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Nature May 2017The development of the nervous system involves a coordinated succession of events including the migration of GABAergic (γ-aminobutyric-acid-releasing) neurons from...
The development of the nervous system involves a coordinated succession of events including the migration of GABAergic (γ-aminobutyric-acid-releasing) neurons from ventral to dorsal forebrain and their integration into cortical circuits. However, these interregional interactions have not yet been modelled with human cells. Here we generate three-dimensional spheroids from human pluripotent stem cells that resemble either the dorsal or ventral forebrain and contain cortical glutamatergic or GABAergic neurons. These subdomain-specific forebrain spheroids can be assembled in vitro to recapitulate the saltatory migration of interneurons observed in the fetal forebrain. Using this system, we find that in Timothy syndrome-a neurodevelopmental disorder that is caused by mutations in the Ca1.2 calcium channel-interneurons display abnormal migratory saltations. We also show that after migration, interneurons functionally integrate with glutamatergic neurons to form a microphysiological system. We anticipate that this approach will be useful for studying neural development and disease, and for deriving spheroids that resemble other brain regions to assemble circuits in vitro.
Topics: Autistic Disorder; Cell Line; Cell Movement; Cells, Cultured; Female; GABAergic Neurons; Glutamic Acid; Humans; Interneurons; Long QT Syndrome; Male; Models, Biological; Neurogenesis; Neurons; Pluripotent Stem Cells; Prosencephalon; Spheroids, Cellular; Synapses; Syndactyly
PubMed: 28445465
DOI: 10.1038/nature22330 -
Nature Jun 2019Experimental models of the human brain are needed for basic understanding of its development and disease. Human brain organoids hold unprecedented promise for this...
Experimental models of the human brain are needed for basic understanding of its development and disease. Human brain organoids hold unprecedented promise for this purpose; however, they are plagued by high organoid-to-organoid variability. This has raised doubts as to whether developmental processes of the human brain can occur outside the context of embryogenesis with a degree of reproducibility that is comparable to the endogenous tissue. Here we show that an organoid model of the dorsal forebrain can reliably generate a rich diversity of cell types appropriate for the human cerebral cortex. We performed single-cell RNA-sequencing analysis of 166,242 cells isolated from 21 individual organoids, finding that 95% of the organoids generate a virtually indistinguishable compendium of cell types, following similar developmental trajectories and with a degree of organoid-to-organoid variability comparable to that of individual endogenous brains. Furthermore, organoids derived from different stem cell lines show consistent reproducibility in the cell types produced. The data demonstrate that reproducible development of the complex cellular diversity of the central nervous system does not require the context of the embryo, and that establishment of terminal cell identity is a highly constrained process that can emerge from diverse stem cell origins and growth environments.
Topics: Cell Line; Cerebral Cortex; Female; Fetus; Humans; Induced Pluripotent Stem Cells; Male; Organoids; Prosencephalon; RNA-Seq; Reproducibility of Results; Single-Cell Analysis; Time Factors; Tissue Culture Techniques; Transcriptome
PubMed: 31168097
DOI: 10.1038/s41586-019-1289-x -
Nature Protocols Sep 2018The ability to generate region-specific three-dimensional (3D) models to study human brain development offers great promise for understanding the nervous system in both...
The ability to generate region-specific three-dimensional (3D) models to study human brain development offers great promise for understanding the nervous system in both healthy individuals and patients. In this protocol, we describe how to generate and assemble subdomain-specific forebrain spheroids, also known as brain region-specific organoids, from human pluripotent stem cells (hPSCs). We describe how to pattern the neural spheroids toward either a dorsal forebrain or a ventral forebrain fate, establishing human cortical spheroids (hCSs) and human subpallial spheroids (hSSs), respectively. We also describe how to combine the neural spheroids in vitro to assemble forebrain assembloids that recapitulate the interactions of glutamatergic and GABAergic neurons seen in vivo. Astrocytes are also present in the human forebrain-specific spheroids, and these undergo maturation when the forebrain spheroids are cultured long term. The initial generation of neural spheroids from hPSCs occurs in <1 week, with regional patterning occurring over the subsequent 5 weeks. After the maturation stage, brain region-specific spheroids are amenable to a variety of assays, including live-cell imaging, calcium dynamics, electrophysiology, cell purification, single-cell transcriptomics, and immunohistochemistry studies. Once generated, forebrain spheroids can also be matured for >24 months in culture.
Topics: Humans; Models, Biological; Organ Culture Techniques; Organogenesis; Organoids; Pluripotent Stem Cells; Prosencephalon; Tissue Engineering
PubMed: 30202107
DOI: 10.1038/s41596-018-0032-7 -
Nature Oct 2023The assembly of cortical circuits involves the generation and migration of interneurons from the ventral to the dorsal forebrain, which has been challenging to study at...
