency Syndrome progression in patient-derived midbrain organoids, validating their relevance. Understanding the extent of human tissue recapitulation in midbrain laboratory models is essential to justify their use as biological proxies.
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Light and electron microscopic structure of Golgi-stained neurons in the vertebrate brain (new rapid Golgi procedure).
After application of a rapid, selective silver impregnation procedure for light (LM) and electron (EM) microscopy, individual neurons are distinguished by a light silver precipitation. The silver content is sufficient that entire nerve cells can be observed light microscopically; on the other hand, electron microscopically the cytological details are still visible. Brains of mice were fixed by phosphate-buffered aldehyde perfusion, and pices of tissue left in a 1% K2Cr2O7 solution for 13 h before impregnation in a 0.5% AgNO3 solution for 2 h. Thick sections (30-50 micron) of the impregnated tissue were cut; from these sections, suitably stained neurons were dissected out and re-embedded for ultrathin sectioning, thereby allowing observations on the same neurons at the EM level. A thin silver deposit was observed along the delimiting neuronal membrane, the microtubules and the smooth ER, including the spinal apparatus of the dendritic spines. The fine cytoplasmic details of the impregnated neurons and the surrounding tissue are well preserved and, therefore, suitable for subsequent determination of synaptic relationships of the impregnated neurons with the adjacent neuronal elements.
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Autophagy Activators Normalize Aberrant Tau Proteostasis and Rescue Synapses in Human Familial Alzheimer's Disease iPSC-Derived Cortical Organoids.
Alzheimer's disease (AD) is the leading cause of dementia worldwide. Nevertheless, its cellular and molecular mechanisms remain incompletely understood, partially due to inadequate disease models. To illuminate early changes in AD, we developed a cerebrocortical organoid (CO) model with improved methodology. Our COs produce excitatory and inhibitory neurons alongside glia, utilizing established isogenic wild-type and diseased human induced pluripotent stem cells (hiPSCs) carrying heterozygous familial AD mutations in PSEN1ΔE9/WT, PSEN1M146V/WT, or APPSwe/WT. In addition to amyloid-beta (Aβ) accumulation, the AD COs display time-progressive loss of monomeric Tau, and accumulation of aggregated high-molecular-weight (HMW) phospho(p)-Tau species (pT181 and pT217). They also exhibit neuronal hyperexcitability reminiscent of early electroencephalography (EEG) clinical findings and synapse loss in AD patient brains. Single-cell RNA-sequencing analyses of AD and WT control COs reveal significant divergent molecular abnormalities in excitatory vs. inhibitory neurons, with several pathways being upregulated in one while downregulated in the other, providing insight into AD phenotypes. Finally, we show that chronic dosing with autophagy activators, including a novel mTOR inhibitor-independent drug candidate, prevents pathologic Aβ and HMW p-Tau accumulation, normalizes hyperexcitability, and rescues synaptic loss in AD COs. Collectively, our results demonstrate this CO model as a useful platform for assessing early features of familial AD pathogenesis and for testing small-molecule candidate therapeutics.