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Cortical organoids model early brain development disrupted by 16p11.2 copy number variants in autism.
Reciprocal deletion and duplication of the 16p11.2 region is the most common copy number variation (CNV) associated with autism spectrum disorders. We generated cortical organoids from skin fibroblasts of patients with 16p11.2 CNV to investigate impacted neurodevelopmental processes. We show that organoid size recapitulates macrocephaly and microcephaly phenotypes observed in the patients with 16p11.2 deletions and duplications. The CNV dosage affects neuronal maturation, proliferation, and synapse number, in addition to its effect on organoid size. We demonstrate that 16p11.2 CNV alters the ratio of neurons to neural progenitors in organoids during early neurogenesis, with a significant excess of neurons and depletion of neural progenitors observed in deletions. Transcriptomic and proteomic profiling revealed multiple pathways dysregulated by the 16p11.2 CNV, including neuron migration, actin cytoskeleton, ion channel activity, synaptic-related functions, and Wnt signaling. The level of the active form of small GTPase RhoA was increased in both, deletions and duplications. Inhibition of RhoA activity rescued migration deficits, but not neurite outgrowth. This study provides insights into potential neurobiological mechanisms behind the 16p11.2 CNV during neocortical development.
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An update on stem cell biology and engineering for brain development.
Two recent technologies, induced-pluripotent stem cells (iPSCs) and direct somatic reprogramming, have shown enormous potential for cell-based therapies against intractable diseases such as those that affect the central nervous system. Already, methods that generate most major cell types of the human brain exist. Whether the cell types are directly reprogrammed from human somatic cells or differentiated from an iPSC intermediate, the overview presented here demonstrates how these protocols vary greatly in their efficiencies, purity and maturation of the resulting cells. Possible solutions including micro-RNA switch technologies that purify target cell types are also outlined. Further, an update on the transition from 2D to 3D cultures and current organoid (mini-brain) cultures are reviewed to give the stem cell and developmental engineering communities an up-to-date account of the progress and future perspectives for modeling of central nervous system disease and brain development in vitro.
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Label-free three-photon imaging of intact human cerebral organoids for tracking early events in brain development and deficits in Rett syndrome.
Human cerebral organoids are unique in their development of progenitor-rich zones akin to ventricular zones from which neuronal progenitors differentiate and migrate radially. Analyses of cerebral organoids thus far have been performed in sectioned tissue or in superficial layers due to their high scattering properties. Here, we demonstrate label-free three-photon imaging of whole, uncleared intact organoids (~2 mm depth) to assess early events of early human brain development. Optimizing a custom-made three-photon microscope to image intact cerebral organoids generated from Rett Syndrome patients, we show defects in the ventricular zone volumetric structure of mutant organoids compared to isogenic control organoids. Long-term imaging live organoids reveals that shorter migration distances and slower migration speeds of mutant radially migrating neurons are associated with more tortuous trajectories. Our label-free imaging system constitutes a particularly useful platform for tracking normal and abnormal development in individual organoids, as well as for screening therapeutic molecules via intact organoid imaging.
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Brain endothelial spheroids and cortical organoids reveal the impact of Toxoplasma gondii lineage and host-phagocyte-pathogen interactions on colonization.
Toxoplasma gondii chronically infects the central nervous system (CNS), but the mechanisms enabling its traversal of the blood-brain barrier (BBB) remain unclear. Here, we investigated BBB penetration using brain endothelial spheroids and cerebral tissue-derived organoids that recapitulate three-dimensional barrier features. We show that T. gondii tachyzoites efficiently colonize spheroids, without detectable barrier disruption or obligatory parasite replication. Following direct transmigration, tachyzoites invaded and replicated within deeper cell layers. Type I strains (RH, CPS) exhibited higher colonization efficiency than type II strains (PRU, ME49), independent of replication. In contrast, when spheroids were exposed to T. gondii-infected dendritic cells (DCs), both strain types were transported similarly into deep cellular layers. Infected DCs adopted an amoeboid-like migratory phenotype that facilitated parasite transport and subsequent dissemination after egress. Colonization was attenuated by ICAM-1 blockade or heparin treatment, while the parasite effector GRA15, despite modulating DC-endothelial adhesion, did not significantly impact intratissue migration. In contrast, deletion of the effector TgWIP markedly reduced the number of infected DCs entering the spheroids. Similar colonization dynamics were observed in murine cerebral organoids. Collectively, these findings highlight spheroid and organoid models as robust systems for uncovering the cellular and molecular mechanisms underlying T. gondii BBB traversal and CNS colonization.
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Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders.
The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.
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A human tissue screen identifies a regulator of ER secretion as a brain-size determinant.
Loss-of-function (LOF) screens provide a powerful approach to identify regulators in biological processes. Pioneered in laboratory animals, LOF screens of human genes are currently restricted to two-dimensional cell cultures, which hinders the testing of gene functions requiring tissue context. Here, we present CRISPR-lineage tracing at cellular resolution in heterogeneous tissue (CRISPR-LICHT), which enables parallel LOF studies in human cerebral organoid tissue. We used CRISPR-LICHT to test 173 microcephaly candidate genes, revealing 25 to be involved in known and uncharacterized microcephaly-associated pathways. We characterized IER3IP1, which regulates the endoplasmic reticulum (ER) function and extracellular matrix protein secretion crucial for tissue integrity, the dysregulation of which results in microcephaly. Our human tissue screening technology identifies microcephaly genes and mechanisms involved in brain-size control.