The assembly of cortical circuits involves the generation and migration of interneurons from the ventral to the dorsal forebrain, which has been challenging to study at inaccessible stages of late gestation and early postnatal human development. Autism spectrum disorder and other neurodevelopmental disorders (NDDs) have been associated with abnormal cortical interneuron development, but which of these NDD genes affect interneuron generation and migration, and how they mediate these effects remains unknown. We previously developed a platform to study interneuron development and migration in subpallial organoids and forebrain assembloids. Here we integrate assembloids with CRISPR screening to investigate the involvement of 425 NDD genes in human interneuron development. The first screen aimed at interneuron generation revealed 13 candidate genes, including CSDE1 and SMAD4. We subsequently conducted an interneuron migration screen in more than 1,000 forebrain assembloids that identified 33 candidate genes, including cytoskeleton-related genes and the endoplasmic reticulum-related gene LNPK. We discovered that, during interneuron migration, the endoplasmic reticulum is displaced along the leading neuronal branch before nuclear translocation. LNPK deletion interfered with this endoplasmic reticulum displacement and resulted in abnormal migration. These results highlight the power of this CRISPR-assembloid platform to systematically map NDD genes onto human development and reveal disease mechanisms.
Topics: Female; Humans; Infant, Newborn; Pregnancy; Cell Movement; CRISPR-Cas Systems; Gene Editing; Interneurons; Neurodevelopmental Disorders; Organoids; Endoplasmic Reticulum; Prosencephalon; Active Transport, Cell Nucleus
PubMed: 37758944
DOI: 10.1038/s41586-023-06564-w -
Nature Biotechnology Jul 2017Three-dimensional cell culture models have either relied on the self-organizing properties of mammalian cells or used bioengineered constructs to arrange cells in an...
Three-dimensional cell culture models have either relied on the self-organizing properties of mammalian cells or used bioengineered constructs to arrange cells in an organ-like configuration. While self-organizing organoids excel at recapitulating early developmental events, bioengineered constructs reproducibly generate desired tissue architectures. Here, we combine these two approaches to reproducibly generate human forebrain tissue while maintaining its self-organizing capacity. We use poly(lactide-co-glycolide) copolymer (PLGA) fiber microfilaments as a floating scaffold to generate elongated embryoid bodies. Microfilament-engineered cerebral organoids (enCORs) display enhanced neuroectoderm formation and improved cortical development. Furthermore, reconstitution of the basement membrane leads to characteristic cortical tissue architecture, including formation of a polarized cortical plate and radial units. Thus, enCORs model the distinctive radial organization of the cerebral cortex and allow for the study of neuronal migration. Our data demonstrate that combining 3D cell culture with bioengineering can increase reproducibility and improve tissue architecture.
Topics: Batch Cell Culture Techniques; Cells, Cultured; Guided Tissue Regeneration; Humans; Neural Stem Cells; Neurogenesis; Organ Culture Techniques; Organoids; Prosencephalon; Tissue Engineering
PubMed: 28562594
DOI: 10.1038/nbt.3906 -
Nature Neuroscience Aug 2019The medial prefrontal cortex (mPFC) contains populations of GABAergic interneurons that play different roles in cognition and emotion. Their local and long-range inputs...
The medial prefrontal cortex (mPFC) contains populations of GABAergic interneurons that play different roles in cognition and emotion. Their local and long-range inputs are incompletely understood. We used monosynaptic rabies viral tracers in combination with fluorescence micro-optical sectioning tomography to generate a whole-brain atlas of direct long-range inputs to GABAergic interneurons in the mPFC of male mice. We discovered that three subtypes of GABAergic interneurons in two areas of the mPFC are innervated by same upstream areas. Input from subcortical upstream areas includes cholinergic neurons from the basal forebrain and serotonergic neurons (which co-release glutamate) from the raphe nuclei. Reconstruction of single-neuron morphology revealed novel substantia innominata-anteromedial thalamic nucleus-mPFC and striatum-anteromedial thalamic nucleus-mPFC circuits. Based on the projection logic of individual neurons, we classified cortical and hippocampal input neurons into several types. This atlas provides the anatomical foundation for understanding the functional organization of the mPFC.
Topics: Animals; Brain Mapping; Cell Count; Hippocampus; Interneurons; Male; Mice; Mice, Inbred C57BL; Parasympathetic Nervous System; Prefrontal Cortex; Prosencephalon; Raphe Nuclei; Serotonergic Neurons; Thalamus; gamma-Aminobutyric Acid
PubMed: 31285615
DOI: 10.1038/s41593-019-0429-9 -
Cell Nov 2017N-methyladenosine (mA), installed by the Mettl3/Mettl14 methyltransferase complex, is the most prevalent internal mRNA modification. Whether mA regulates mammalian brain...
N-methyladenosine (mA), installed by the Mettl3/Mettl14 methyltransferase complex, is the most prevalent internal mRNA modification. Whether mA regulates mammalian brain development is unknown. Here, we show that mA depletion by Mettl14 knockout in embryonic mouse brains prolongs the cell cycle of radial glia cells and extends cortical neurogenesis into postnatal stages. mA depletion by Mettl3 knockdown also leads to a prolonged cell cycle and maintenance of radial glia cells. mA sequencing of embryonic mouse cortex reveals enrichment of mRNAs related to transcription factors, neurogenesis, the cell cycle, and neuronal differentiation, and mA tagging promotes their decay. Further analysis uncovers previously unappreciated transcriptional prepatterning in cortical neural stem cells. mA signaling also regulates human cortical neurogenesis in forebrain organoids. Comparison of mA-mRNA landscapes between mouse and human cortical neurogenesis reveals enrichment of human-specific mA tagging of transcripts related to brain-disorder risk genes. Our study identifies an epitranscriptomic mechanism in heightened transcriptional coordination during mammalian cortical neurogenesis.
