Pluripotent stem cells show a remarkable ability to self-organize and differentiate in vitro in three-dimensional aggregates, known as organoids or organ spheroids, and to recapitulate aspects of ...human brain development and function. Region-specific 3D brain cultures can be derived from any individual and assembled to model complex cell-cell interactions and to generate circuits in human brain assembloids. Here I discuss how this approach can be used to understand unique features of the human brain and to gain insights into neuropsychiatric disorders. In addition, I consider the challenges faced by researchers in further improving and developing methods to probe and manipulate patient-derived 3D brain cultures.
Genetic perturbations of cortical development can lead to neurodevelopmental disease, including autism spectrum disorder (ASD). To identify genomic regions crucial to corticogenesis, we mapped the ...activity of gene-regulatory elements generating a single-cell atlas of gene expression and chromatin accessibility both independently and jointly. This revealed waves of gene regulation by key transcription factors (TFs) across a nearly continuous differentiation trajectory, distinguished the expression programs of glial lineages, and identified lineage-determining TFs that exhibited strong correlation between linked gene-regulatory elements and expression levels. These highly connected genes adopted an active chromatin state in early differentiating cells, consistent with lineage commitment. Base-pair-resolution neural network models identified strong cell-type-specific enrichment of noncoding mutations predicted to be disruptive in a cohort of ASD individuals and identified frequently disrupted TF binding sites. This approach illustrates how cell-type-specific mapping can provide insights into the programs governing human development and disease.
Display omitted
•Single-cell RNA and chromatin profiling charts human corticogenesis•Distinct TFs underlie neurogenesis and gliogenesis regulatory programs•Lineage-determining TFs adopt an active chromatin state early in differentiation•Neural networks prioritize noncoding de novo mutations in autism spectrum disorder
A single-cell atlas of gene expression and chromatin accessibility of human developing cortex during mid-gestation reveals lineage-determining transcription factors for human corticogenesis and identifies prioritized mutations for autism spectrum disorder.
The differentiation of pluripotent stem cells in three-dimensional cultures can recapitulate key aspects of brain development, but protocols are prone to variable results. Here we differentiated ...multiple human pluripotent stem cell lines for over 100 d using our previously developed approach to generate brain-region-specific organoids called cortical spheroids and, using several assays, found that spheroid generation was highly reliable and consistent. We anticipate the use of this approach for large-scale differentiation experiments and disease modeling.
The construction of the human nervous system is a distinctly complex although highly regulated process. Human tissue inaccessibility has impeded a molecular understanding of the developmental ...specializations from which our unique cognitive capacities arise. A confluence of recent technological advances in genomics and stem cell-based tissue modeling is laying the foundation for a new understanding of human neural development and dysfunction in neuropsychiatric disease. Here, we review recent progress on uncovering the cellular and molecular principles of human brain organogenesis in vivo as well as using organoids and assembloids in vitro to model features of human evolution and disease.
The complexities of human brain development and neuropsychiatric disease are being illuminated through advances in genomics and stem cell-based models.
The biology of the human brain, and in particular the dynamic interactions between the numerous cell types and regions of the central nervous system, has been difficult to study due to limited access ...to functional brain tissue. Technologies to derive brain organoids and assembloids from human pluripotent stem cells are increasingly utilized to model, in progressively complex preparations, the crosstalk between cell types in development and disease. Here, we review the use of these human cellular models to study cell–cell interactions among progenitors, neurons, astrocytes, oligodendrocytes, cancer cells, and non-central nervous system cell types, as well as efforts to study connectivity between brain regions following controlled assembly of organoids. Ultimately, the promise of these patient-derived preparations is to uncover previously inaccessible features of brain function that emerge from complex cell–cell interactions and to improve our mechanistic understanding of neuropsychiatric disorders.
Human pluripotent stem cell-derived brain organoids produce a diversity of cell types that interact with each other in a complex 3D environment.Combining organoids resembling distinct areas into assembloids can be used to model aspects of interactions that occur between regions in the human brain.Organoids can be supplemented with non-central nervous system-derived cell types, including microglia and endothelial cells, to study the interplay of nervous system cells with immune cells and blood vessels.Patient-derived organoids can be genetically manipulated or infected with pathogens and subsequently used as tools for studying disease processes in a human context.
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.
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 Ca
1.2 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.
Human stem-cell-derived models provide the promise of accelerating our understanding of brain disorders, but not knowing whether they possess the ability to mature beyond mid- to late-fetal stages ...potentially limits their utility. We leveraged a directed differentiation protocol to comprehensively assess maturation in vitro. Based on genome-wide analysis of the epigenetic clock and transcriptomics, as well as RNA editing, we observe that three-dimensional human cortical organoids reach postnatal stages between 250 and 300 days, a timeline paralleling in vivo development. We demonstrate the presence of several known developmental milestones, including switches in the histone deacetylase complex and NMDA receptor subunits, which we confirm at the protein and physiological levels. These results suggest that important components of an intrinsic in vivo developmental program persist in vitro. We further map neurodevelopmental and neurodegenerative disease risk genes onto in vitro gene expression trajectories to provide a resource and webtool (Gene Expression in Cortical Organoids, GECO) to guide disease modeling.
Investigating human oligodendrogenesis and the interaction of oligodendrocytes with neurons and astrocytes would accelerate our understanding of the mechanisms underlying white matter disorders. ...However, this is challenging because of the limited accessibility of functional human brain tissue. Here, we developed a new differentiation method of human induced pluripotent stem cells to generate three-dimensional brain organoids that contain oligodendrocytes as well as neurons and astrocytes, called human oligodendrocyte spheroids. We found that oligodendrocyte lineage cells derived in human oligodendrocyte spheroids transitioned through developmental stages similar to primary human oligodendrocytes and that the migration of oligodendrocyte lineage cells and their susceptibility to lysolecithin exposure could be captured by live imaging. Moreover, their morphology changed as they matured over time in vitro and started myelinating neurons. We anticipate that this method can be used to study oligodendrocyte development, myelination, and interactions with other major cell types in the CNS.
Self-organizing neural organoids represent a promising in vitro platform with which to model human development and disease
. However, organoids lack the connectivity that exists in vivo, which limits ...maturation and makes integration with other circuits that control behaviour impossible. Here we show that human stem cell-derived cortical organoids transplanted into the somatosensory cortex of newborn athymic rats develop mature cell types that integrate into sensory and motivation-related circuits. MRI reveals post-transplantation organoid growth across multiple stem cell lines and animals, whereas single-nucleus profiling shows progression of corticogenesis and the emergence of activity-dependent transcriptional programs. Indeed, transplanted cortical neurons display more complex morphological, synaptic and intrinsic membrane properties than their in vitro counterparts, which enables the discovery of defects in neurons derived from individuals with Timothy syndrome. Anatomical and functional tracings show that transplanted organoids receive thalamocortical and corticocortical inputs, and in vivo recordings of neural activity demonstrate that these inputs can produce sensory responses in human cells. Finally, cortical organoids extend axons throughout the rat brain and their optogenetic activation can drive reward-seeking behaviour. Thus, transplanted human cortical neurons mature and engage host circuits that control behaviour. We anticipate that this approach will be useful for detecting circuit-level phenotypes in patient-derived cells that cannot otherwise be uncovered.
PDF