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Brain organoids in environmental neurotoxicology: applications, mechanisms, and future perspectives.
The advent of human induced pluripotent stem cell (hiPSC)-derived brain organoids represents a significant advance in environmental neurotoxicology, propelling the discipline toward human-relevant, mechanistic, and predictive in vitro paradigms. This review explores the utility of brain organoids in environmental neurotoxicology, which uniquely address critical limitations of traditional models by recapitulating key aspects of human brain development, including three-dimensional (3D) cytoarchitecture, multilineage cellular heterogeneity, and functional network activity. This review systematically elaborates on their construction principles, unique advantages in neurotoxicological research, and the significant progress made in elucidating mechanisms of toxicity. Notably, brain organoids exhibit enhanced sensitivity in identifying the subtle adverse outcomes of chronic, low-dose exposures to environmental contaminants, often eluding conventional approaches. Their key advantage lies in the greater capacity to deconstruct complex toxicological pathways, enabling precise tracing of adverse outcome pathways (AOPs). Future development requires enhancing model complexity through vascularization, promoting automation and standardization, and integrating artificial intelligence (AI) for data analysis. Concurrently, establishing sustained ethical oversight and standardized frameworks is essential to ultimately advance the field toward more precise and efficient hazard identification and risk characterization.Highlights1. Human brain organoids bridge species gaps for more human-relevant neurotoxicity assessment.2. Brain organoids effectively recapitulate chronic, low-dose and mixture environmental risks.3. Integrating brain organoid data into the AOP frameworks enhances mechanistic understanding.4. Coupling brain organoids with organ-on-a-chip and AI advances next-generation risk assessment.
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Probing impaired neurogenesis in human brain organoids exposed to alcohol.
The fetal brain is highly vulnerable to ethanol exposure, which can trigger various long-term neuronal disabilities and cognitive dysfunctions. However, a comprehensive understanding of fetal brain development under ethanol exposure is challenging due to the limitations of animal models. Here, we propose a human induced pluripotent stem cell (hiPSC)-based 3D brain organoid model, and explore the mechanisms underlying neural dysfunctions in prenatal alcohol exposure (PAE) in vitro. Brain organoids were examined to resemble brain organogenesis in vivo at early stages during gestation, with specific features of neuronal differentiation, brain regionalization, and cortical organization. With ethanol exposure, the brain organoids displayed attenuated neurite outgrowth and skewed neural maturation. Transcriptome analysis identified a series of new markedly altered genes and enriched pathways, such as GSX2, RSPO2, and the Hippo signaling pathway. These genes or pathways, to our knowledge, were reported to be involved in ethanol-induced impaired neurogenesis for the first time. Our new findings might facilitate better understanding of the various postnatal neural disorders observed in individuals with PAE.
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EpCAM Is a Surface Marker for Enriching Anterior Pituitary Cells From Human Hypothalamic-Pituitary Organoids.
Human stem cell-derived organoid culture enables the in vitro analysis of the cellular function in three-dimensional aggregates mimicking native organs, and also provides a valuable source of specific cell types in the human body. We previously established organoid models of the hypothalamic-pituitary (HP) complex using human pluripotent stem cells. Although the models are suitable for investigating developmental and functional HP interactions, we consider that isolated pituitary cells are also useful for basic and translational research on the pituitary gland, such as stem cell biology and regenerative medicine. To develop a method for the purification of pituitary cells in HP organoids, we performed surface marker profiling of organoid cells derived from human induced pluripotent stem cells (iPSCs). Screening of 332 human cell surface markers and a subsequent immunohistochemical analysis identified epithelial cell adhesion molecule (EpCAM) as a surface marker of anterior pituitary cells, as well as their ectodermal precursors. EpCAM was not expressed on hypothalamic lineages; thus, anterior pituitary cells were successfully enriched by magnetic separation of EpCAM+ cells from iPSC-derived HP organoids. The enriched pituitary population contained functional corticotrophs and their progenitors; the former responded normally to a corticotropin-releasing hormone stimulus. Our findings would extend the applicability of organoid culture as a novel source of human anterior pituitary cells, including stem/progenitor cells and their endocrine descendants.
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Inferring and perturbing cell fate regulomes in human brain organoids.
Self-organizing neural organoids grown from pluripotent stem cells1-3 combined with single-cell genomic technologies provide opportunities to examine gene regulatory networks underlying human brain development. Here we acquire single-cell transcriptome and accessible chromatin data over a dense time course in human organoids covering neuroepithelial formation, patterning, brain regionalization and neurogenesis, and identify temporally dynamic and brain-region-specific regulatory regions. We developed Pando-a flexible framework that incorporates multi-omic data and predictions of transcription-factor-binding sites to infer a global gene regulatory network describing organoid development. We use pooled genetic perturbation with single-cell transcriptome readout to assess transcription factor requirement for cell fate and state regulation in organoids. We find that certain factors regulate the abundance of cell fates, whereas other factors affect neuronal cell states after differentiation. We show that the transcription factor GLI3 is required for cortical fate establishment in humans, recapitulating previous research performed in mammalian model systems. We measure transcriptome and chromatin accessibility in normal or GLI3-perturbed cells and identify two distinct GLI3 regulomes that are central to telencephalic fate decisions: one regulating dorsoventral patterning with HES4/5 as direct GLI3 targets, and one controlling ganglionic eminence diversification later in development. Together, we provide a framework for how human model systems and single-cell technologies can be leveraged to reconstruct human developmental biology.