Topics: Animals; Cell Cycle; Gene Expression Regulation; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Humans; Methylation; Methyltransferases; Mice; Mice, Knockout; Neural Stem Cells; Neurogenesis; Organoids; Prosencephalon; RNA Processing, Post-Transcriptional; RNA Stability; RNA, Messenger
PubMed: 28965759
DOI: 10.1016/j.cell.2017.09.003 -
American Journal of Medical Genetics.... Jun 2018Holoprosencephaly (HPE) is a primary disorder of neural induction and patterning of the rostral neural tube resulting in noncleavage of the forebrain with failure to... (Review)
Review
Holoprosencephaly (HPE) is a primary disorder of neural induction and patterning of the rostral neural tube resulting in noncleavage of the forebrain with failure to form two separate distinct hemispheres. The spectrum of HPE is very broad and encompasses various neuropathological phenotypes of different severity. The recent literature has demonstrated that the phenotypic variability of HPE ranges from aprosencephaly-atelencephaly, at the most severe end, to milder forms such as the "middle interhemispheric variant" of HPE at the less severe end of the spectrum. Between them, different intermediate forms demonstrate a continuum in a wide phenotypic spectrum rather than well-defined categories. Although the term "HPE" suggests a disorder affecting only the prosencephalon, other brain structures are involved, underlining the complexity of the malformation. Because of close spatiotemporal interactions and common signaling pathways contributing to the development of both brain and face, concomitant facial and ocular anomalies are associated with brain malformation. In this review, the characteristic neuropathological features of the various forms of HPE are described as well as their associated brain, face, and ocular malformations, to delineate the different phenotypes.
Topics: Anencephaly; Brain; Central Nervous System; Dandy-Walker Syndrome; Eye Abnormalities; Face; Holoprosencephaly; Humans; Prosencephalon; Spinal Cord
PubMed: 30182440
DOI: 10.1002/ajmg.c.31623 -
International Journal of Molecular... May 2021A prolonged developmental timeline for GABA (γ-aminobutyric acid)-expressing inhibitory neurons (GABAergic interneurons) is an amplified trait in larger, gyrencephalic... (Review)
Review
A prolonged developmental timeline for GABA (γ-aminobutyric acid)-expressing inhibitory neurons (GABAergic interneurons) is an amplified trait in larger, gyrencephalic animals. In several species, the generation, migration, and maturation of interneurons take place over several months, in some cases persisting after birth. The late integration of GABAergic interneurons occurs in a region-specific pattern, especially during the early postnatal period. These changes can contribute to the formation of functional connectivity and plasticity, especially in the cortical regions responsible for higher cognitive tasks. In this review, we discuss GABAergic interneuron development in the late gestational and postnatal forebrain. We propose the protracted development of interneurons at each stage (neurogenesis, neuronal migration, and network integration), as a mechanism for increased complexity and cognitive flexibility in larger, gyrencephalic brains. This developmental feature of interneurons also provides an avenue for environmental influences to shape neural circuit formation.
Topics: Animals; Animals, Newborn; Female; Gestational Age; Interneurons; Pregnancy; Prosencephalon; gamma-Aminobutyric Acid
PubMed: 34066025
DOI: 10.3390/ijms22105113 -
PLoS Biology Nov 2022The striatum links neuronal circuits in the human brain, and its malfunction causes neuronal disorders such as Huntington's disease (HD). A human striatum model that...
The striatum links neuronal circuits in the human brain, and its malfunction causes neuronal disorders such as Huntington's disease (HD). A human striatum model that recapitulates fetal striatal development is vital to decoding the pathogenesis of striatum-related neurological disorders and developing therapeutic strategies. Here, we developed a method to construct human striatal organoids (hStrOs) from human pluripotent stem cells (hPSCs), including hStrOs-derived assembloids. Our hStrOs partially replicated the fetal striatum and formed striosome and matrix-like compartments in vitro. Single-cell RNA sequencing revealed distinct striatal lineages in hStrOs, diverging from dorsal forebrain fate. Using hStrOs-derived assembloids, we replicated the striatal targeting projections from different brain parts. Furthermore, hStrOs can serve as hosts for striatal neuronal allografts to test allograft neuronal survival and functional integration. Our hStrOs are suitable for studying striatal development and related disorders, characterizing the neural circuitry between different brain regions, and testing therapeutic strategies.
Topics: Humans; Organoids; Pluripotent Stem Cells; Corpus Striatum; Neostriatum; Prosencephalon
PubMed: 36395338
DOI: 10.1371/journal.pbio.3001868